The concept of telomeres as being the end-part of eukaryotic chromosomes was first described by H. J. Muller and B. McClintock. Their pioneering work opened the path for multiple new researches and assays on a thrilling subject, with implications for various domains such as aging, replication, immortality, and cancer. Yeast has been a model of choice to study telomere length, senescence, telomerase activity, telomere cloning, and sequencing with important new techniques being discovered in this species and adapted afterward for other organisms. The main functions of telomeres include the protection of the genome from deletions, recombination, and degradation, and they are therefore essential for genome stability. Their maintenance is assured by a specific enzyme (telomerase) and it is of vital interest for the organism to maintain their length and specific structure. Multiple assays have been described to analyze telomere length and for yeast, Southern blot analysis of terminal restriction fragments (TRFs) remains one of the most popular ones to get a global picture of the state of telomeres in a given experimental setting. However, growth phenotypes (senescence) and fine-structure analyses of the chromosome terminal DNA are also becoming increasingly important.

OBJECTIVE

To evaluate the psychosocial well-being of 9-18-year-old IVF children. IVF alters fetal programming, with consequences for physical functioning. This also may apply to behavior. Behavior and socioemotional functioning of children born after IVF and spontaneously from parents with former fertility problems were studied, to control for parental factors and investigate the role of IVF on the children's functioning.

METHODS

Child Behavior CheckList (CBCL) and Teacher Report Form (TRF) reports of 139 IVF and 143 control children.

METHODS

VU University Medical Center, Amsterdam.

METHODS

Children's age ranged from 9 to 18 years; mean age 13.60 (+/-2.12) years in IVF and 13.51 (+/-2.11) years in controls.

METHODS

Child's behavior and socioemotional functioning reported by parents (CBCL) and teachers (TRF).

RESULTS

The CBCL and TRF mean scores were within the normal range. Scores of IVF parents versus controls were lower on the total problems scale, externalizing scale and syndrome scales thought problems, attention problems, aggressive and rule-breaking behavior. Less IVF children had scores in the borderline/clinical range on these scales. On the TRF, a tendency was found for lower scores on the externalizing scale in IVF children compared with controls. More IVF children scored in the borderline/clinical range on the syndrome scale withdrawn/depressed behavior.

CONCLUSIONS

Overall, behavior, and socioemotional functioning of 9-18-year-old IVF children is normal. The reduced behavior of externalizing nature reported by the parents, and teacher ratings of more withdrawn/depressed behavior need further study.

OBJECTIVE

To evaluate the epidemiology of attention problems using parent, teacher, and youth informants among a nationally representative Turkish sample.

METHODS

The children and adolescents, 4 to 18 years old, were selected from a random household survey. Attention problems derived from the Child Behavior Checklist (CBCL) (N = 4,488), Teacher Report Form (TRF) (N = 2,360), and the Youth Self Report (YSR) (N = 2,206) were examined.

RESULTS

The CBCL and TRF attention problems scores were higher among young male children, whereas the YSR reported scores were higher among older adolescents without a gender effect. The CBCL and YSR scores were also higher by urban residence.

CONCLUSIONS

Compared with other European samples, our national sample had higher mean attention problems scores than the Scandinavian but lower mean scores than the former Soviet Union samples. In addition to elucidating the profile of attention problems in Turkey, our results also contribute to understanding the comparative global epidemiology of attention problems.

T cell-replacing factor (TRF) is known to play a critical role in the regulation of B cell growth and differentiation. In this study, the role of TRF in the expression of mRNA for both IgM and IgG1 class was investigated. The TRF was purified from cellfree supernatants from a T cell hybridoma, B151K12. RNA was isolated from chronic B cell leukemia (BCL1) cells, DNP-KLH-primed B cells, or normal B cells cultured with or without LPS, and LPS plus TRF or LPS plus BSF-1. The steady state level of isotype-specific mRNA was assessed by Northern blot analysis with a mu-specific or a gamma 1-specific probe. It was demonstrated that BCL1 and purified B cells cocultured with TRF expresses increased levels (twofold and fourfold, respectively) of secreted forms of mu mRNA. Purified B cells from DNP-KLH-primed mice also expressed increased levels (twofold to fourfold) of mu as well as gamma 1 mRNA for secreted form by stimulation with TRF. Total expression of mu mRNA, however, was approximately threefold higher than that of gamma 1 mRNA. The stimulation of normal B cells with LPS plus TRF induced an increase in the levels of mu mRNA and gamma 1 mRNA expression, fourfold and threefold, respectively. However, the levels of gamma 1 mRNA expression was one-third of that induced in B cells stimulated with LPS plus BSF-1. These results indicate that TRF preferentially induces increased levels of secreted type of mu mRNA and induces less gamma 1 mRNA than BSF-1. The differential role of TRF from BSF-1 in the expression of Ig mRNA will be discussed.

Fc fragments derived from human and murine Ig have been shown to possess adjuvant properties. The spleen cell population(s) affected by the Fc fragments is the T lymphocyte. That Fc fragments do not act directly on the B lymphocyte was shown by the fact that in a system where T cell help was substituted for by TRF and using TI antigens, no adjuvant effect was observed. The adjuvant signal provided by the Fc fragments is needed early in the culture period, because it was reduced by approximately 50% when delayed by 24 hr. When the addition of Fc was delayed by 72 hr, the antibody response was not significantly increased over the culture that received antigen only.

The objective of this study was to optimize a novel tocotrienol (TRF)-rich self emulsified drug delivery system (SEDDS). In the first part, an unusual phenomenon was investigated. It was observed that by substituting Tween 80 with Cremophor EL in the SEDDS it was possible to emulsify>55% TRF (by weight of the formulation) into submicron (<200 nm) emulsion. With Tween, only 17.5% of the loaded TRF could be emulsified into crude emulsion. The superiority of Cremophor was attributed to the special arrangement of the surfactant at the oil/water interface, which was confirmed by modelling and docking studies. In the second part of this study, the composition of the secondary ingredients in the TRF-rich SEDDS were optimized by the modified Multisimplex approach. SEDDS were manufactured at pre-defined step-size and tested for their dissolution behavior. Testing was performed sequentially until the optimum composition that can emulsify 50% of the loaded TRF into a stable<150 nm submicron emulsion was obtained. Optimization end-point was identified when the "membership value" approached 1, which was confirmed by a second Multisimplex run. Overall, this study demonstrated the utility of docking studies and the Multisimplex approach in product development when little is known about the experimental "design space".

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).

Leaf Area Index (leaf area per unit ground area, LAI) is a key driver of forest productivity but has never previously been measured directly at the landscape scale in tropical rain forest (TRF). We used a modular tower and stratified random sampling to harvest all foliage from forest floor to canopy top in 55 vertical transects (4.6 m(2)) across 500 ha of old growth in Costa Rica. Landscape LAI was 6.00 +/- 0.32 SEM. Trees, palms and lianas accounted for 89% of the total, and trees and lianas were 95% of the upper canopy. All vertical transects were organized into quantitatively defined strata, partially resolving the long-standing controversy over canopy stratification in TRF. Total LAI was strongly correlated with forest height up to 21 m, while the number of canopy strata increased with forest height across the full height range. These data are a benchmark for understanding the structure and functional composition of TRF canopies at landscape scales, and also provide insights for improving ecosystem models and remote sensing validation.

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.

Recently, shortened telomere length and increased telomerase activity have been demonstrated in various human cancers. In the study reported here, we ascertained whether gene changes are characteristic of pancreatic cancers. Hamster duct carcinomas and cell lines were investigated by Southern blot analysis for telomere restriction fragment (TRF) length and by the telomeric repeat amplification protocol (TRAP) assay for telomerase activity. Comparison with normal pancreas and spleen revealed shortened TRF length and markedly increased telomerase activity in primary pancreatic duct carcinomas induced by the rapid-production model as well as in a transplantable carcinoma and the cell lines. The enzyme level was 86.0-215.7 times the low levels found in control pancreas and spleen tissues. Late-passage Syrian hamster embryo cells, known to be immortalized and tumorigenic, had shorter TRFs than the original cells in primary culture did. These results indicate that hamster pancreatic duct carcinoma cells are immortalized, with the potential for proliferation ad infinitum, and provide a model for basic therapeutic research into the substances targeting telomerase.

We have developed a method to isolate and analyze nascent human reticulocytes in peripheral blood for the presence of micronuclei (MN). For a very short time peripheral reticulocytes show residual expression of the transferrin receptor. Using immunomagnetic separation of cells expressing the transferrin receptor, a population of immature reticulocytes (Trf-Ret) was isolated from peripheral blood. In humans, the spleen actively removes micronucleated erythrocytes but during the short lifetime of the isolated Trf-Ret only a fraction (less than about 20%) of the MN-containing reticulocytes will have been eliminated. Cells were stained with the fluorescent dyes Thiazole Orange for RNA and Hoechst 33342 for DNA and analyzed by flow cytometry and fluorescence microscopy. Baseline frequencies of MN-Trf-Ret on a group of healthy donors were found to be 1.1% for males and 1.4% for females; however, the gender difference was not significant. The frequency of MN-Trf-Ret in the studied group increased with age, and was dependent on blood group. In three donors studied over 4 months, the baseline level remained stable. In cancer patients treated with radiation or chemotherapy, the frequency of MN-Trf-Ret increased 10- to 20-fold after 1-4 days, depending on the treatment. A high correlation between flow and manual analysis of MN-Trf-Ret was seen. We believe the method has a high potential as a sensitive and rapid method for biological monitoring in presumed exposed groups and individuals.

OBJECTIVE

A 10-year-old boy presented with cleft palate, hepatopathy, cholecystolithiasis, myopathy, coagulopathy, hyperlipidemia, hypoglycemia, hyperuricemia, short stature, obesity, hypothyroidism, microcephaly and mild intellectual disability. The multi-systemic manifestation involving certain distinct clinical features prompted us to search for a subtype of congenital disorders of glycosylation (CDG).

METHODS

The patient was screened for CDG by examining the distribution of transferrin (TRF) and apolipoprotein C-III (ApoC-III) sialylated isoforms using isoelectric focusing of serum. This was followed by spectrophotometric measurement of phosphoglucomutase 1 (PGM1) activity in fibroblasts and molecular analysis including sequencing and PCR-RFLP of PGM1 gene. Selected bioinformatics tools were used to evaluate the data.

RESULTS

Increased relative levels of di-, mono- and asialotransferrin reflected a defect of N-glycosylation in the patient. Markedly decreased activity of PGM1 corresponding to less than 5% of control´s was found. Sequencing of PGM1 gene revealed the presence of two heterozygous missense mutations c.1010C>T (p.T337M) and c.1508G>A (p.R503Q), whose pathogenicity was confirmed by in silico analysis.

CONCLUSIONS

We report the first Czech patient with a glycosylation disorder due to PGM1 deficiency. Compared to the described cases, no dilated cardiomyopathy was noted in our patient. However, he suffered from a mild neurological impairment, which is an uncommon feature that extends the phenotype associated with PGM1-CDG. Lactose-rich diet, which was previously reported to have ameliorated the clinical symptoms in some PGM1-CDG patients, did not result in any improvement in our patient.

In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length.

Three PPD-reactive long-term cultured helper T cell clones were established from Mycobacterium tuberculosis (Tbc)-primed spleen cells. Clones B11.15 and B12.F were derived from C57BL/6 mice, and clone D-2 was originated from DBA/2Ha mice that have an X-linked recessive inheritance of T cell-replacing factor (TRF) unresponsiveness. Proliferative responses of these cloned T cells were induced by stimulation with PPD in a dose-dependent manner only when I-A-subregion compatible antigen-presenting cells (APC) were present. These three T cell clones have distinct helper functions in B cell activation. Clone B11.15 activated DNP-primed B cells to induce anti-DNP IgM plaque-forming cell (PFC) responses only when high amounts of PPD (5 micrograms) were added to the culture in a major histocompatibility complex (MHC)-unrestricted manner (factor-mediated interaction), whereas stimulation with a low amount of DNP-PPD (0.05 microgram) was ineffective. On the other hand, clone D-2 triggered B cells in the presence of a low amount of DNP-PPD in a MHC-restricted manner (cognate interaction). Significant helper activity of D-2, however, was not observed in the presence of high amounts of PPD. Clone B12.F was able to activate B cells in the presence of either DNP-PPD or PPD. Moreover, both B11.15 and B12.F produced helper factor(s) such as TRF by stimulation with high amounts of PPD in the presence of syngeneic APC, whereas D-2 did not produce measurable helper factor(s) under the same conditions. These results suggest that at least three distinctly functioning PPD-reactive helper T cells can be generated by active immunization with Tbc in vivo. T-B cell interaction between distinctly functioning T cell clones and B cells from (DBA/Ha X C57BL/6) (DB6)F1 male or female mice was then examined. B cells from DB6F1 female mice were triggered by both B11.15 and B12.F in a factor-mediated manner and were also activated with B12.F or D-2 in cognate manner. On the other hand, B cells from DB6F1 male mice, which are TRF low responders, were activated by B12.F or D-2 only through cognate interaction, and they failed to cooperate with B12.F or B11.15 in factor-mediated manner. These findings further suggest that B cells can be triggered by at least two distinct helper T cell subpopulations via respective pathways (cognate interaction and factor-mediated interaction).(ABSTRACT TRUNCATED AT 400 WORDS)

Methanol extracts of several rat tissues (hypothalamus, amygdala, the rest of the forebrain, brain stem and pancreas) were partially purified in SP-cation exchange chromatography and measured in TRF radioimmunoassay. It was found that hypothalamic and amygdaloid TRF contents were highest during the light period of the day, but brain stem TRF had an opposite rhythm. Hypothalamic TRF content rose after 10 min by 14% (p less than 0.05) when the rats were exposed to transfer stress. During short-time immobilization stress tests, hypothalamic TRF rose by 41% (p less than 0.05) after a 10-sec immobilization. Cold exposure did not produce any changes in hypothalamic TRF contents within 45 min. Handling stress and time of day appear to be the most important factors influencing the hypothalamic TRF content and have to be taken into account when measuring TRF.

Growth characteristics, karyotype changes, and telomere length variations were analyzed during the life span of 12 anchorage-independent clones isolated from a xeroderma pigmentosum fibroblast strain. After an initial period of comparable active growth, all the clones showed a decline in the growth rate and finally entered a phase of replicative senescence; however, the number of population doublings and the time required to enter senescence varied among the clones. Repeated cytogenetic analyses during culture propagation showed the appearance of chromosome anomalies, mainly telomeric association (tas) and unbalanced translocations. In all the clones the percentage of abnormal mitoses increased with culture passage, but reached different levels (from less than 10% to about 100%). This finding indicates that the replicative block may be associated with differently altered cytogenetic patterns. Specific chromosome arms (5p, 16q, 19q, and 20q) were preferentially involved in tas, suggesting that alterations in chromosome ends may occur which predispose to fusion. In some clones it was possible to demonstrate the origin of marker chromosomes from the evolution of tas. Telomere length analysis by Southern blotting on DNA samples prepared from 7 clones and from the parental cell lines showed that the terminal restriction fragment (TRF) profiles were homogeneous in senescent parental cells and in the clones during the last part of their life in culture, regardless of the degree of karyotype abnormalities. The homogeneity of the TRF profiles supports the hypothesis of a critical telomere length at senescence.

Age is one of the key parameters in establishing a physical characteristic profile of an individual. For biological evidence left in crime scenes such as blood, saliva, hair, etc, the evidence owner's age can be determined only by DNA extracted from these materials. Previous researches have found that there are certain DNA regions with specialized characteristic and function called telomere being able to predict age. The present study was to determine the correlation between telomere length and age as well as the effect of sex on the correlation and to create linear regression equation for age estimation in Thai population for forensic purposes. Blood samples obtained from unrelated healthy Thai fresh cadavers without anatomical organ abnormalities were used in this study. All cadaver subjects underwent the postmortem examination in jurisdiction of the Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, and Institute of Forensic Medicine, Police General Hospital. Fifty blood samples from both sexes of all ages divided into 6 groups for equal age distribution (0-11, 12-23, 24-35, 36-47, 48-59, and 60 years old and older) were collected for a total of 100 samples. The extracted genomic DNA samples were then subjected to telomere length estimation by terminal restriction fragment (TRF) assay. The results showed that the mean TRF length was inversely correlated with age (r = -0.625), and sex did not have a statistically significant influence on the association between age and mean TRF length (P>> 0.05). The obtained linear regression equation was y = 113.538 ± 9.604 - 0.012 × (R = 0.391; P < 0.001). However, the correlation was too low to be used for age estimation with high certainty and a possible reason for this in part would be the postmortem DNA degradation at some level, even using fresh cadaver blood, and other biological factors such as ethnicity and DNA sources. Roughly, those individuals who had a mean TRF length longer than 6.3 kilobase (kb), between 5.5 and 6.3 kb, and shorter than 5.5 kb aged younger than 28 years, 30 to 44 years, and older than 46 years, respectively (P < 0.01). As a preliminary study, this study highlighted that telomere length could act as a useful biomarker of aging in human population and might be used for rough age estimation in a Thai population. However, further studies with a larger sample size and/or in living human bloods as well as other cell types are recommended to support the results of this study.

OBJECTIVE

To characterize the influence of neurological lesion level on the cardiorespiratory and ventilatory responses of two groups of paraplegic athletes during incremental exercise on a treadmill and in the usual conditions for wheelchair exercise.

METHODS

Cardioventilatory responses evaluated in two groups of paraplegic wheelchair sportsmen designated as high paraplegic athletes (HPA) and low paraplegic athletes (LPA). After 2 min of data collection at rest and 3 min of warm-up at 4 km x h(-1), treadmill speed was increased by 1 km x h(-1) every minute until exhaustion. During this test, ventilation and its components, as well as respiratory exchanges, were measured breath by breath (C.P.X. Medical Graphics) every minute by taking the mean of the last 20 s of each increment.

RESULTS

Spirometric values presented no significant differences between groups. At rest, no significant difference was observed between the two groups for all cardiorespiratory and ventilatory values obtained during the treadmill test. At submaximal exercise, all variables increased with the augmentation in workload. With the exception of R, there were no significant differences in the classic cardiorespiratory parameters (VO2, VCO2, HR, VE) between the two groups of paraplegics. For the ventilatory parameters, we observed significant differences between the two groups, with values of f and It/<em>Trf</em> significantly higher (0.01<P<0.001) and values of <em>Trf</em> and Vt significantly lower (0.01<P<0.001) for HPA versus LPA. We observed changes in breathing pattern, ie, in f, Vt, Trc and It/Trc, were significantly different between groups, with significantly higher values of f and It/Trc for HPA. We noted a ventilatory disturbance which was manifested by values of breathing frequency and tidal volume during exercise that were significantly different between groups. During maximal exercise, we observed no significant differences between the two groups concerning cardiorespiratory and ventilatory values. Despite the absence of significant differences, the more linear time course of the ensemble of HPA flows, the achievement of a greater number of work loads, and the higher maximal values indicate a better capacity for adaptation to exercise in the group of lower thoracic paraplegics.

CONCLUSIONS

These results raise questions about the influence of neurological level and further research is needed to define with more precision the capacities of readaptation of the different cardiovascular and respiratory functions, as well as the training methods best adapted to the optimization of physical capacities.

This study investigated the effects of palm tocotrienol-rich fraction (TRF) on aortic proatherosclerotic changes in rats fed with a high methionine diet. Forty-two male Wistar rats were divided into six groups. The first group was the control (fed with a basal diet). Another five groups were fed with 1% methionine diet for 10 weeks. From week 6 onward, folate (8 mg/kg diet) or palm TRF (30, 60, and 150 mg/kg diets) was added into the diet of the last four rat groups, respectively. The high methionine diet raised the plasma total homocysteine and aortic lipid peroxidation, which were reduced by the palm TRF and folate supplementations. Plasma nitric oxide was reduced in the high methionine group compared to the control (3.72 ± 0.57 versus 6.65 ± 0.53 μ mol/L, P < 0.05), which reduction was reversed by the palm TRF (60 and 150 mg/kg) and folate supplementations. The increased aortic vascular cell adhesion molecule-1 expression in the methionine group (2.58 ± 0.29) was significantly reduced by the folate (1.38 ± 0.18) and palm TRF at 150 mg/kg (1.19 ± 0.23). Palm TRF was comparable to folate in reducing high methionine diet-induced plasma hyperhomocysteinemia, aortic oxidative stress, and inflammatory changes in rats.

OBJECTIVE

To determine the significance of young maternal age, family adversity and maternal behaviour during mother-toddler interaction in the prediction of child disruptive behaviour at age eight.

METHODS

From an ongoing longitudinal study of infants at risk for later psychopathology (n = 362), 72 young mothers aged between 15 and 24 y (median 22 y) at first birth were compared with 197 primiparous older mothers ranging in age from 25 to 41 y (median 29 y). Family adversity at childbirth was assessed using a modified version of Rutter's Family Adversity Index (FAI) and measures of child disruptive behaviour at age eight were obtained using Achenbach's Teacher Report Form (TRF). An observational procedure was used to assess maternal behaviour during mother-child interaction at the age of 2 y.

RESULTS

Young mothers encountered more adverse family characteristics and were more inadequate, restrictive and more negative during interaction with their toddlers. Their school-aged children showed higher scores on all disruptive behaviour scales of the TRF. Hierarchical regression analyses revealed that family adversity and maternal behaviour during toddler interaction could account for most of the association between early motherhood and child disruptive behaviour.

CONCLUSIONS

The impact of young motherhood on child mental health is not confined to teenage mothers and is mainly attributed to psychosocial and interactional factors.