The vitamin E family of compounds is divided into two subgroups, tocopherols and tocotrienols. However, tocotrienols display more potent apoptotic activity in mammary cancer cells. Although the mechanism(s) mediating tocotrienol-induced apoptosis is presently unknown, apoptosis is carried out by activation of initiator caspases (caspase-8 or -9) that subsequently activate effector caspases (caspase-3, -6, or -7). Studies were conducted to determine whether tocotrienol-induced apoptosis is mediated by activation of the caspase-8 and/or caspase-9 pathway. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained on serum-free media. Treatment with tocotrienol-rich-fraction of palm oil (TRF) and g-tocotrienol, but not a-tocopherol, induced a dose-dependent decrease in +SA cell viability. TRF- and g-tocotrienol-induced cell death resulted from apoptosis, as determined by DNA fragmentation and positive TUNEL assay staining. Additional studies showed that treatment with 50 mM TRF or 20 mM g-tocotrienol increased intracellular activity and levels of processed caspase-8 and -3 but not caspase-9. Furthermore, treatment with specific caspase-8 or -3 inhibitors, but not caspase-9 inhibitor, completely blocked the tocotrienol-induced apoptosis in +SA cells. These findings demonstrate that tocotrienol-induced apoptosis in +SA mammary cancer cells is mediated through activation of the caspase-8 signaling pathway and is independent of caspase-9 activation.

OBJECTIVE

To investigate the relationship of telomerase activity, telomere length, and DNA ploidy in high grade prostatic intraepithelial neoplasia (PIN).

METHODS

Tissue samples were carefully microdissected to obtain adenocarcinoma or PIN-containing tissue free of cancer. Telomerase activity was measured using the PCR-based telomeric repeat amplification protocol (TRAP). Telomere length was estimated from Southern blots of telomere restriction fragments (TRFs). DNA ploidy of PIN and carcinoma was determined by image analysis of adjacent Feulgen stained tissue sections.

RESULTS

Telomerase activity was found in 4 of 25 samples (16%) of high grade PIN. All telomerase positive PIN foci had a diploid DNA content. Although 5 of 25 samples (25%) of high grade PIN foci analyzed were DNA aneuploid, none of these demonstrated telomerase activity. Telomerase positive foci of prostate carcinoma (69% of all cancer foci analyzed) displayed heterogeneity in TRF length, with a mean TRF length two kilobase pairs shorter than that of telomerase negative specimens.

CONCLUSIONS

Telomerase activity is present in a low percentage of high-grade PIN foci, which are diploid by DNA content measurements.

The Child Behavior Checklist's applicability to a Hispanic sample was assessed by an examination of the instrument's internal consistency and concurrent validity. The CBCL and TRF were administered to a community sample representative of children of Puerto Rico aged 4 to 16. Cronbach's alpha was used to assess the internal consistency of empirically derived scales. The relation of CBCL and TRF scores to clinical diagnosis, adaptive functioning, and need for services served as indicators of the concurrent validity of the instrument's Spanish version. The results indicate that the total behavior problem scores on the instruments are good continuous measures of maladjustment for children in Puerto Rico. A child with high values on the scales has a high probability of being classified as a case by a psychiatrist. High levels of internal consistency were found in most subscales. Only scales comprising low prevalence problems showed poor internal consistency.

BACKGROUND

Menopause is associated with significant hormonal changes that result in increased total body fat and abdominal fat, amplifying the risk for metabolic syndrome and diseases such as diabetes, cardiovascular disease and cancer in postmenopausal women. Intermittent fasting regimens hold significant health benefit promise for obese humans, however, regimens that include extreme daytime calorie restriction or daytime fasting are generally associated with hunger and irritability, hampering long-term compliance and adoption in the clinical setting. Time-restricted feeding (TRF), a regimen allowing eating only during a specific period in the normal circadian feeding cycle, without calorie restriction, may increase compliance and provide a more clinically viable method for reducing the detrimental metabolic consequences associated with obesity.

METHODS

We tested TRF as an intervention in a mouse model of postmenopausal obesity. Metabolic parameters were measured using Clinical Laboratory Animal Monitoring System (CLAMS) and we carried out glucose tolerance tests. We also stained liver sections with oil red O to examine steatosis and measured gene expression related to gluconeogenesis.

RESULTS

Preexisting metabolic disease was significantly attenuated during 7 weeks of TRF. Despite having access to the same high fat diet (HFD) as ad libitum fed (ALF) mice, TRF mice experienced rapid weight loss followed by a delayed improvement in insulin resistance and a reduced severity of hepatic steatosis by having access to the HFD for only 8h during their normal nocturnal feeding period. The lower respiratory exchange ratio in the TRF group compared with the ALF group early in the dark phase suggested that fat was the predominant fuel source in the TRF group and correlated with gene expression analyses that suggested a switch from gluconeogenesis to ketogenesis. In addition, TRF mice were more physically active than ALF fed mice.

CONCLUSIONS

Our data support further analysis of TRF as a clinically viable form of intermittent fasting to improve metabolic health due to obesity.

Telomere length was evaluated by terminal repeat fragment method (TRF) in 50 patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) arising from MDS and in 21 patients with untreated primary AML to ascertain, whether telomere erosion was associated with progression of MDS towards overt leukemia. Heterogeneity of TRF among MDS FAB subgroups (P=0.004) originated from its shortening in increased number of patients during progression of the disease. Chromosomal aberrations were present in 32% MDS patients with more eroded telomeres (P=0.022), nevertheless a difference between mean TRF in the subgroups with normal and abnormal karyotype diminished during progression of MDS. A negative correlation between individual TRF and IPSS value (P=0.039) showed that telomere dynamics might serve as a useful prognostic factor for assessment of an individual MDS patient's risk and for decision of an optimal treatment strategy.

The bacterial community of Zostera marina-inhabited bulk sediment vs. root-associated bacteria was investigated by terminal restriction fragment length polymorphism and sequencing, and the spatial extension of the oxygen loss from roots was determined by oxygen microsensors. Extensive oxygen loss was found in the tip region of the youngest roots, and most of the rhizoplane of Z. marina roots was thus anoxic. A significant difference between the bacterial communities associated with the roots and bulk sediment was found. No significant differences were found between differently aged root-bundles. Terminal restriction fragments (TRFs) assigned to sulfate-reducing Deltaproteobacteria showed a relative mean distribution of 12% and 23% of the PCR-amplified bacterial community in the bulk-sediment at the two sites, but only contributed <2% to the root-associated communities. TRFs assigned to Epsilonproteobacteria showed a relative mean distribution of between 5% and 11% in the root-associated communities of the youngest root bundle, in contrast to the bulk-sediment where this TRF only contributed <1.3%. TRFs assigned to Actinobacteria and Gammaproteobacteria also seemed important first root-colonizers, whereas TRFs assigned to Deltaproteobacteria became increasingly important in the root-associated community of the older root bundles. The presence of the roots thus apparently selects for a distinct bacterial community, stimulating the growth of potential symbiotic Epsilon- and Gammaproteobacteria and/or inhibiting the growth of sulfate-reducing Deltaproteobacteria.

To examine the influence of oxidative stress on oxidative protein damage, we studied 51 young Type 1 diabetic patients clinically free of complications and 48 healthy normolipidaemic age-matched controls. We determined: (1) plasma carbonyl (PCO), plasma total thiol (T-SH), and nitrotyrosine (NT) levels as markers of oxidative protein damage; (2) plasma lipid hydroperoxide (LHP), and nitric oxide (NO) levels as markers of oxidative stress; (3) plasma total antioxidant capacity (TAO), ceruloplasmin (Cp), transferrin (TRF), unsaturated iron binding capacity (UIBC), erythrocyte glutathione (GSH), and erythrocyte superoxide dismutase (SOD) as markers of free radical scavengers. There were no significant differences in the levels of these markers between prepubertal diabetic patients and the controls. The levels of both of PCO and LHP were increased in adolescent and young adult Type 1 diabetic patients with respect to their controls. In the adolescent group, patient versus control values for PCO were 1.04+/-0.067 versus 0.67+/-0.0274 nmol/mg and for LHP they were 2. 10+/-1.09 versus 1.00+/-0.4 nmol/mg. In the young adult group, patient versus control values for PCO were 0.99+/-0.054 versus 0. 66+/-0.02 nmol/mg and for LHP they were 1.96+/-0.78 versus 1.15+/-0. 4 nmol/mg. TAO levels were significantly decreased in adolescent diabetic patients compared to their controls (0.92+/-0.27 vs. 1. 86+/-0.37) and in young adult diabetic patients compared to their controls (0.80+/-0.27 vs. 2.11+/-0.54 nmol/mg). T-SH was not different between diabetic patients and the controls. Serum NT, NO, and erythrocyte SOD levels were not different either between three groups of diabetic patients or between the patients and their controls. We attribute this lack of difference to limited disease duration. Changes in markers of oxidative stress other than NT, NO, and SOD observed in adolescent and young adult early stage Type 1 diabetic patients contribute to the imbalance in the redox status of the plasma. We attribute this imbalance to metal-catalyzed protein oxidation in both groups of Type 1 diabetic patients clinically free from complications.

The soaring prevalence of obesity and diabetes is associated with an increase in comorbidities, including elevated risk for cardiovascular diseases (CVDs). CVDs continue to be among the leading causes of death and disability in the United States. While increased nutritional intake from an energy-dense diet is known to disrupt metabolic homeostasis and contributes to the disease risk, circadian rhythm disruption is emerging as a new risk factor for CVD. Circadian rhythms coordinate cardiovascular health via temporal control of organismal metabolism and physiology. Thus, interventions that improve circadian rhythms are prospective entry points to mitigate cardiometabolic disease risk. Although light is a strong modulator of the neural circadian clock, time of food intake is emerging as a dominant agent that affects circadian clocks in metabolic organs. We discovered that imposing a time-restricted feeding (TRF) regimen in which all caloric intakes occur consistently within ≤ 12 h every day exerts many cardiometabolic benefits. TRF prevents excessive body weight gain, improves sleep, and attenuates age- and diet-induced deterioration in cardiac performance. Using an integrative approach that combines Drosophila melanogaster (fruit fly) genetics with transcriptome analyses it was found that the beneficial effects of TRF are mediated by circadian clock, ATP-dependent TCP/TRiC/CCT chaperonin and mitochondrial electron transport chain components. Parallel studies in rodents have shown TRF reduces metabolic disease risks by maintaining metabolic homeostasis. As modern humans continue to live under extended periods of wakefulness and ingestion events, daily eating pattern offers a new potential target for lifestyle intervention to reduce CVD risk.

tRNA-derived RNA fragments (tRFs), non-coding single-stranded RNAs with 14-35 nt in length, were found to play important roles in gene regulation, even in carcinogenesis. In this study, we investigated the expression of tRF-Leu-CAG in human non-small cell lung cancer (NSCLC) and its function in the cell proliferation and cell cycle of NSCLC. The expression level of tRF-Leu-CAG was detected in NSCLC tissues, cell lines, and sera. tRF-Leu-CAG RNA levels were higher in NSCLC tumor tissues than in normal tissues, and also upregulated in NSCLC cell lines. A significant relationship was observed between stage progression and tRF-Leu-CAG in NSCLC sera. We found that in H1299 cells, inhibition of tRF-Leu-CAG suppressed cell proliferation and impeded cell cycle. AURKA was also repressed with the knockdown of tRF-Leu-CAG. Thus, our study revealed that tRF-Leu-CAG may be involved in regulating AURKA and could be a new diagnostic marker and potential therapeutic target in NSCLC.

This study concerns the second, more clinically oriented, part of our epidemiological project. The prevalence of child psychiatric disorders in random samples of 8- and 11-year-old children is assessed by using standardized parent- and child-interviews. The relationships between different measures of child psychopathology are determined. Furthermore, additional validity measures of the Child Behavior Checklist (CBCL) and Teacher Report Form (TRF) developed by Achenbach, are provided. Of the 153 8- and 11-year-old children selected through a two-stage sampling procedure, 116 (76%) children and their parents were intensively clinically assessed. The correlation between CBCL and direct child assessment is .42, whereas the correlation between the TRF and direct child assessment is .28. The correlation between TRF and CBCL is .26. The implications of the low correlation between raters who saw children is different situations are emphasized. The weight given to reports from different sources varies with the kind of problem the child exhibits. It is concluded that more research is needed to investigate the relative value of different types of data for different conditions. Seven percent of the 8- and 11-year-olds were judged severely disordered, whereas for 26% the clinicians judged the child to be moderately or severely disordered. The high prevalence rates found in our study compared with others is partly attributed to the arbitrary nature of clinical judgement. ANOVAs and discriminant function analysis were performed to obtain those CBCL items that showed the best discrimination between children who were clinically judged disordered and children from the normative comparison group. Many items found to be good discriminators of clinical status in this study were among the best discriminators of referral status in the earlier reported part of the study. A number of family and social factors were found to be associated with psychiatric disorder. These findings support those in other studies.

The differentiation of B cells in the in vitro PFC-response to red blood cell antigen proceeds through 2 phases. Antigen-reactive B cells acquire the ability to interact with helper T cells in the first phase. This phase is controlled by macrophages through a mediator that they release (Interleukin 1 ([Il-1]). B cells convert into antibody-secreting cells (PFC) in the second phase, which is controlled by helper T cells or by a mediator that they release (T cell-replacing factors [TRF]). This is demonstrated in experiments in which Il-1 increases the number of B cells capable of responding to T cell help. The majority of antigen-reactive B cells reaches that state of differentiation within 40 hr of incubation with Il-1. After this time, the response of B cells depends no longer on the presence of Il-1 but on the presence of T cells or TRF. The presented data suggest that antigen-primed helper T cells (but not unprimed T cells) induce the release of Il-1 by macrophages, thereby also influencing the early phase of B cell differentiation.

BACKGROUND

Details of mechanisms regulating telomere length are poorly understood in human hematopoietic cells.

METHODS

Gene expression for TRFs and TIN2 was studied in hematopoietic cell lines, blood or bone marrow cells from acute leukemia and in normal leukocyte fractions.

RESULTS

The telomeres were longest in normal leukocytes, shorter in patient samples and still shorter in malignant hematopoietic cell lines. TRF1 mRNA, TRF2 mRNA and TIN2 mRNA in three myeloblastic cell lines and six lymphoblastic cell lines were significantly less abundant than in the corresponding normal cell types. In patients with acute myeloid leukemia, expression of these gene was also less than in normal cells. In additional studies in culture, HL-60 cells with initially high telomerase activity and low expression of TRF mRNA and TIN2 mRNA differentiated into granulocytic and monocytic cells with low telomerase activity and high expression of these mRNAs.

CONCLUSIONS

In hematopoietic carcinogenesis, gene expression for suppressors of telomerase activity, such as TRF and TIN2, is decreased. These genes might hold promise for gene therapy against cancer.

Telomeres of a specific length are essential for continuous cell proliferation. The length of telomeres must be maintained by telomerase action and the telomeric DNA-repeat binding protein must be protected. Therefore, there seems to be a relationship between cell immortality due to telomerase activity and telomeric DNA-repeat binding protein. We examined telomerase activity and the expression of telomeric-repeat binding factor 1 and 2 (TRF1 and TRF2) in gastric cancer. Telomerase activity was semi-quantified using the f-TRAP technique in 53 cancerous and non-cancerous gastric tissue specimens. TRF1 and TRF2 were also studied using an immunohistochemical method to determine the frequency of these factors in cell nuclei. Telomerase activity was observed in 79.2% of the cancerous tissue and in 39.6% of the non-cancerous tissue. The average semi-quantitative values for telomerase activity were 67.3 total product generated (TPG) unit/microg protein in cancerous tissue and 6.0 TPG unit/microg protein in non-cancerous tissue. Moreover, T0/1 tumor had the same incidence of telomerase activity as T2 or deeper tumors. These results indicated that the activation of telomerase begins at an early stage of carcinogenesis. TRF1 and TRF2 were detected in 45.1% and 42.9% of the cancerous tissue and in 70.6% and 65.6% of the non-cancerous tissue, respectively. In addition, low positive staining ratios were found for TRF1 and TRF2 when cancer had more deeply invaded. However, telomerase activity did not correlate with either TRF1 or TRF2. These findings suggest that optimal conditions for efficient telomerase are produced as cancer progresses, via suppression of TRFs.

BACKGROUND

Obligate bacterial symbionts alter the diets of host animals in numerous ways, but the ecological roles of facultative bacterial residents that colonize insect guts remain unclear. Carabid beetles are a common group of beneficial insects appreciated for their ability to consume insect prey and seeds, but the contributions of microbes to diet diversification in this and similar groups of facultative granivores are largely unknown.

RESULTS

Using 16S rRNA gene clone libraries and terminal restriction fragment (tRF) length polymorphism analyses of these genes, we examined the bacterial communities within the guts of facultatively granivorous, adult Harpalus pensylvanicus (Carabidae), fed one of five dietary treatments: 1) an untreated Field population, 2) Seeds with antibiotics (seeds were from Chenopodium album), 3) Seeds without antibiotics, 4) Prey with antibiotics (prey were Acheta domesticus eggs), and 5) Prey without antibiotics. The number of seeds and prey consumed by each beetle were recorded following treatment. Harpalus pensylvanicus possessed a fairly simple gut community of approximately 3-4 bacterial operational taxonomic units (OTU) per beetle that were affiliated with the Gammaproteobacteria, Bacilli, Alphaproteobacteria, and Mollicutes. Bacterial communities of the host varied among the diet and antibiotic treatments. The field population and beetles fed seeds without antibiotics had the closest matching bacterial communities, and the communities in the beetles fed antibiotics were more closely related to each other than to those of the beetles that did not receive antibiotics. Antibiotics reduced and altered the bacterial communities found in the beetle guts. Moreover, beetles fed antibiotics ate fewer seeds, and those beetles that harbored the bacterium Enterococcus faecalis consumed more seeds on average than those lacking this symbiont.

CONCLUSIONS

We conclude that the relationships between the bacterium E. faecalis and this factultative granivore's ability to consume seeds merit further investigation, and that facultative associations with symbiotic bacteria have important implications for the nutritional ecology of their hosts.

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein <em>TRF</em>1 and its associated proteins. However, the link between <em>TRF</em>1 and inhibition of telomerase elongation of telomeres remains elusive because <em>TRF</em>1 has no direct effect on telomerase activity. We have previously identified one Pin2/<em>TRF</em>1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-<em>TRF</em>1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and <em>TRF</em>1 responsible for the PinX1-<em>TRF</em>1 interaction, we show that the <em>TRF</em> homology domain of <em>TRF</em>1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous <em>TRF</em>1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by <em>TRF</em>1 and provides a critical link between <em>TRF</em>1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.

Two in vitro B cell tumor lines have been used to characterize and partially purify a lymphokine, or family of lymphokines, from monoclonal helper T cell immune response supernatants. These lymphokines induce the pre-B-like 70Z/3 tumor cell to synthesize Ig L chains and express complete Ig molecules on its cell surface, and cause the mature B cell-like WEHI-279 tumor cell to increase its ratio of secretory to membrane mu production, begin high rate Ig secretion, and then die. Most of the activity responsible for these changes co-purifies during five different separation procedures, implying the existence of a discrete molecule or closely related class of molecules able to mediate all of these effects. The molecules active in these systems appear distinct from the other lymphokines IL 1, IL 2, G/M-CSA, TRF, IFN, BCGF, and the activity variously termed IL 3/BPA/PSF/HCGF/MCGF, etc. We call these B cell-differentiating molecules BMF, or B cell maturation factor(s). The BMF molecules are mildly acidic (pI 5 to 6 in various conditions), extremely hydrophobic, probably heterogeneously glycosylated glycoproteins, with an apparent m.w. of 50,000 to 55,000 by gel permeation chromatography and 16,000 by SDS-PAGE. BMF has been purified approximately 3000-fold by three sequential chromatographic steps, with the use of the B tumor line assay systems. BMF molecules thus purified also cause normal resting splenic B cells to mature to the state of active Ig secretion.

The Cuatro Cienegas basin (Coahuila, México) is a composite of different water systems in the middle of the desert with unusually high levels of endemism and diversity in different taxa. Although the diversity of macrobiota has been well described, little is known about the diversity and distribution of microorganisms in the oligotrophic ponds. Here we describe the extent and distribution of diversity found in aquatic prokaryotic communities by analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes and phylogenetic analysis of cloned genes. Twelve locations within the basin were sampled. Among all the samples, we found a total of 117 operational taxonomic units (OTUs) using T-RFLPs, which ranged in any single sample from four to 49. OTU richness and Shannon diversity indices for different sites varied, but none were particularly high. 16S rRNA gene sequence data showed 68 different phylotypes among 198 clones. The most abundant phylotypes were Gamma- and Betaproteobacteria, and extreme halophiles. The differences among sites were significant; 45 TRFs were found only once, and 37% of the total diversity was represented by differences between sites, suggesting high beta-diversity. Further studies are needed to test whether this is a direct consequence of environmental heterogeneity in the basin.

T191, a monoclonal antibody reactive with the T200 common leukocyte antigen, profoundly inhibits an early event(s) associated with alpha-immunoglobulin M (alpha IgM)/T cell replacing factor (TRF) or alpha IgM/recombinant interleukin 1 and 2 (rIL 1 and rIL 2)-induced tonsillar B cell proliferation. Kinetic analysis of T191-mediated inhibition indicated that the antibody exerts its effect within 12 to 24 hr of the initiation of cultures and rapidly loses its activity thereafter. Small resting B cells are most sensitive to T191 inhibition, whereas B cells with increasing buoyant density (presumably reflecting stages of increased activation) become progressively T191 insensitive. Analysis of RNA synthesis subsequent to alpha IgM crosslinking of surface immunoglobulin demonstrated that T191 reduced [3H]uridine incorporation by up to 38% during the first 20 hr of culture. In contrast to the effects seen with alpha IgM stimulated B cells, T191 had no inhibitory effect upon phorbol myristate acetate-induced B cell proliferation. The inhibitory effect upon B cell proliferation observed with T191 is not unique among other alpha-T200 antibodies. Four of five previously described alpha-T200 monoclonal antibodies had similar inhibitory effects (82 to 57% maximum inhibition of [3H]thymidine incorporation). However, 13.3, an alpha-T200 monoclonal antibody previously shown to block natural killer (NK) cell-mediated killing was without effect. Likewise, those antibodies capable of inhibiting B cell proliferation failed to block NK-mediated cytolysis. Antibody binding experiments together with proliferation inhibition studies suggest that all of the monoclonal antibodies tested recognized distinct epitopes on the T200 antigen. Both observations are of significance because they demonstrate that the effects seen with anti-T200 antibodies represent an interference with highly specific functional regions on the T200 molecules.

We have investigated the effect of iron on the expression of transferrin receptors (TrfRs) and ferritin chains in cultures of human peripheral blood monocytes maturing to macrophages. Monocyte-macrophage maturation is associated with a gradual rise of Trf-binding capacity in the absence of cell proliferation. At all culture times, treatment with ferric ammonium citrate induces a dose-dependent rise of the Trf-binding level as compared with nontreated cells. Scatchard analysis revealed that this phenomenon is due to an increase in receptor number rather than an alteration in ligand-receptor affinity. Biosynthesis experiments indicated that the rise in number of TrfRs is due to an increase of receptor synthesis, which is associated with a sustained elevation of the TrfR RNA level. The up-regulation of TrfR synthesis is specific in that expression of other macrophage membrane proteins is not affected by iron addition. Conversely, addition of an iron chelator induced a slight decrease of TrfR synthesis. The expression of heavy and light ferritin chains at RNA and protein levels was markedly more elevated in cultured macrophages than in fresh monocytes, thus suggesting modulation of ferritin genes at transcriptional or post-transcriptional levels. Addition of iron salts to monocyte-macrophage cultures sharply stimulated ferritin synthesis but only slightly enhanced the level of ferritin RNA, thus indicating a modulation at the translational level. These results suggests that in cultured human monocytes-macrophages, iron up-regulates TrfR expression, thus in sharp contrast to the negative feedback reported in a variety of other cell types. These observations may shed light on the mechanism(s) of iron storage in tissue macrophages under normal conditions and possibly on the pathogenesis of diseases characterized by abnormal iron storage.

The acute phase response involves changes in serum concentrations of a number of liver-synthesized proteins. Among these are C-reactive protein (CRP), ferritin (FER), transferrin (Trf) and ceruloplasmin (Cp). Determination of serum CRP, FER, Trf, and Cp was performed in 52 patients with inoperable head and neck cancer (n=11), inoperable esophageal cancer (n=10), rectal cancer (n=9; operation was performed=5, inoperable=4), and lung cancer (n=22), all of whom were treated with radical radiotherapy (RT). Post-radiotherapy CRP levels were significantly higher compared to the preradiotherapy levels (p<0.001). We found decreased serum Trf levels during the irradiation period, while acute-phase proteins such as CRP, FER, and Cp levels increased during the RT period. Further studies on the roles of other acute phase reactants and the above mentioned parameters in a large-patients-with cancer group during radiotherapy are required to understand the role of markers, which are altered during radiotherapy.

The evaluation of receptor ligand interactions is important in the field of drug discovery and development. Currently these interactions are typically measured with cumbersome (low throughput) radiolabels. Higher throughput screens are available such as fluorescent measurements of G-protein coupled receptor-induced Ca2+ increases or fluorescence anisotropy, yet these have limited applicability and/or low signal to noise. Hence, there is a need to develop more widely applicable and more sensitive labels that can be used to monitor ligand-receptor interactions. Lanthanides provide an attractive alternative to the traditional labels used for monitoring ligand-receptor interactions. The incorporation of lanthanide labels into traditional assays used to assess receptor-ligand interactions can make these assays more affordable, less time consuming and amenable to automation. Lanthanides can be coupled to ligands and provide strong luminescent signals that can be detected using time-resolved fluorescence (TRF) methods. This approach takes advantage of the long fluorescence lifetime of the lanthanide and can detect less than one attomole of europium in a multiwell plate sample. This short review provides a basic introduction into lanthanides and TRF and describes some of the recent assays which have utilized lanthanides as labels to assess ligand-receptor interactions.

OBJECTIVE

To demonstrate that chronic hyperoxia induces single-stranded breaks in chromosomal telomeres as a measure of oxidative DNA damage in cultured RPE cells.

METHODS

RPE340 cells were cultured in 40% and 20% (control) O(2). DNA damage was assessed by mean terminal restriction fragment (TRF) length, and the S1 nuclease assay was used to determine the frequency of single-strand breaks in telomeric DNA. The degree of oxidative stress in cells was estimated by flow cytometric analysis of reactive oxygen intermediate (ROI)-induced 2',7'-dichlorodihydrofluorescein diacetate fluorescence and Northern blot analysis of heme oxygenase-1 (HO-1) mRNA induction.

RESULTS

The mean TRF length of cells grown in 40% O(2) shortened at a faster rate than those grown in 20% O(2). The S1 nuclease assay showed that the accelerated mean TRF length shortening was due to an increased accumulation of single-stranded breaks in telomeric DNA. The degree of ROI production and HO-1 mRNA induction was greater in cells treated with 40% than 20% O(2), an effect that was also larger in old than young passaged cells.

CONCLUSIONS

RPE340 cells in vitro grown in chronic hyperoxia exhibited evidence of DNA damage with accelerated telomeric shortening via an increased accumulation of single-strand breaks in telomeric DNA. These changes could provide insight into aging of RPE cells by oxidative DNA damage.