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PubMed2002

The telomeric length and heterogeneity decrease with age in normal human oral keratinocytes.

We have examined the telomere length in NHOK explanted from 28 donors between the ages of 21 and 84 years. Genomic DNA was isolated from exponentially replicating NHOK and digested with HinFI to yield terminal restriction fragments (TRF). The TRF length ranged from 4.1 to 7.0 kbp with a mean of 5.3 +/- 0.8 kbp, which was significantly shorter than that (8.9 +/- 1.0 kbp) of normal human oral fibroblasts (NHOF). The TRF length was inversely correlated to the increase of donor age in NHOK (m=-23 bp per year; r=-0.60; P<0.001). Also, the heterogeneity of TRF length in cultured NHOK decreased with increased donor age (r=-0.38, P<0.05). These data indicated that clonogenic NHOK cells had replicated in situ and showed a progressive shortening of TRF length. The short telomere length and decreased telomeric length heterogeneity in immortalized cells suggested that there is a critical minimum for cell survival.

Adult↗
PubMed1998

Temporal sampling requirements for the tracer kinetics modeling of breast disease.

The physiological parameters measured in the tracer kinetics modeling of data from a dynamic contrast-enhanced magnetic resonance (MR) breast exam (blood flow-extraction fraction product [FE], volume of the extracellular extravascular space [Ve], and blood volume [Vb]) may enable non-invasive diagnosis of breast cancer. One of the factors that compromises the accuracy and precision of the parameter estimates, and therefore their diagnostic potential, is the temporal resolution of the MR scans used to measure contrast agent (gadolinium-diethylenetriamine pentaacetic acid [Gd-DTPA]) concentration in an artery (arterial input function [AIF]) and in the tissue (tissue residue function [TRF]). Using computer simulations, we have examined, for several AIF widths, the errors introduced into estimates of tracer kinetic parameters in breast tissue due to insufficient temporal sampling. Temporal sampling errors can be viewed as uncertainties and biases in the parameter estimates introduced by the uncertainty in the relative alignments of the AIF, TRF, and sampling grid. These effects arise from the model's inherent sensitivity to error in either the AIF or TRF, which is dependent on the values of the tracer kinetic parameters and increases with AIF width. Based on the results of the simulations, to ensure that the error in FE and Ve will be under 10% of their true values, we recommend a rapid bolus injection of contrast agent (approximately 10 s), that the AIF be sampled every second, and that the TRF be sampled every 16 s or less. An accurate measurement of Vb requires that the TRF be sampled at least every 4 s. The results of these investigations can be used to set minimum dynamic imaging rates for tracer kinetics modeling of the breast.

Breast↗
PubMed2000

Analysis of telomere length and thymic output in fast and slow/non-progressors with HIV infection.

There are two models for CD4+ T-cell depletion leading to AIDS: a kinetic model and an immune suppression model. In the kinetic model, direct cell killing due to viral replication results in a continuous demand for CD4+ T-cells, which eventually exhausts their capacity for renewal by proliferative mechanisms. In the immune suppression model, CD4+ T-cell decline is due to an indirect global inhibitory effect of the virus on uninfected immune cell function. In order to address differences in the two models, we investigated proliferative history and thymic output in PBMC from the GRIV cohort of fast (FP) and slow/non-progressors (S/NP), and uninfected controls. Proliferative history and thymic output were assessed by measurement of mean telomeric restriction fragment (TRF) length and T-cell receptor Rearrangement Excision Circles (TREC) levels, respectively. Mean TRF lengths were significantly shorter in S/NP (n = 93, 7.59 +/- 0.11 kb) and FP (n = 42, 7.25 +/- 0.15 kb) compared to controls (n = 35, 9.17 +/- 0.19 kb). Mean TRF length in PBMC (n = 9, 7.32 +/- 0.31 kb) and CD4+ enriched fractions (n = 9, 7.41 +/- 0.30 kb) from a subset of non-GRIV HIV-1 infected samples were also significantly smaller than PBMC (n = 8, 9.77 +/- 0.33 kb) and CD4+ fractions (n = 8, 9.41 +/- 0.32 kb) from uninfected controls. Rates of telomeric shortening, however, were similar among S/NP (n = 93, -45 +/- 20 bp/yr), FP (n = 42, -41 +/- 14 bp/yr) and controls (-29 +/- 17 bp/yr). Paralleling differences observed in mean TRF length, TREC levels were significantly reduced in S/NP (n = 10, 3,433 +/- 843 mol/mu and FP (n = 8, 1,193 +/- 413) compared to controls (n = 15, 22,706 +/- 5,089), indicative of a defect in thymopoiesis in HIV-1 infection. When evaluated in the context of reduced thymopoiesis, the difference in mean TRF length between S/NP and controls (1.58 +/- 0.30 kb) is similar to that observed between memory and naïve T-cells (1.4 +/- 0.1 kb), and may reflect perturbations in the peripheral T-cell population due to a decline in thymic output of naïve T-cells rather than increased turnover. Based on the different clinical criteria used to select S/NP and FP, the sight difference in TREC between these two groups suggests the threshold for pathogenesis as a result of naïve T-cell depletion may be quite low, and incremental increases in thymic output may yield substantial clinical results. Future studies regarding therapeutic vaccination, specifically with HIV-1 Tat targeted anti-immunosuppressive vaccines, should address the defect in thymic output in HIV-1 infection by using TREC analysis as a rapid method for biological evaluation.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Tocotrienols from palm oil as effective inhibitors of protein oxidation and lipid peroxidation in rat liver microsomes.

Tocotrienols from palm oil showed significant ability to inhibit oxidative damage induced by ascorbate-Fe2+ and photosensitization, involving different mechanisms, in rat liver microsomes. The tocotrienol-rich fraction from palm oil (TRF), being tried as a more economical and efficient substitute for alpha-tocopherol, showed time- and concentration-dependent inhibition of protein oxidation as well as lipid peroxidation. It was more effective against protein oxidation. The extent of inhibition by TRF varied with different peroxidation products such as conjugated dienes, lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS). Among the constituents of TRF, gamma-tocotrienol was the most effective followed by its alpha- and delta-isomers. In general, at a low concentration of 5 microM, TRF was able to prevent oxidative damage to significant extent (37% inhibition of protein oxidation and 27-30% of lipid peroxidation at 1 h of incubation). The protective ability of TRF (30.1% at 5 microM with TBARS formation) was significantly higher than that of the dominant form of vitamin E, alpha-tocopherol (16.5% under same conditions). Hence our studies indicate that this fraction from palm oil can be considered as an effective natural antioxidant supplement capable of protecting cellular membranes against oxidative damage.

Animals↗
PubMedDate not supplied

Cloning of complementary DNA encoding T-cell replacing factor and identity with B-cell growth factor II.

Proliferation and maturation of antigen-stimulated B cells are regulated by several soluble factors derived from macrophages and T cells. These soluble factors are functionally divided into two groups: B-cell growth factor (BCGF), thought to be involved in B-cell proliferation; and B-cell differentiation factor (BCDF), responsible for maturation of activated B cells into immunoglobulin-secreting cells. This classification needs to be re-examined in the light of the recent cloning of complementary DNA encoding IgG1 induction factor (interleukin-4, IL-4) from the 2.19 mouse T-cell line. Recombinant IL-4 has BCGF and BCDF activities and affects B cells, T cells and mast cells (refs 7, 8; our unpublished data). Another well-characterized B-cell factor is T-cell replacing factor (TRF), which, when secreted by the murine T-cell hybridoma B151K12, is defined by two activities: induction of IgM secretion by BCL1 leukaemic B-cell line; and induction of secondary anti-dinitrophenol (DNP) immunoglobulin G (IgG) synthesis in vitro by DNP-prime B cells. Although TRF from B151K12 was classified as BCDF, purified TRF has BCGF-II activity. To elucidate the molecular properties of TRF we isolated cDNA encoding TRF from the 2.19 T-cell line and report here the structure and multiple activities of this lymphokine.

Animals↗
PubMed1977

Mechanism of T-cell help in the immune response to soluble protein antigens II. Reconstitution of primary and secondary in vitro immune responses to dinitrophenyl-carrier conjugates by T-cell-replacing factor.

Spleen cells of dinitrophenyl keyhole limpet hemocyanin (DNPKLH) primed and boosted mice produced a nonantigen-specific helper factor upon in vitro challenge with DNPKLH. This helper factor displays all of the biological characteristics so far described for TRF produced by allogeneic or Concanavalin A stimulation of mouse spleen cells. It restores the primary anti-SRBC response in nude spleen cultures following the same kinetics of action as T-cell-replacing factor (TRF). Conversely, TRF restores the primary in vitro immune response of nude spleen cultures to DNPKLH. TRF also restores the secondary anti-hapten IgG response of T-cell-deprived spleen cell cultures derived from DNPKLH primed and boosted mice. Here the need for carrier specificity is fully overcome. The data therefore suggest that TRF, as a nonantigen-specific maturation signal, is involved in the primary and secondary immune responses to both particulate and soluble antigens.

Animals↗
PubMed1984

Both interleukin 2 and a second T cell-derived factor in EL-4 supernatant have activity as differentiation factors in IgM synthesis.

B cells cultured with anti-IgM, BSF-p1, and B15-TRF will differentiate into high rate IgM-synthesizing cells in the presence of supernatants from EL-4 cells that have been induced with phorbol myristate acetate. These supernatants contain two molecular species (EL-TRFs) that have differentiative activity. One co-migrates with interleukin 2 (IL-2) and its activity is blocked by antibody to the IL-2 receptor. Furthermore, molecularly cloned IL-2, at concentrations of 100 U/ml or more, expresses such EL-TRF activity. The EL-TRF activity of cloned IL-2 can also be inhibited by antibody to the IL-2 receptor. The other material with EL-TRF activity has a molecular weight of approximately 32,000. This material lacks IL-2 activity. Antibody to the IL-2 receptor does not impair its function. B cells stimulated with anti-IgM and BSF-p1, with or without B15-TRF, express determinants that react with two monoclonal antibodies which recognize distinct epitopes on the T cell IL-2 receptor. These determinants are present at much lower density (approximately 100-fold) on stimulated B cells that on HT-2 cells, an IL-2-dependent T cell line. Very small amounts of [3H]IL-2 (less than 1,000 molecules per cell) bind to activated B cells. These results indicate that IL-2 binds to a receptor on appropriately prepared B cells and causes them to differentiate into high rate IgM-synthesizing cells. The physiologic significance of the B cell differentiative activity of IL-2 remains to be investigated.

Animals↗
PubMed1994

Intragastric vs oral feeding: effect on the thermogenic response to feeding in lean and obese subjects.

The aim of this study was to observe the effects of oropharyngeal stimulation on thermogenic response to feeding (TRF) in obese and healthy individuals. Resting energy expenditure was measured in eight normal-weight (BMI, in kg/m2: 22.9 +/- 1.7) and nine obese subjects (BMI: 36.5 +/- 7.2), once after the ingestion of a standardized meal and once after the intragastric administration of the same, blenderized meal. In control subjects, TRF was lower after intragastric than after oral feeding: 5.6 +/- 1.4% vs 8.0 +/- 1.8% of the ingested energy for intragastric vs oral feeding, respectively (P < 0.01), but in obese subjects no difference occurred (6.5 +/- 3.0% vs 6.1 +/- 2.0%). In obese subjects the response over 6 h after the oral meal was lower than in/control subjects (P < 0.01). Intragastric TRF was not different between the two groups. This study confirms our previous observation that TRF has two components in humans, and suggests that oropharyngeal stimulation elicits a greater TRF in normal-weight than in obese individuals.

Adolescent↗
PubMed2006

Leukocyte telomere length and cardiovascular disease in the cardiovascular health study.

The telomere length of replicating somatic cells is inversely correlated with age and has been reported to be associated cross-sectionally with cardiovascular disease (CVD). Leukocyte telomere length, as expressed by mean terminal restriction fragment (TRF) length, was measured in 419 randomly selected participants from the Cardiovascular Health Study, comprising a community-dwelling cohort recruited in four US communities. The authors investigated associations between TRF length and selected measures of subclinical CVD/risk factors for CVD (data were collected at the 1992/1993 clinic visit) and incident CVD (ascertained through June 2002). In these participants (average age = 74.2 years (standard deviation, 5.2)), mean TRF length was 6.3 kilobase pairs (standard deviation, 0.62). Significant or borderline inverse associations were found between TRF length and diabetes, glucose, insulin, diastolic blood pressure, carotid intima-media thickness, and interleukin-6. Associations with body size and C-reactive protein were modified by gender and age, occurring only in men and in participants aged 73 years or younger. In younger (but not older) participants, each shortened kilobase pair of TRF corresponded with a threefold increased risk of myocardial infarction (hazard ratio = 3.08, 95% confidence interval: 1.22, 7.73) and stroke (hazard ratio = 3.22, 95% confidence interval: 1.29, 8.02). These results support the hypotheses that telomere attrition may be related to diseases of aging through mechanisms involving oxidative stress, inflammation, and progression to CVD.

Aged↗
PubMed1981

Immunological, physical, and chemical evidence for the identity of brain and kidney post-proline cleaving-enzyme.

Recent studies from this laboratory have suggested a similarity, if not identity, of thyrotropin releasing factor (TRF) deamidase and post-proline cleaving enzyme. Bovine brain TRF deamidase was purified to homogeneity and used to elicit antibodies to the enzyme. These antibodies were used to demonstrate identical immunological reactivity between rat brain TRF deamidase and rat kidney post-proline cleaving enzyme. In addition, both proteins exhibit a molecular weight of 75,000, and have identical Km, values for the synthetic substrate pGlu-(N-benzyl-L-His)-Pro-beta-naphthylamide and identical K1 values for TRF and luteinizing hormone releasing factor as inhibitors. Finally, and enzymes exhibit the same sensitivities to inhibition by mercury, iodoacetamide, N-ethylmaleimide, and 5, 5'-dithiobis-(2-nitrobenzoic acid). These results strongly suggest that brain TRF deamidase and kidney post-proline cleaving enzyme are identical.

Animals↗
PubMed2006

Insulin resistance, oxidative stress, hypertension, and leukocyte telomere length in men from the Framingham Heart Study.

Insulin resistance and oxidative stress are associated with accelerated telomere attrition in leukocytes. Both are also implicated in the biology of aging and in aging-related disorders, including hypertension. We explored the relations of leukocyte telomere length, expressed by terminal restriction fragment (TRF) length, with insulin resistance, oxidative stress and hypertension. We measured leukocyte TRF length in 327 Caucasian men with a mean age of 62.2 years (range 40-89 years) from the Offspring cohort of the Framingham Heart Study. TRF length was inversely correlated with age (r = -0.41, P < 0.0001) and age-adjusted TRF length was inversely correlated with the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) (r =-0.16, P = 0.007) and urinary 8-epi-PGF(2alpha) (r = -0.16, P = 0.005) - an index of systemic oxidative stress. Compared with their normotensive peers, hypertensive subjects exhibited shorter age-adjusted TRF length (hypertensives = 5.93 +/- 0.042 kb, normotensives = 6.07 +/- 0.040 kb, P = 0.025). Collectively, these observations suggest that hypertension, increased insulin resistance and oxidative stress are associated with shorter leukocyte telomere length and that shorter leukocyte telomere length in hypertensives is largely due to insulin resistance.

Adult↗
PubMed2003

Telomere length study in celiac disease.

OBJECTIVES: Telomeres are important structures that are critical for maintaining chromosomal integrity and cell surveillance. The aim of this study was to analyze telomere length in patients with celiac disease (CD), a multifactorial disorder with a strong genetic component that exhibits genomic instability and cancer predisposition, particularly T-cell lymphomas. METHODS: Telomere length measured by telomere restriction fragments (TRF) was studied in small intestinal biopsy (SIB) samples and peripheral blood lymphocytes (PBL) from 20 untreated CD patients, distributed according to the clinical form as four asymptomatic, five monosymptomatic, and 11 polysymptomatic individuals. We also analyzed TRF from normal peripheral blood lymphocytes and normal biopsy samples as normal controls. RESULTS: TRF evaluation showed a significant telomere shortening in SIB samples from CD patients (4.21 +/- 0.29 Kb) compared to PBL from the same individuals (9.17 +/- 0.35 Kb) (p < 0.0001), independently of clinical form. Mean TRF peak values from normal biopsy samples were significantly higher (8.33 +/- 0.38 Kb) than those observed in CD biopsy samples (p < 0.001). No differences between TRF values in CD-PBL and normal peripheral blood lymphocytes (8.89 +/- 0.37Kb) were found. CONCLUSIONS: Our findings in patients with CD, a disorder in which the gluten-induced mucosal injury could accelerate telomere shortening, would increase the process of end-to-end fusions resulting in chromosomal changes, supports the hypothesis that genomic instability and telomere reduction may play a role in the cancer predisposition observed in these patients.

Adult↗
PubMedDate not supplied

Characterization of the anti-cancer activity of transferrin-adriamycin conjugates.

The anthracycline anti-cancer drug adriamycin (Adr) was coupled to human transferrin (Trf) by using a glutaraldehyde technique. The effect of Trf-Adr conjugates and unconjugated Adr on human cells was determined by using normal peripheral blood mononuclear cells and chronic myelogenous K562 cells. Cytotoxicity was determined by using an assay that measures the conversion of a tetrazolium salt (MTT) into a purple product (formazan) by mitochondrial dehydrogenases in viable cells. We found that free Adr at a concentration of 1 x 10(-7) had little effect on K562 cells, while Trf-Adr conjugates inhibited 75% of cellular activity. When normal peripheral blood mononuclear cells were tested against Trf-Adr conjugates, the 50% inhibitory concentration was found to be 1.4-1.7 x 10(-6) M, at which concentration greater than 85% of K562 cells were inhibited. Interactions of Trf-Adr conjugates with plasma membrane energy-producing systems are the proposed mechanisms of cytotoxicity.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2006

Arterial baroreflex function does not influence telomere length in kidney of rats.

AIM: To investigate the relationship between arterial baroreflex (ABR) function and telomere length in kidney of rats. METHODS: Stroke-prone spontaneously hypertensive rats (SHR-SP) and sinoaortic denervated rats (SAD) were used as models with depressed arterial baroreflex. In the first experiments, SHR-SP rats were examined at the age of 24 weeks for both sexes and 40 weeks for female rats. In the second experiments, SAD rats were studied 4 and 35 weeks after SAD operation. Blood pressure was continuously recorded for 4 h in a conscious state. After the determination of baroreflex sensitivity (BRS), the terminal restriction fragment (TRF) of rat kidney was analyzed using Southern blot. RESULTS: The TRF length was found shorter in: a) male SHR-SP compared with age-matched female SHR-SP; b) female SHR-SP 40 weeks of age compared with 24 weeks of age; c) in rats 35 weeks after operation compared with rats 4 weeks post operation in both sham-operated and SAD rats. CONCLUSION: In SHR-SP, the TRF length did not correlate with BRS. In addition, SAD did not affect TRF length at either 4 or 35 weeks post-surgery. It may be concluded that baroreflex function does not influence the terminal restriction fragment (TRF) length in rats.

Animals↗
PubMed2000

Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis.

The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662-1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.

Bacteria↗
PubMed1991

Decreased thermogenic response to food with intragastric vs. oral feeding.

To examine the effects of sensory stimulations associated with eating on postprandial energy expenditure, thermogenic response to food (TRF) was measured in nine subjects after ingestion of a test meal and after intragastric injection of the same pureed meal through a nasogastric tube. A third measure was made after ingestion of water and a fourth after chewing the meal without deglutition. Each measurement lasted 6 h. Intragastric injection of the meal elicited a lower TRF than oral ingestion in every subject, and this difference was seen whether TRF was calculated from the pretest energy expenditure (PTEE) or from energy expenditure measured after water ingestion (EEW) (175 +/- 57 vs. 83 +/- 32 and 209 +/- 68 vs. 106 +/- 45 kJ for PTEE and EEW, respectively; P less than 0.05 for each test). In both tests, changes in respiratory quotient, plasma glucose, and insulin were similar. Sensory stimulation by the meal without deglutition did not induce a significant change in energy expenditure. These results suggest that TRF has two components in humans, one of which is dependent on preabsorptive sensory stimulations. Lack of change in substrate oxidations between oral and intragastric feeding suggests that TRF related to preabsorptive stimulations does not depend on the preferential use of fatty acids or glucose as a source of fuel.

Adult↗
PubMed2000

Increased thermogenic response to food and fat oxidation in female athletes: relationship with VO(2 max).

The thermogenic response to food (TRF) and substrate oxidation were studied in 12 endurance-trained and 13 untrained female subjects. Energy expenditure and substrate oxidation were calculated by indirect calorimetry before and for 6 h after an oral test meal and after the same meal given intragastrically on a separate occasion. The TRF was calculated after the oral meal, the obligatory component after the intragastric meal (OTRF), and the facultative component from the difference between the two. VO(2 max) was measured on a treadmill and body composition by underwater weighing. The TRF and OTRF were significantly higher in trained than in untrained subjects: 223 +/- 63 vs. 185 +/- 50 kJ/6 h (P < 0.03) and 174 +/- 38 vs. 131 +/- 37 kJ/6 h (P < 0.01) for the TRF and OTRF in trained vs. untrained subjects, respectively. Multiple regression analysis showed that maximum O(2) consumption (VO(2 max)), but not percentage of body fat, was significantly related to OTRF (r =0.68, P < 0.01). Trained subjects had higher fatty acid oxidation than untrained subjects before (0.6 vs. 0.4 mg. kg(-1). min(-1), P < 0.05) and after the oral meal (13 +/- 6 vs. 8 +/- 4 g/6 h P < 0.05). These results demonstrate that 1) TRF is higher in trained than in untrained women; 2) this is due to a higher cost of nutrient digestion, absorption and storage; 3) the difference is related to higher VO(2 max); and 4) fatty acid oxidation is greater in trained women in both the postabsorptive and postprandial states. These observations suggest that endurance training induces metabolic changes that favor leanness.

Adipose Tissue↗
PubMed1975

Renal blood flow distribution during steady-state exercise and exhaustion in conscious dogs.

To determine whether renal blood flow is reduced or redistributed during exercise, we measured total renal flow (TRF) and intrarenal flow distribution (IRFD) in nine dogs. They ran on a motor-driven treadmill at 3-8 mph at grades of 8-15% for an average of 35 min. We measured aortic pressure, heart rate, stroke volume, and cardiac output (CO) via chronically implanted catheters and an electromagnetic flow probe. We injected 15-mum radiolabeled microspheres (85Sr, 141Ce, and 51Cr) via a left atrial catheter during resting control, steady state (SS) and exhaustive (EE) exercise; measured their distribution by gamma spectrometry; and determined TRF as % CO and as ml/100 g per min. We determined IRFD for the outer and inner cortex and the outer medulla. TRF as %CO dropped (P less than 0.05) during both levels of exercise: from 10.2 +/- 0.7% to 3.9 +/- 0.4% (SS) and 3.4 +/- 0.6% (EE). TRF in ml/100 g per min did not change significantly from control (228 +/- 30 ml/100 g per min). IRFD was unchanged with exercise, remaining at about 80, 20, and 3% of TRF for the outer and inner cortex and outer medulla, respectively. We conclude that blood flow is not diverted from the kidneys during severe exercise in the dog.

Animals↗