We investigated whether changes in iron metabolism and the transferrin receptor (TRF-R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara-C). Treatment with 100 nM ara-C for 48h reduced thymidine uptake and increased the surface expression of the TRF-R on leukaemic blasts derived from 13/16 (81%) patients and on the HL-60 and U-937 cell lines. Whereas intracellular non-haem iron was strongly depleted 24 h after ara-C addition, TRF-R up-regulation and recovery of intracellular non-haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara-C and the iron chelator desferioxamine (DSF) on the growth of HL-60 and U-937 cells. We found that desferioxamine strongly potentiated the effects of ara-C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara-C and an iron chelator in terms of antileukaemic effects.

The ends of human chromosomes consist of a specialized structure, the telomere, composed of repeats of TTAGGG making up a total of 5-15 kilobase pairs, depending on age and proliferative activity of the tissue. The major function of telomeres is to provide stability to chromosomes and protect underlying unique coding sequences from degradation. There is a loss of telomeric sequences following every cell division estimated to be between 50 and 65 basepairs/cell division in human fibroblasts and embryonic kidney cells in vitro. This loss is due to the fact that DNA replication is incomplete for one strand at each telomere end. In lower eukaryotes there is a compensation mechanism provided by the enzyme telomerase, which is inactive in human somatic cells. Telomerase activation has also been detected in vitro immortalized human cells. In this study we analyzed renal cell carcinoma for the occurrence of telomere shortening using the probe (TTAGGG)4. Southern blots of HinfI-digested DNA revealed a shortening of mean telomere restriction fragment (TRF) length of 0.4 to 2.5 kilobase pairs in 2 or 3 intratumoral samples in all 10 tumors analyzed. No obvious intratumoral heterogeneity was found in mean TRF length values. However, heterogeneity was shown by the occurrence of at least two separate peak TRF values in 7 of 10 tumors, indicating the presence of different tumor cell clones. A conflicting observation was made when we evaluated the intensity of the hybridization signals, where three of the tumors showed an increase in hybridization signals despite concomitant TRF reduction. We found no correlation between tumor size and calculated tumor cell divisions undergone. In two tumors, the calculated cell division cycles were unrealistically low compared to the tumor size. These data suggest that telomerase activation might occur in human renal cell carcinoma.

The present study employed mass sequencing of small RNA libraries to identify the repertoire of small noncoding RNAs expressed in normal CD4(+) T cells compared to cells transformed with human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma (ATLL). The results revealed distinct patterns of microRNA expression in HTLV-1-infected CD4(+) T-cell lines with respect to their normal counterparts. In addition, a search for virus-encoded microRNAs yielded 2 sequences that originated from the plus strand of the HTLV-1 genome. Several sequences derived from tRNAs were expressed at substantial levels in both uninfected and infected cells. One of the most abundant tRNA fragments (tRF-3019) was derived from the 3' end of tRNA-proline. tRF-3019 exhibited perfect sequence complementarity to the primer binding site of HTLV-1. The results of an in vitro reverse transcriptase assay verified that tRF-3019 was capable of priming HTLV-1 reverse transcriptase. Both tRNA-proline and tRF-3019 were detected in virus particles isolated from HTLV-1-infected cells. These findings suggest that tRF-3019 may play an important role in priming HTLV-1 reverse transcription and could thus represent a novel target to control HTLV-1 infection.

OBJECTIVE

Small noncoding RNAs, a growing family of regulatory RNAs that includes microRNAs and tRNA fragments, have recently emerged as key players in many biological processes, including viral infection and cancer. In the present study, we employed mass sequencing to identify the repertoire of small noncoding RNAs in normal T cells compared to T cells transformed with human T-cell leukemia virus type 1 (HTLV-1), a retrovirus that causes adult T-cell leukemia/lymphoma. The results revealed a distinct pattern of microRNA expression in HTLV-1-infected cells and a tRNA fragment (tRF-3019) that was packaged into virions and capable of priming HTLV-1 reverse transcription, a key event in the retroviral life cycle. These findings indicate tRF-3019 could represent a novel target for therapies aimed at controlling HTLV-1 infection.

Time-restricted feeding ameliorates the deleterious effects of a high-fat diet on body weight and metabolism in young adult mice. Because obesity is highly prevalent in the middle-aged population, this study tested the hypothesis that time-restricted feeding alleviates the adverse effects of a high-fat diet in male middle-aged (12months) mice. C57BL6/J mice were fed one of three diets for 21-25weeks: 1) high-fat diet (60% total calories from fat) ad-libitum (HFD-AL), 2) HFD, time-restricted feeding (HFD-TRF), and 3) low-fat diet (10% total calories from fat) ad-libitum (LFD-AL) (n=15 each). HFD-TRF mice only had food access for 8h/day during their active period. HFD-TRF mice gained significantly less weight than HFD-AL mice (~20% vs 55% of initial weight, respectively). Caloric intake differed between these groups only during the first 8weeks and accounted for most but not all of their body weight difference during this time. TRF of a HFD lowered glucose tolerance in terms of incremental area under the curve (iAUC) (p<0.02) to that of LFD-AL mice. TRF of a HFD lowered liver weight (p<0.0001), but not retroperitoneal or epididymal fat pad weight, to that of LFD-AL mice. Neither HFD-AL nor HFD-TRF had any effect on performance in the novel object recognition or object location memory tests. Circulating corticosterone levels either before or after restraint stress were not affected by diet. In conclusion, TRF without caloric restriction is an effective strategy in middle-aged mice for alleviating the negative effects of a HFD on body weight, liver weight, and glucose tolerance.

BACKGROUND

Breast milk contains many immunomodulatory factors (soluble CD14 (sCD14), IgA and cytokines) with the potential to influence infant immune development.

OBJECTIVE

To determine if changes in breast milk omega-3 polyunsaturated fatty acid (n-3 PUFA) composition as a result of maternal dietary fish oil supplementation during pregnancy can modify levels of these immunological parameters in breast milk.

METHODS

In a randomized controlled trial, 83 atopic women received either 4 g fish oil capsules (containing 3.7 g n-3 PUFA) (n = 40) or 4 g olive oil capsules (n = 43) from 20 weeks gestation until delivery. Breast milk was collected 3 days post-partum and fatty acids were analysed by gas liquid chromatography and IgA, sCD14 and cytokines (IL-5, IL-6, IL-10, TNF-alpha and IFN-gamma) were quantitated by ELISA or time resolved fluorescence (TRF).

RESULTS

Omega-3 docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) levels were significantly higher (P < 0.001) in breast milk from women supplemented with fish oil (n = 33, DHA mean 1.15%, SD 0.47% and EPA mean 0.16%, SD 0.07%) than in samples from the control group (n = 40, DHA mean 0.50%, SD 0.17% and EPA mean 0.05%, SD 0.02%). Breast milk arachidonic acid (AA; 20:4n-6) levels were significantly lower (P = 0.045) in the fish oil group (mean 0.55%, SD 0.12%) compared with the control group (mean 0.61%, SD 0.14%). Breast milk IgA was positively correlated with DHA (P = 0.046) and 22:5n-3 (P = 0.003), but inversely correlated with linoleic acid (LA; 18:2n-6) (P=0.034). Levels of sCD14 were also positively correlated with 22:5n-3 (P=0.009). Cytokines involved in IgA synthesis (IL-10 and IL-6) were also significantly correlated with both IgA and n-3 PUFA levels, although there were no differences in the levels of breast milk IgA, sCD14 or cytokines between study groups.

CONCLUSIONS

Supplementation with fish oil during pregnancy significantly alters early post-partum breast milk fatty acid composition. omega-3 PUFA levels were positively associated with IgA and sCD14 levels, suggesting a relationship between fatty acid status and mucosal immune function.

Disruption of the circadian rhythm contributes to obesity. This study tested the hypothesis that time-restricted feeding (TRF) reduces high-fat diet-induced increase in adiposity. Male C57BL/6 mice were fed the AIN93G or the high-fat diet ad libitum (ad lib); TRF of the high-fat diet for 12 or 8hours during the dark cycle was initiated when high-fat diet-fed mice exhibited significant increases in body weight. Energy intake of the TRF 12-hour group was not different from that of the high-fat ad lib group, although that of the TRF 8-hour group was slightly but significantly lower. Restricted feeding of the high-fat diet reduced body fat mass and body weight compared with mice fed the high-fat diet ad lib. There were no differences in respiratory exchange ratio (RER) among TRF and high-fat ad lib groups, but the RER of these groups was lower than that of the AIN93G group. Energy expenditure of the TRF groups was slightly but significantly lower than that of the high-fat ad lib group. Plasma concentrations of ghrelin were increased in TRF groups compared with both AIN93G and high-fat ad lib groups. Elevations of plasma concentrations of insulin, leptin, monocyte chemoattractant protein-1, and tissue inhibitor metalloproteinase-1 by high-fat ad lib feeding were reduced by TRF to the levels of mice fed the AIN93G diet. In conclusion, TRF during the dark cycle reduces high-fat diet-induced increases in adiposity and proinflammatory cytokines. These results indicate that circadian timing of food intake may prevent obesity and abate obesity-related metabolic disturbance.

Telomeres comprise the protective caps of natural chromosome ends and function in the suppression of DNA damage signaling and cellular senescence. Therefore, techniques used to determine telomere length are important in a number of studies, ranging from those investigating telomeric structure to effects on human disease. Terminal restriction fragment (TRF) analysis has for a long time shown to be one of the most accurate methods for quantification of absolute telomere length and range from a number of species. As this technique centers on standard Southern blotting, telomeric DNA is observed on resulting autoradiograms as a heterogeneous smear. Methods to accurately determine telomere length from telomeric smears have proven problematic, and no reliable technique has been suggested to obtain mean telomere length values. Here, we present TeloTool, a new program allowing thorough statistical analysis of TRF data. Using this new method, a number of methodical biases are removed from previously stated techniques, including assumptions based on probe intensity corrections. This program provides a standardized mean for quick and reliable extraction of quantitative data from TRF autoradiograms; its wide application will allow accurate comparison between datasets generated in different laboratories.

Three experiments were conducted to evaluate chromium picolinate (CrPic) in growing-finishing pigs. Treatments were replicated four times within each experiment with three pigs per replicate in Exp. 1 and four pigs per replicate in Exp. 2 and 3. Average initial weights were 37.8, 30.5, and 22.4 kg in Exp. 1, 2, and 3, respectively. In Exp. 1, the basal corn-soybean meal diet (B, 120% NRC Lys) was supplemented with 0, 25, 50, 100, or 200 ppb of Cr from CrPic. Daily gain was increased (Cr cubic, P < .02) and serum cholesterol decreased (Cr cubic, P < .08) by addition of CrPic. In Exp. 2, the basal diet was supplemented with 0, 100, 200, 400, or 800 ppb of Cr from CrPic. Daily gain and ADFI were decreased (Cr linear, P < .05) by CrPic. Serum cholesterol also was decreased (Cr quadratic, P < .05) by CrPic. Longissimus muscle area (LMA) and percentage of muscling (MUS) were increased (Cr quadratic, P < .01) and 10th rib fat (TRF) was decreased (Cr quadratic, P < .01) by CrPic. In Exp. 3, pigs were allotted to the following treatments: 1) B, 2) B + 1,467 ppb of picolinate (Pic), 3) B + 200 ppb of Cr from CrCl3.6H2O, 4) B + 1,467 ppb of Pic + 200 ppb of Cr from CrCl3.6H2O, 5) B + 100 ppb of Cr from CrPic, or 6) B + 200 ppb of Cr from CrPic. Longissimus muscle area and MUS were increased (P < .01) and TRF decreased (P < .01) in pigs fed CrPic but not in pigs fed CrCl3.6H2O and(or) Pic.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

Telomere length (TL) is commonly measured using quantitative PCR (qPCR). Although, easier than the southern blot of terminal restriction fragments (TRF) TL measurement method, one drawback of qPCR is that it introduces greater measurement error and thus reduces the statistical power of analyses. To address a potential source of measurement error, we consider the effect of well position on qPCR TL measurements.

METHODS

qPCR TL data from 3,638 people run on a Bio-Rad iCycler iQ are reanalyzed here. To evaluate measurement validity, correspondence with TRF, age, and between mother and offspring are examined.

RESULTS

First, we present evidence for systematic variation in qPCR TL measurements in relation to thermocycler well position. Controlling for these well-position effects consistently improves measurement validity and yields estimated improvements in statistical power equivalent to increasing sample sizes by 16%. We additionally evaluated the linearity of the relationships between telomere and single copy gene control amplicons and between qPCR and TRF measures. We find that, unlike some previous reports, our data exhibit linear relationships. We introduce the standard error in percent, a superior method for quantifying measurement error as compared to the commonly used coefficient of variation. Using this measure, we find that excluding samples with high measurement error does not improve measurement validity in our study.

CONCLUSIONS

Future studies using block-based thermocyclers should consider well position effects. Since additional information can be gleaned from well position corrections, rerunning analyses of previous results with well position correction could serve as an independent test of the validity of these results.

This study compared behavioral and emotional problems reported by parents and teachers in Chinese urban and rural samples and demographically similar American samples. Parents of 469 6-to-13-year-old children of each nationality completed the Child Behavior Checklist (CBCL). Teachers completed the Teacher's Report Form (TRF). Cross-cultural differences were generally modest in magnitude. Chinese children scored higher on TRF Delinquent Behavior and Anxious/Depressed syndromes, and on Internalizing. American children scored higher on CBCL Aggressive Behavior and TRF Attention Problems syndromes. Boys exhibited more externalizing behaviors across both cultures. The mean correlation between parent and teacher ratings was .36 in the Chinese sample and .29 in the American sample, a nonsignificant difference. Findings indicate considerable similarity between problems reported for children in very different societies.

Recent evidences have highlighted an influence of micronutrients in the maintenance of telomere length (TL). In order to explore whether diet-related telomere shortening had any physiological relevance and was accompanied by significant damage in the genome, in the present study, TL was assessed by terminal restriction fragment (TRF) analysis in peripheral blood lymphocytes of 56 healthy subjects for which detailed information on dietary habits was available and data were compared \with the incidence of nucleoplasmic bridges (NPBs), a marker of chromosomal instability related to telomere dysfunction visualised with the cytokinesis-blocked micronucleus assay. To increase the capability to detect even slight impairment of telomere function, the incidence of NPBs was also evaluated on cells exposed in vitro to ionising radiation. Care was taken to control for potential confounding factors that might influence TL, viz. age, hTERT genotype and smoking status. Data showed that higher consumption of vegetables was related with significantly higher mean TL (P = 0.013); in particular, the analysis of the association between micronutrients and mean TL highlighted a significant role of antioxidant intake, especially beta-carotene, on telomere maintenance (P = 0.004). However, the diet-related telomere shortening did not result in associated increased spontaneous or radiation-induced NPBs. The distribution of TRFs was also analysed and a slight prevalence of radiation-induced NPBs (P = 0.03) was observed in subjects with higher amount of very short TRFs (<2 kb). The relative incidence of very short TRFs was positively associate with ageing (P = 0.008) but unrelated to vegetables consumption and daily intake of micronutrients, suggesting that the degree of telomere erosion related with low dietary intake of antioxidants observed in this study was not so extensive to lead to chromosome instability.

This paper links mass trajectories with telomere dynamics to construct theoretical models of successful and unsuccessful aging in human beings. It couples parameters of telomere length in somatic cells, as expressed by the terminal restriction fragment (TRF), at birth and the rate of telomere attrition thereafter with nonlinear models of somatic growth to predict the probability of surviving disease free, based on the assumption that telomere length in replicating somatic cells is a surrogate indicator of aging determinants in humans. The models capture aspects of individual variation in successful and unsuccessful aging and the long-term consequences of rapid growth early in life.

We are studying novel tocotrienols, which have a number of activities that might interfere with the formation of atherosclerotic plaques, including hypocholesterolemic, antioxidant, anti-inflammatory and antiproliferation effects. This study compared the effects of alpha-tocopherol, the tocotrienol-rich fraction (TRF(25)) and didesmethyl tocotrienol (d-P(25)-T3) of rice bran on the pathogenesis of atherosclerotic lesions in C57BL/6 apolipoprotein (apo)E-deficient (-/-) mice. These mice are an excellent model because they become hyperlipidemic even when they consume a low fat diet and they develop complex atherosclerotic lesions similar to those of humans. These compounds were also tested in wild-type C57BL/6 apoE (+/+) and (+/-) mice fed low or high fat diets. When a high fat diet was supplemented with alpha-tocopherol, TRF(25) or d-P(25)-T3 and fed to mice (+/+) for 24 wk, atherosclerotic lesion size was reduced 23% (P = 0.33), 36% (P = 0.14) and 57% (P < 0.02), respectively, and in mice (+/-) fed for 18 wk, lesions were reduced by 19% (P = 0.15), 28% (P < 0.01) and 33% (P < 0.005), respectively, compared with mice fed a control diet. A low fat diet did not cause atherosclerotic lesions in these mice. The low fat diet supplemented with TRF(25) or d-P(25)-T3 fed to apoE-deficient (-/-) mice for 14 wk decreased atherosclerotic lesion size by 42% (P < 0.04) and 47% (P < 0.01), respectively, whereas alpha-tocopherol supplementation resulted in only an 11% (P = 0.62) reduction. These results demonstrate the superior efficacy of tocotrienols compared with alpha-tocopherol. Although tocotrienols decreased serum triglycerides, total and LDL cholesterol levels, the decreases in atherosclerotic lesions seem to be due to the other activities. Serum tocol concentrations in various groups are also described. This is the first report of a significant reduction in the atherosclerotic lesion size in all three genotypes of apoE mice fed a novel tocotrienol (d-P(25)-T3) of rice bran. Dietary tocotrienol supplements may provide a unique approach to promoting cardiovascular health.

The effects of a sudden but sustained exposure to cold (1 to 6C) on serum TSH, thyroxine (T4) and triiodothyronine (T3) (all measured by radioimmunoassay), pituitary TSH concentration, pituitary TSH secretory responsiveness to hypothalamic extract or synthetic thyrotropin-releasing hormone (TRH) in vitro as well as in vivo, and the changes of the thyrotropin-releasing activity in three TRF-rich hypothalamic areas were determined. In normal animals, serum TSH underwent a series of oscillations, first rising then returning to the basal levels, then rising again, whereas serum T4 and T3 increased within 2 h of cold exposure and remained elevated. Pituitary TSH concentration and the in vitro pituitary responsiveness declined after an initial elevation, whereas the in vivo responsiveness to TRH was diminished throughout the whole exposure to cold. Thyroid-blocked animals with steady, low levels of serum T4 and T3 showed a step by step increase of serum TSH levels and no changes in the other parameters. It is therefore assumed that the decrease of TSH secretion following the initial rise is due to a feedback inhibition by the increased levels of thyroid hormones as is the decreased pituitary responsiveness of TRH in vivo. The pituitary responsiveness in vitro seems to be determined by TSH pituitary concentrations, the changes of which are probably also secondary to the changes of the thyroid hormone levels. The mechanism of the second rise of serum TSH levels is not clear. Thyrotropin-releasing factor (TRF) activity was higher after 2 and 24 h of cold exposure in the median eminence and after 8 h in the anterior hypothalamus-preoptic area, but lower after 8 and 24 h in the dorsomedial hypothalamus. Since the changes of TRF activity in the median eminence coincided with the elevated serum TSH, they are assumed to reflect increased TRF production and secretion. The significance of the TRF changes in the other two areas is not clear.

The synthesis and release of human prolactin (hPRL) in the human fetus was assessed by radioimmunoassay analysis of the content and concentration of hPRL in 82 pituitary glands and the concentration of serum hPRL in 47 fetuses of gestational age 68 days to term. Fetal hPRL exhibited parallelism with the reference standard (Lewis 203-1). hPRL was detected by 68 days of gestation (10 wk), the earliest fetal pituitary gland studied; 8 out of 33 pituitaries had a prolactin (PRL) content above 2.0 ng between 10-15 wk gestation. The mean ocntent of PRL in the pituitary gland increased sharply from 14.8 plus or minus 4.6 ng at 15-19 wk to 405 plus or minus 142 ng at 20-24 wk and 542 plus or minus ng at 25-29 wk gestation. By term, the mean content was 2,039 plus or minus 459 (range 493-3,689) and the mean concentration 15.9 plus or minus 2.4 ng/mg (range 7-20). There was a significant positive correlation (P less than 0.001) between the hPRL and human growth hormone (hGH) content of fetal pituitary glands; at term the hPRL/hGH ratio was 1/290. The concentration of serum hPRL between 12 and 24 wk ranged from 2.9 to 67 ng/ml, mean 19.5 plus or minus 2.5 ng/ml )n = 21); by 26 wk fetal serum hPRL increased sharply and attained levels of 300-500 ng/ml in late gestation. At delivery, the mean plasma concentration of hPRL was 167 plus or minus 14.2 ng/ml in 36 umbilical venous specimens and 111.8 plus or minus 12.3 ng/ml in the matched maternal venous specimens. No correlation between serum hPRL and the pituitary content or concentration of hPRL was demonstrable in 12 matched fetal specimens. In five anencephalic infants, umbilical venous hPRL levels were between 65 and 283 ng/ml. In two anencephalic infants, thyrotropin releasing factor (TRF) (200 mug IV) evoked a rise in serum hPRL in one patient from 43 to 156 ng/ml at 30 min, and in the other from 65 to 404 ng/ml at 120 min. In both patients, plasma thyroid-stimulating hormone (TSH) rose from undetectable base-line levels to peak levels of 97 and 380 muU/ml, respectively. The pattern of change in serum hPRL in the human fetus contrasts sharply with that of serum hGH, luteinizing hormone, or follicle-stimulating hormone. These observations in the fetus and in anencephalic infants suggest that the striking elevation of serum PRL in the fetus is neither mediated by a putative PRL releasing factor or by TRF, nor is a consequence of suppression or absence of PRL release inhibiting factor alone, as a functional hypothalamus is not required to attain the high PRL concentration at term. Several lines of evidence support the view that high plasma estrogen levels characteristic of gestation act directly on the fetal anterior hypophysis to stimulate PRL secretion or to sensitize the secretory mechanism of the lactotrope, increasing its responsiveness to other stimuli.

The biologic activities of helper T cell-replacing factors derived from concanavalin A-stimulated murine T cells (TRF-T) and from lipopolysaccharide-activated macrophages (TFR-M) have been compared. TRF-T stimulates immune responses to heterologous erythrocyte antigens (SRBC and BRBC) in T cell-depleted spleen cultures but not in macrophage-depleted spleen cultures. TRF-M stimulates immune responses in both T cell-depleted and macrophage-depleted spleen cultures. Under conditions where LPS stimulates the release of TRF-M from cultures of activated macrophages, TRF-t has no effect on TFR-M production. Thus. TRF-T does not appear to function by stimulating the release of TRF-M from macrophages. In macrophage-depleted spleen cultures, saturating concentrations of TRF-T and TRF-M when mixed together exhibit striking synergistic effects on the induction of immune responses to erythrocyte antigens. The kinetics of the synergistic effects of TRF-M and TRF-T are consistent with an effect of TRF-M on the production of TRF-T sensitive B cells.

Telomeres progressively shorten with age in somatic cells in culture and in vivo because DNA replication results in the loss of sequences at the 5' ends of double-stranded DNA. Whereas somatic cells do not express the enzyme, telomerase, which adds repeated telomere sequences to chromosome ends, telomerase activity is detected in immortalised and tumour cells in vitro and in primary tumour tissues. This represents an important difference between normal cells and cancer cells, suggesting that telomere shortening causes cellular senescence. Hybrids between immortal cells and normal cells senesce, indicating that immortal cells have lost, mutated or inactivated genes that are required for the programme of senescence in normal cells. Genes involved in the senescence programme have been mapped to over ten different genetic loci using microcell fusion to introduce human chromosomes and restore the senescence programme. Multiple pathways of cellular senescence have also been demonstrated by chromosome transfer, indicating that the functions of the mapped senescence genes are probably different. One possibility is that one or more of these senescence genes may suppress telomerase activity in immortal cells, resulting in telomere shortening and cellular senescence. To test this hypothesis, telomerase activity and the length of terminal restriction fragments (TRFs) have been examined in microcell hybrids. Re-introduction of a normal chromosome 3 into the renal cell carcinoma cell line RCC23, which has the short arm of chromosome 3, restored cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells containing the introduced chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to the parental RCC23. Thus, restoration of cellular senescence by chromosome 3 is associated with repression of telomerase function in RCC cells. This evidence suggests that telomerase suppression is one of several pathways involved in immortalisation.

OBJECTIVE

To our knowledge, none of the articles has been published in the literature which analyse the alterations of saliva in different stages of chronic renal failure (CRF). The objective of this study has been to analyse the flow, pH and biochemical composition of saliva in patients with moderate-severe CRF (M-S CRF) and with terminal renal failure (TRF).

METHODS

The study group was formed of 50 CRF patients: 22 with M-S CRF and 28 with TRF included in a haemodialysis programme. A control group with similar characteristics was established (n=64). Saliva samples were taken from both patients and controls to determine the salivary pH and biochemical composition.

RESULTS

Whole salivary flow was similar in patients and controls. Salivary concentrations of creatinine, urea, sodium, potassium, chloride and alpha-amylase were significantly higher in patients than in controls. Calcium concentration was significantly lower in patients than in controls. Creatinine, urea and potassium concentrations were significantly higher in patients with M-S CRF than in those with TRF. The salivary pH was significantly higher in patients than in controls.

CONCLUSIONS

Salivary composition in patients with CRF is conditioned by the stage of renal failure. The relationship between these biochemical parameters and the oral health status has still not been definitively clarified.

More boys than girls with attention deficit hyperactivity disorder (ADHD) receive treatment. One explanation for this bias may be that boys score higher on disruptive behavior scales than girls. Although this was supported by findings in clinical samples, recent studies in nonreferred samples showed that boys and girls with ADHD are similar with respect to their levels of disruptive behavior as reported by their mother. In this report, we investigate whether the difference in treatment rate is associated with higher teacher problem scores in boys with ADHD than in girls with ADHD. Data were obtained from mothers and teachers in a nonreferred sample of 283 boys and 291 girls with and without ADHD. Children were selected when they scored either low (controls) or high (probands) on attention problems. Mothers completed DSM-IV interviews, Child Behavior Checklists (CBCL) and the Conners Rating Scale (CRS). Teachers filled in the Teacher Report Form (TRF), and the CRS. Boys and girls with ADHD had similar levels of psychiatric illness and school impairment (such as being held back, special class placement and learning problems) by mother report. Mothers reported similar levels of aggression and attention problems in boys and girls with ADHD. In contrast, teachers consistently rated boys with ADHD as having higher scores on reports of attention problems and aggression than girls with ADHD. Gender differences vary across settings: boys and girls with ADHD are rated as behaving differently at school, but not at home. The higher level of teacher reported problem behavior at school may explain the high male-female ratio for ADHD in clinical settings. These findings have implications for the results of genetic studies that rely on referred samples, as these studies may give a distorted view of sex differences in the population.

We report a novel quantum-dot-conjugated graphene, i.e., hybrid SiO2-coated quantum dots (HQDs)-conjugated graphene, for targeted cancer fluorescent imaging, tracking, and monitoring drug delivery, as well as cancer therapy. The hybrid SiO2 shells on the surface of QDs not only mitigate its toxicity, but also protect its fluorescence from being quenched by graphene. By functionalizing the surface of HQDs-conjugated graphene (graphene-HQDs) with transferrin (Trf), we developed a targeted imaging system capable of differential uptake and imaging of cancer cells that express the Trf receptor. The widely used fluorescent antineoplastic anthracycline drug, doxorubicin (DOX), is adsorbed on the surface of graphene and results in a large loading capacity of 1.4 mg mg(-1). It is advantageous that the new delivery system exhibits different fluorescence color in between graphene-HQDs and DOX in the aqueous core upon excitation at a same wavelength for the purpose of tracking and monitoring drug delivery. This simple multifunctional nanoparticle system can deliver DOX to the targeted cancer cells and enable us to localize the graphene-HQDs and monitor intracellular DOX release. The specificity and safety of the nanoparticle conjugate for cancer imaging, monitoring, and therapy has been demonstrated in vitro.

Transferrin, the major plasma iron carrier, mediates iron entry into cells through interaction with its receptor. Several in vitro studies have demonstrated that transferrin plays an essential role in lymphocyte division, a role attributed to its iron transport function. In the present study we used hypotransferrinaemic (Trf(hpx/hpx)) mice to investigate the possible involvement of transferrin in T lymphocyte differentiation in vivo. The absolute number of thymocytes was substantially reduced in Trf(hpx/hpx) mice, a result that could not be attributed to increased apoptosis. Moreover, the proportions of the four major thymic subpopulations were maintained and the percentage of dividing cells was not reduced. A leaky block in the differentiation of CD4(-) CD8(-) CD3(-) CD44(-) CD25(+) (TN3) into CD4(-) CD8(-) CD3(-) CD44(-) CD25(-) (TN4) cells was observed. In addition, a similar impairment of early thymocyte differentiation was observed in mice with reduced levels of transferrin receptor. The present study demonstrates, for the first time, that transferrin itself or a pathway triggered by the interaction of transferrin with its receptor is essential for normal early T-cell differentiation in vivo.

Small regulatory RNAs (sRNAs) are universally distributed in all three domains of life, Archaea, Bacteria, and Eukaryotes. In bacteria, sRNAs typically function by binding near the translation start site of their target mRNAs and thereby inhibit or activate translation. In eukaryotes, miRNAs and siRNAs typically bind to the 3'-untranslated region (3'-UTR) of their target mRNAs and influence translation efficiency and/or mRNA stability. In archaea, sRNAs have been identified in all species investigated using bioinformatic approaches, RNomics, and RNA-Seq. Their size can vary significantly between less than 50 to more than 500 nucleotides. Differential expression of sRNA genes has been studied using northern blot analysis, microarrays, and RNA-Seq. In addition, biological functions have been unraveled by genetic approaches, i.e., by characterization of designed mutants. As in bacteria, it was revealed that archaeal sRNAs are involved in many biological processes, including metabolic regulation, adaptation to extreme conditions, stress responses, and even in regulation of morphology and cellular behavior. Recently, the first target mRNAs were identified in archaea, including one sRNA that binds to the 5'-region of two mRNAs in Methanosarcina mazei Gö1 and a few sRNAs that bind to 3'-UTRs in Sulfolobus solfataricus, three Pyrobaculum species, and Haloferax volcanii, indicating that archaeal sRNAs appear to be able to target both the 5'-UTR or the 3'-UTRs of their respective target mRNAs. In addition, archaea contain tRNA-derived fragments (tRFs), and one tRF has been identified as a major ribosome-binding sRNA in H. volcanii, which downregulates translation in response to stress. Besides regulatory sRNAs, archaea contain further classes of sRNAs, e.g., CRISPR RNAs (crRNAs) and snoRNAs.

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.

Guillain-Barré syndrome (GBS) is an acute polyneuropathy with a variable degree of weakness that reaches its maximal severity within 4 weeks. The disease is mostly preceded by an infection and generally runs a monophasic course. Both intravenous immunoglobulin (IVIg) and plasma exchange (PE) are effective in GBS. Rather surprisingly, steroids alone are ineffective. Mainly for practical reasons, IVIg usually is the preferred treatment. GBS can be subdivided in the acute inflammatory demyelinating polyneuropathy (AIDP), the most frequent form in the western world; acute motor axonal neuropathy (AMAN), most frequent in Asia and Japan; and in Miller-Fisher syndrome (MFS). Additionally, overlap syndromes exist (GBS-MFS overlap). About 10% of GBS patients have a secondary deterioration within the first 8 weeks after start of IVIg. Such a treatment-related fluctuation (TRF) requires repeated IVIg treatment. About 5% of patients initially diagnosed with GBS turn out to have chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) with acute onset (A-CIDP). It is yet unknown whether GBS patients who remain able to walk ('mildly affected GBS patients'), or patients with MFS, also benefit from IVIg. Despite current treatment, GBS remains a severe disease, as about 25% of patients require artificial ventilation during a period of days to months, about 20% of patients are still unable to walk after 6 months and 3-10% of patients die. Additionally, many patients have pain, fatigue or other residual complaints that may persist for months or years. Pain can also be very confusing in making the diagnosis, especially when it precedes the onset of weakness. Advances in prognostic modelling resulted in the development of a simple prognostic scale that predicts the chance for artificial ventilation, already at admission; and in an outcome scale that can be used to determine the chance to be able to walk unaided after 1, 3 or 6 months. GBS patients with a poor prognosis potentially might benefit from a more intensified treatment. A larger increase in serum IgG levels after standard IVIg treatment (0.4 g/kg/day for 5 consecutive days) seems to be related with an improved outcome after GBS. This was one of the reasons to start the second course IVIg trial (SID-GBS trial) in GBS patients with a poor prognosis. This study is currently going on. The international GBS outcome study (IGOS) is a new worldwide prognostic study that aims to get further insight in the (immune)pathophysiology and outcome of GBS, both in children and adults. Hopefully these and other studies will further help to improve the understanding and especially the outcome in patients with GBS.