Competence, emotional and behavioural problems were examined in 256 school-children, aged 13-17 years, in Novosibirsk, Russia, using the Youth Self-Report (YSR), the Child Behavior Checklist (CBCL) and the Teacher's Report Form (TRF). The internal consistency of syndrome scales for all three instruments was generally adequate. Interrater agreements for the problem scales were higher for girls than for boys in all combinations of informants. These findings are discussed in terms of gender role development. Gender effects on the scales' scores were more numerous and stronger than age. No SES differences were found. Russian children reported more problems than children of other nationalities did. Compared with American children, Russians were lower on competencies and higher on Somatic complaints and self-reported behavioural problems. On competence scales, Russian children in an unselected sample scored themselves lower than American children in a clinical sample. It is supposed that low self-esteem may be the first effect of the unfavourable development in Russian adolescents.

The precise temporal and spatial expressions of specific transcription regulation factors (TRF) have long been considered essential for spermatogenesis. Recently, it has been speculated that mammals have evolved more specialised TRF genes. In the human, the TAF7L gene may be essential for maintenance of spermatogenesis. In this study, we investigated the possible role of the TAF7L gene located on the X chromosome in testicular function and spermatogenic failure. In a case-controlled retrospective study, we recruited 16 infertile males with consistent, nonobstructive azoospermia and with normal serum follicle-stimulating hormone (FSH) levels. Twenty age-matched men with normal spermatogenesis with the same ethnic background (Caucasian) were recruited as controls. Their genomic DNA was screened for sequence changes in the coding regions and part of the flanking introns of the TAF7L gene by direct sequencing. Amino acid sequence was compared with the NCBI standard sequence (BC043391). Semen analysis and hormone evaluation were performed. We observed six sequence variations in four patients, consisting of two point mutations, one each in exon 9 and 13 and one six-basepair deletion in exon 13 with concomitant changes in amino acid. One additional nucleotide exchange was observed in intron 8. Most of these changes were also found in eight controls with the exception of changes in exon 13. A meta-analysis including the present study and literature data suggests a possible association of the point mutation in exon 13 with infertility. There was no association or relationship with reproductive hormones. In conclusion, the sequence variants in the cDNA sequence observed are common polymorphisms. The changes in intron 8 appear novel. We report for the first time that most of the alterations are not associated with gonadal dysfunction, while the sequence variant in exon 13 may represent a risk factor for spermatogenic failure.

Colorectal cancer (CRC) is the third most common malignance. Although great efforts have been made to understand the pathogenesis of CRC, the underlying mechanisms are still unclear. It is now clear that more than 90% of the total genome is actively transcribed, but lack of protein-coding potential. The massive amount of RNA can be classified as housekeeping RNAs (such as ribosomal RNAs, transfer RNAs) and regulatory RNAs (such as microRNAs [miRNAs], PIWI-interacting RNA [piRNAs], tRNA-derived stress-induced RNA, tRNA-derived small RNA [tRFs] and long non-coding RNAs [lncRNAs]). Small non-coding RNAs are a group of ncRNAs with the length no more than 200 nt and they have been found to exert important regulatory functions under many pathological conditions. In this review, we summarize the biogenesis and functions of regulatory sncRNAs, such as miRNAs, piRNA and tRFs, and highlight their involvements in cancers, particularly in CRC.

Asn-linked oligosaccharides terminating with the sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha (S4GGnM) are present on the glycoprotein hormones lutropin and thyrotropin, pro-opiomelanocortin, and tissue factor pathway inhibitor. The peptide motif ProXaaArg/Lys (PXR/K), which is recognized by a PXR/K-specific GalNAc-transferase, is present in each of these glycoproteins 6-9 residues NH2-terminal to an Asn glycosylation site. Both the PXR/K-specific GalNAc-transferase and a GalNAc beta 1,4GlcNAc beta 1,2Man alpha (GGnM)-4-sulfotransferase are required for synthesis of the S4GGnM sequence. Glycoproteins which do not contain the PXR/K motif but bear Asn-linked oligosaccharides terminating with GGnM or sialic acid alpha 2,3/6GGnM have also been described, suggesting a distinct GalNAc-transferase may be responsible for their synthesis. We have examined a number of tissues and cultured cell lines for the transfer of sulfate to the trisaccharide acceptor GGnM and transfer of GalNAc to oligosaccharide acceptors on protein which do, human chorionic gonadotropin (hCG), and do not, transferrin (Trf), contain the PXR/K motif. The PXR/K-specific GalNAc-transferase and the GGnM-4-sulfo-transferase are expressed in salivary gland, pituitary, lacrimal gland, kidney, and brain, and in the cell lines AtT-20, 293, SHSY5Y, and alpha T3. In contrast Bowes, EL-4, and B16L6 cell extracts transferred GalNAc to oligosaccharides acceptors on Trf but not on hCG. A number of tissues and cell lines displayed transfer of GalNAc to both hCG and to Trf suggesting that two distinct GalNAc-transferases were present. The GGnM-4-sulfotransferase was expressed in tissues and cell lines which expressed the PXR/K-specific GalNAc-transferase but not in cell lines expressing exclusively the Trf-specific GalNAc-transferase. Thus, the PXR/K-specific GalNAc-transferase and the GGnM-4-sulfotransferase are coordinately expressed in a number of tissues other than pituitary. The Trf-specific GalNAc-transferase may account for the presence of beta 1,4-linked GalNAc on glycoproteins which do not contain the PXR/K motif.

An increase in mRNA encoding the secretory form of the mu heavy (H) chain (mus) and in the ratio of mus mRNA to the mRNA encoding the membrane form of the microH chain (micron) occurs in normal B cells stimulated with anti-IgM and BSF-p1 together with the two B cell differentiation factors B15-TRF and EL-TRF. Stimulation of cells with anti-IgM and BSF-p1 with either B15- or EL-TRF causes no change in mus levels or mus/micron ratios. The requirements for induction of high rate IgM synthesis in normal B cells stimulated with anti-IgM were precisely the same as those required for elevation of mus, mRNA levels and for increase in mus/micron mRNA ratios. A very close correlation also exists between induction of mRNA for J chain and increase in mus mRNA levels. Similarly, the increase in J chain protein concentration and percent of cells with cytoplasmic IgM were correlated to each other and to levels of mus and J chain mRNA. These results indicate that elevation of mus mRNA and mus/micron mRNA ratios occur in normal B cells only upon commitment to IgM synthesis, and reaffirm the close relation between IgM synthesis and the presence of J chain.

An 8-month old male with Cushing's disease is presented; his clinical presentation and appearance were typical of infants with glucocorticoid excess. Concentrations of cortisol, 17-hydroxyprogesterone, and adrenal androgens were strikingly elevated. High doses of dexamethasone did not suppress the excretion of urinary free cortisol or 17-hydroxycorticoids, and administration of ACTH elicited no further rise in plasma cortisol. Responses of LH, FSH, and PRL to iv LRF and TRF were appropriate for age, but neither TSH nor ACTH rose significantly. Plasma ACTH values were elevated to 700 pg/ml. An intracranial mass lesion superior and anterior to the sella turcica was demonstrated by computerized axial tomography and angiography. An inoperable pituitary adenoma was a massive surrounding fibroblastic reaction was found at craniotomy. The pathological diagnosis of an ACTH-producing pituitary adenoma was confirmed by immunohistochemistry and by the in vitro secretion of ACTH by cells cultured from the tumor.

BACKGROUND

Cowpea [Vigna unguiculata (L.) Walp.] is one of the most important food and forage legumes in the semi-arid tropics because of its ability to tolerate drought and grow on poor soils. It is cultivated mostly by poor farmers in developing countries, with 80% of production taking place in the dry savannah of tropical West and Central Africa. Cowpea is largely an underexploited crop with relatively little genomic information available for use in applied plant breeding. The goal of the Cowpea Genomics Initiative (CGI), funded by the Kirkhouse Trust, a UK-based charitable organization, is to leverage modern molecular genetic tools for gene discovery and cowpea improvement. One aspect of the initiative is the sequencing of the gene-rich region of the cowpea genome (termed the genespace) recovered using methylation filtration technology and providing annotation and analysis of the sequence data.

METHODS

CGKB, Cowpea Genespace/Genomics Knowledge Base, is an annotation knowledge base developed under the CGI. The database is based on information derived from 298,848 cowpea genespace sequences (GSS) isolated by methylation filtering of genomic DNA. The CGKB consists of three knowledge bases: GSS annotation and comparative genomics knowledge base, GSS enzyme and metabolic pathway knowledge base, and GSS simple sequence repeats (SSRs) knowledge base for molecular marker discovery. A homology-based approach was applied for annotations of the GSS, mainly using BLASTX against four public FASTA formatted protein databases (NCBI GenBank Proteins, UniProtKB-Swiss-Prot, UniprotKB-PIR (Protein Information Resource), and UniProtKB-TrEMBL). Comparative genome analysis was done by BLASTX searches of the cowpea GSS against four plant proteomes from Arabidopsis thaliana, Oryza sativa, Medicago truncatula, and Populus trichocarpa. The possible exons and introns on each cowpea GSS were predicted using the HMM-based Genscan gene predication program and the potential domains on annotated GSS were analyzed using the HMMER package against the Pfam database. The annotated GSS were also assigned with Gene Ontology annotation terms and integrated with 228 curated plant metabolic pathways from the Arabidopsis Information Resource (TAIR) knowledge base. The UniProtKB-Swiss-Prot ENZYME database was used to assign putative enzymatic function to each GSS. Each GSS was also analyzed with the Tandem Repeat Finder (TRF) program in order to identify potential SSRs for molecular marker discovery. The raw sequence data, processed annotation, and SSR results were stored in relational tables designed in key-value pair fashion using a PostgreSQL relational database management system. The biological knowledge derived from the sequence data and processed results are represented as views or materialized views in the relational database management system. All materialized views are indexed for quick data access and retrieval. Data processing and analysis pipelines were implemented using the Perl programming language. The web interface was implemented in JavaScript and Perl CGI running on an Apache web server. The CPU intensive data processing and analysis pipelines were run on a computer cluster of more than 30 dual-processor Apple XServes. A job management system called Vela was created as a robust way to submit large numbers of jobs to the Portable Batch System (PBS).

CONCLUSIONS

CGKB is an integrated and annotated resource for cowpea GSS with features of homology-based and HMM-based annotations, enzyme and pathway annotations, GO term annotation, toolkits, and a large number of other facilities to perform complex queries. The cowpea GSS, chloroplast sequences, mitochondrial sequences, retroelements, and SSR sequences are available as FASTA formatted files and downloadable at CGKB. This database and web interface are publicly accessible at http://cowpeagenomics.med.virginia.edu/CGKB/.

Telomere repeat factor 1 (<em>TRF</em>1) regulates the steady-state length of chromosomes, whereby its overexpression results in telomere shortening while dominant negative <em>TRF</em>1 mutations can lead to telomere elongation, which is linked to cell immortalization/transformation. Although present in the nucleus at mammalian chromosomal ends during interphase and mitosis, nothing is known of the mechanism by which <em>TRF</em>1 enters the nucleus or how its nuclear levels may be regulated and the relevance of this, in turn, to telomere length and cell immortalization. Here we examine the nuclear import mechanism of <em>TRF</em> by expressing and purifying a recombinant <em>TRF</em>1-GFP (green fluorescent protein) fusion protein that is functional in terms of being able to bind telomeric DNA specifically as shown using a novel, quantitative double-label gel mobility shift assay. We quantitate the ability of <em>TRF</em>1-GFP to accumulate in the nucleus using real time confocal laser scanning microscopy, showing that the nuclear import pathway of <em>TRF</em>1 is mediated by importin (Imp) beta1 and Ran. Imp beta is shown to bind directly to <em>TRF</em>1 with nanomolar affinity using native gel electrophoretic and fluorescence polarization (FP) approaches; FP experiments also demonstrate that Imp beta residues 1-380 are responsible for <em>TRF</em>1 binding. Intriguingly, when dimerized to Imp beta, Imp alpha was found to inhibit Imp beta-mediated nuclear accumulation, although not affecting Imp beta binding to <em>TRF</em>1. The study represents the first elucidation of the nuclear transport mechanism of <em>TRF</em>1; that its nuclear import is mediated directly by Imp beta but inhibited by Imp alpha may represent a novel regulatory mechanism, with potential relevance to oncogenesis.

We sought to determine if serum total iron-binding capacity (TIBC) is equivalent to serum transferrin (TRF) so that a low-cost colorimetric chemical assay for unsaturated iron-binding capacity (UIBC) could be substituted for a high-cost immunologic assay for TRF. Our study design included independent and blinded measurements of UIBC, serum iron, and TRF concentrations in human serum samples. Data from five independent correlation studies carried out at three different Quest Diagnostics laboratories were combined into one data set containing 570 paired results for TIBC and TRF. r2 was 0.941 when three outliers were eliminated from the 570-sample data set. Scatter about the regression line was fully accounted for by the CVs for the TIBC and TRF assays. When each test is measured precisely and without bias, the ratio of TIBC (mumol/L) to TRF (g/L) in SI units is close to the theoretically expected value of 25.0.

BACKGROUND

Little evidence is available on factors associated with persistence and change of psychopathology, and little is known about the predictive value of factors regarding change once problem behaviours exist. This study aims to evaluate change in level of scores of empirically derived problem patterns and to study factors that influence this change for children and adolescents referred to mental health services.

METHODS

A referred sample (N = 1,652), aged 4 to 18 years at initial assessment, was followed up after a mean interval of 6.2 years. We used standardised information from parents, teachers and subjects, including the CBCL, YSR and TRF at both assessments.

RESULTS

Subjects at follow-up scored significantly above the expected mean norm scores, although for most scores improvement was found. The strongest predicting factor for time 2 psychopathology was the corresponding time 1 score, odds ratios ranging from 1.6 to 21.7. Males and children older at intake improved more than females and younger children, respectively.

CONCLUSIONS

Few child, family and treatment-related factors had additional predictive value over and above earlier psychopathology, and their contribution to the prediction of outcome was small. Findings indicate continuity of behavioural and emotional problems in clinically referred children and adolescents, and these problems should be viewed as chronic conditions. Girls referred for behavioural and emotional problems may form a group especially at risk for poor outcome.

In the present study the neuropsychological, academic and social-emotional profiles were examined in Neurofibromatosis type 1 (NF1) children.

METHODS

17 NF1 children (ages 7-11) with NF1 without serious medical problems and with a full scale IQ (FSIQ) above 70.

METHODS

Wechsler Intelligence Scale for Children-Revised (WISC-R), academic tests and an exhaustive neuropsychological test battery were administered in all children. Parents and teachers filled out the Child Behavioural Checklist (CBCL) and Teacher Report Form (TRF), respectively, the NF1 children the Experienced Competence Scale for Children (ECSC).

CONCLUSIONS

Nearly 50% (8/17) of the children showed learning disabilities, when corrected for IQ in the academic evaluations. Isolated impaired literacy skills, particularly spelling problems, were most frequent (4/8), whereas a pure arithmetic learning disability was rare (1/8). Three children presented both learning disabilities. Results on academic and neuropsychological tests did not fit the well-known types of learning disabilities -- nonverbal learning disability (NLD) and dyslexia. Nearly all NF1 children showed visual perceptual and executive dysfunctions. In this study, teachers more frequently reported behavioural problems in NF1 children than parents, as opposed to literature data in a general population. The correspondence of the perception of internalizing problems between the children and teachers was greater than between children and their parents. No correlation was found between the performances on the WISC-R, specific neuropsychological results, academic performances and behavioural problems. The Deficiency in Attention, Motor and Perception (DAMP) concept seems most appropriate in order to describe the neuropsychological deficits and their repercussions on behavioural and academic performances seen in NF1 children.

Bacteremia results in significant morbidity and mortality, especially among patient populations that are immunocompromised. Broad-spectrum antibiotics are administered to patients suspected to have bloodstream infections that are awaiting diagnosis that depends on blood culture analysis. Significant delays in identification of pathogens can result, primarily due to the dependence on growth-based identification systems. To address these limitations, we took advantage of terminal restriction fragment (TRF) length polymorphisms (T-RFLP) due to 16S ribosomal DNA (rDNA) sequence diversity to rapidly identify bacterial pathogens directly from positive blood culture. TRF profiles for each organism were determined by sizing fragments from restriction digests of PCR products derived from two sets of 16S rDNA-specific fluorescent dye-labeled primers. In addition, we created a TRF profile database (TRFPD) with 5899 predicted TRF profiles from sequence information representing 2860 different bacterial species. TRF profiles were experimentally determined for 69 reference organisms and 32 clinical isolates and then compared against the predicted profiles in the TRFPD. The predictive value of the profiles was found to be accurate to the species level with most organisms tested. In addition, identification of 10 different genera was possible with profiles comprising two or three TRFs. Although it was possible to identify Enterobacteriaceae by using a profile of three TRFs, the similarity of the TRF profiles of these organisms makes differentiation of species less reliable with the current method. The ability to rapidly (i.e., within approximately 8 h) identify bacteria from blood cultures has potential for reducing unnecessary use of broad-spectrum antibiotics and promoting more timely prescription of appropriate antibiotics.

BACKGROUND

Microvascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses.

METHODS

C3H/HeN mice were infected intravenously with a sublethal dose of R. conorii. At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1μg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs.

RESULTS

In the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in vivo. We also have provided direct evidence that rickettsial infection sensitizes human ECs to the translocation of exogenous ANG in a compartmentalized pattern at different times post-infection. Typically, exogenous ANG translocates into the nucleus at 24 hrs and to the cytoplasm at 72 hrs post-infection. The ANG cytoplasmic translocation enhances phosphorylation and destabilization of VE-cadherin and attenuates endothelial barrier function. Of note, deep sequencing analysis detected tRFs, mostly derived from the 5'-halves of host tRNAs, that are induced by ANG. Northern hybridization validates the two most abundantly cloned tRFs derived from tRNA-ValGTG and tRNA-GlyGCC, in both mouse tissues and human cells. Bioinformatics analysis predicted that these tRFs may interact with transcripts associated with the endothelial barrier, the host cell inflammatory response, and autophagy.

CONCLUSIONS

Our data provide new insight into the role of compartmentalized ANG during SFG rickettsioses, and highlight its possible mediation through tRFs.

Food restriction is associated with a number of endocrine disturbances. We validated the experimental conditions for several house-keeping genes and determined the effects of 12 day 50% food restriction on hypothalamic and pituitary transcription of genes involved in different neuroendocrine systems, using real-time quantitative polymerase chain reaction (PCR). A total of 7 nuclear receptors and 12 neuropeptides and peptide hormones were investigated in the dorsal and ventral hypothalamus and the pituitary gland in rats. In the hypothalamus, food restriction reduced mRNA levels of estrogen receptor alpha (ERalpha), progesterone receptor, glucocorticoid receptor, thyroid hormone receptor alpha and beta, pro-opiomelanocortin (POMC), growth hormone-releasing factor (GHRF), corticotropin-releasing factor (CRF), thyrotropin-releasing factor (TRF), somatostatin, and increased that of neuropeptide Y (NPY). In the pituitary, the treatment reduced growth hormone (GH), luteinizing hormone beta (LHbeta) and thyrotropin beta, but increased ERalpha mRNA levels. The study provides a map of how food restriction affects the regulation of a number of transcripts involved in neuroendocrine control.

The cholesterol-suppressive action of the tocotrienol-rich-fraction (TRF) of palm oil may be due to the effect of its constituent tocotrienols on beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase activity. The tocotrienols, modulate HMG-CoA reductase activity via a post-transcriptional mechanism. As a consequence small doses (5-200 ppm) of TRF-supplemented diets fed to experimental animals lower serum cholesterol levels. These findings led us to evaluate the safety and efficacy of large supplements of TRF and its constituents. Diets supplemented with 50, 100, 250, 500, 1000, or 2000 ppm of TRF, alpha-tocopherol, alpha-tocotrienol, gamma-tocotrienol, or 6-tocotrienol were fed to chickens for 4 wk. There were no differences between groups or within groups in weight gain, or in feed consumption at the termination of the feeding period. Supplemental TRF produced a dose-response (50-2000 ppm) lowering of serum total and LDL cholesterol levels of 22% and 52% (P < 0.05), respectively, compared with the control group. alpha-Tocopherol did not affect total or LDL-cholesterol levels. Supplemental alpha-tocotrienol within the 50-500 ppm range produced a dose-response lowering of total (17%) and LDL (33%) cholesterol levels. The more potent gamma and delta isomers yielded dose-response (50-2,000 ppm) reductions of serum total (32%) and LDL (66%) cholesterol levels. HDL cholesterol levels were minimally impacted by the tocotrienols; as a result, the HDL/LDL cholesterol ratios were markedly improved (123-150%) by the supplements. Serum triglyceride levels were significantly lower in sera of pullets receiving the higher supplements. The safe dose of various tocotrienols for human consumption might be 200-1000 mg/d based on this study.

Compared the effectiveness of discriminating attention deficit/hyperactivity disorder (ADHD) subtypes using the Parent Rating Scale (PRS) and Teacher Rating Scale (TRS) of the Behavior Assessment System for Children (BASC) and the Parent Report Form and Teacher Report Form (TRF) of the Achenbach Child Behavior Checklist (CBCL). To determine the extent to which these scales measured similar behaviors, Pearson Product-Moment Correlations were computed for the parent scales (PRS and CBCL) and for the teacher scales (TRS and TRF). Results indicated that correlations were significant for a number of scales. Discriminant analysis does not suggest a strong advantage of either measure in differentiating children with ADHD from those who do not meet criteria for ADHD, except for the BASC TRS which has better predictive ability for children who do not meet ADHD criteria. For subtypes of ADHD, and specifically the ADHD: Predominantly Inattentive subtype, however, results would favor the use of the BASC PRS and TRS.

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbb(th3/+)), hereditary hemochromatosis (Hfe(-/-), Hjv(-/-), and Tfr2(Y245X/Y245X)), hypotransferrinemia (Trf(hpx/hpx)), heterozygous transferrin receptor 1 deficiency (Tfrc(+/-)) and iron refractory iron deficiency anemia (Tmprss6(-/-) and Tmprss6(hem8/hem8)). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.

In the vascular endothelium of human beings, telomere length is negatively related while the frequency of aneuploidy is positively related to donor age. Both in culture and in vivo the frequency of aneuploidy increases as telomere length is shortened. In this study we explored the relation between telomere length and aneuploidy in cultured human umbilical vein endothelial cells (HUVEC) by: (a) karyotype analysis and fluorescent in situ hybridization (FISH), (b) measurement of the terminal restriction fragments (TRF), and (c) assessment of replicative senescence by the expression of beta-galactosidase. Of 8 HUVEC strains, 7 cell strains lost chromosome 13, as shown by metaphase analysis and FISH of interphase cells. Five strains gained chromosome 11. In addition, five HUVEC strains became hypotetraploid shortly after the loss of chromosome 13. The loss of chromosome 13 was observed as early as PD 20, when mean TRF length was greater than 9 kb and the percentage of cells positive for beta-galactosidase was relatively low. The almost uniform loss of chromosome 13 suggests that this unique type of aneuploidy of HUVEC is the result of a progressive expression of clones with survival advantage.

Multiple myeloma is a hematological cancer of plasma B cells and remains incurable. Two major subtypes of myeloma, hyperdiploid MM (HMM) and non-hyperdiploid MM (NHMM), have distinct chromosomal alterations and different survival outcomes. Transcription factors (TrFs) have been implicated in myeloma oncogenesis, but their dysregulation in myeloma subtypes are less studied. Here, we developed a TrF-pathway coexpression analysis to identify altered coexpression between two sample types. We apply the method to the two myeloma subtypes and the cell cycle arrest pathway, which is significantly differentially expressed between the two subtypes. We find that TrFs MYC, nuclear factor-κB and HOXA9 have significantly lower coexpression with cell cycle arrest in HMM, co-occurring with their overactivation in HMM. In contrast, TrFs ESR1 (estrogen receptor 1), SP1 and E2F1 have significantly lower coexpression with cell cycle arrest in NHMM. SP1 chromatin immunoprecipitation targets are enriched by cell cycle arrest genes. These results motivate a cooperation model of ESR1 and SP1 in regulating cell cycle arrest, and a hypothesis that their overactivation in NHMM disrupts proper regulation of cell cycle arrest. Cotargeting ESR1 and SP1 shows a synergistic effect on inhibiting myeloma proliferation in NHMM cell lines. Therefore, studying TrF-pathway coexpression dysregulation in human cancers facilitates forming novel hypotheses toward clinical utility.

Telomeres are specialized structures consisting of repeat arrays of TTAGGGn located at the ends of chromosomes. They are essential for chromosome stability and, in the majority of normal somatic cells, telomeres shorten with each cell division. Most immortalized cell lines and tumours reactivate telomerase to stabilize the shortening chromosomes. Telomerase activation is regarded as a central step in carcinogenesis and, here, we demonstrate telomerase activation in premalignant skin lesions and also in all forms of skin cancer. Telomerase activation in normal skin was a rare event, and among 16 samples of normal skin (one with a history of chronic sun exposure) 12.5% (2 out of 16) exhibited telomerase activity. One out of 16 (6.25%) benign proliferative lesions, including viral and seborrhoeic wart samples, had telomerase activity. In premalignant actinic keratoses and Bowen's disease, 42% (11 out of 26) of samples exhibited telomerase activity. In the basal cell carcinoma and cutaneous malignant melanoma (CMM) lesions, telomerase was activated in 77% (10 out of 13) and 69% (22 out of 32) respectively. However, only 25% (3 out of 12) of squamous cell carcinomas (SCC) had telomerase activity. With the exception of one SCC sample, telomerase activity in a positive control cell line derived from a fibrosarcoma (HT1080) was not inhibited when mixed with the telomerase-negative SCC or CMM extracts, indicating that, overall, Taq polymerase and telomerase inhibitors were not responsible for the negative results. Mean telomere hybridizing restriction fragment (TRF) analysis was performed in a number of telomerase-positive and -negative samples and, although a broad range of TRF sizes ranging from 3.6 to 17 kb was observed, a relationship between telomerase status and TRF size was not found.

GLUT4 (glucose transporter 4) plays important roles in glucose homoeostasis in vivo. GLUT4 expression and function are diminished in diabetic human and animal subjects. The goal of the present study is to develop a cell-based assay for identifying negative regulators of GLUT4 translocation as potential targets for the treatment of Type 2 diabetes. Traditional GLUT4 translocation assays performed in differentiated myocytes or adipocytes are difficult to perform, particularly in HTS (high-throughput screening) mode. In the present study, we stably co-expressed c-Myc and eGFP [enhanced GFP (green fluorescent protein)] dual-tagged recombinant GLUT4 with recombinant IRS1 (insulin receptor substrate 1) in HEK-293 cells (human embryonic kidney cells) (HEK-293.IRS1.GLUT4 cells). Insulin treatment stimulated both glucose uptake and GLUT4 translocation in these cells. GLUT4 translocation is quantified by a TRF (time-resolved fluorescence) assay in a 96-well HTS format. TRF assays confirmed insulin-stimulated GLUT4 translocation, which can be inhibited by PI3K (phosphoinositide 3-kinase) or Akt [also called PKB (protein kinase B)] inhibitors. Treatment with palmitate increased IRS1 serine phosphorylation and reduced insulin-stimulated Akt phosphorylation and GLUT4 translocation, indicating insulin resistance. Knockdown of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and PTP1B (protein tyrosine phosphatase 1B) gene expression by siRNA (small interfering RNA) treatment significantly increased GLUT4 translocation only in cells treated with palmitate but not in untreated cells. Similar results were obtained on treatment with siRNA of JNK1 (c-Jun N-terminal kinase 1), S6K1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) and PKC(theta) (protein kinase C theta). In summary, we have established and validated a novel GLUT4 translocation assay that is optimal for identifying negative regulators of GLUT4 translocation. In combination with more physiologically relevant secondary assays in myotubes and adipocytes, this assay system can be used to identify potential novel therapeutic targets for the treatment of Type 2 diabetes.

Neoplastic cells frequently have an increased number of transferrin receptors. Coupling transferrin to an anti-neoplastic drug has the potential to overcome multidrug resistance (MDR). The purpose of this study was to examine the distribution and action of doxorubicin-transferrin conjugate (DOXTRF) in a leukaemia cell line (HL60), a multidrug-resistant leukaemia cell line (HL60ADR) and a normal tissue cell line (human fibroblasts). The intracellular accumulation of DOX and DOX-TRF was monitored by direct fluorescence. More DOX-TRF than free DOX was delivered to the tumour cells, and consecutively the levels of DNA double-strand breaks and apoptosis increased even in the multidrug-resistant cell line. In the normal tissue cell line, DOX-TRF did not accumulate, and therefore, the levels of DNA double-strand breaks and apoptosis did not increase. Cell viability was determined using the MTT assay. The IC(50) for DOX-TRF was lower than the IC(50) value for the free drug in both leukaemia cell lines. The IC(50) values for the HL60 cells were 0.08 microM for DOX and 0.02 microM for DOX-TRF. The IC(50) values for HL60ADR cells were 7 microM for DOX and 0.035 microM for DOX-TRF. In conclusion, DOX-TRF was able to overcome MDR in the leukaemia cell lines while having only a very limited effect on normal tissue cells.

The anti-tumour and anti-cholesterol impacts of tocotrienol-rich fraction (TRF) were investigated in rats treated with the chemical carcinogen 7,12-dimethylbenz [alpha]anthracene (DMBA), which is known to induce mammary carcinogenesis and hypercholesterolaemia. DMBA administration to rats was associated with the appearance of multiple tumours on mammary glands after 6 months. Alkaline phosphatase (ALP) and glutathione-S-transferase (GST) are used as marker enzymes to monitor the severity of carcinogenesis. Although no tumours were visible on livers, hepatic ALP and GST activities of DMBA-treated rats were profoundly elevated in comparison to enzyme activities of normal control rats. Feeding of TRF (10 mg/kg body weight/day) for 6 months, isolated from rice bran oil (RBO), to DMBA-administered rats, reduced the severity and extent of neoplastic transformation in the mammary glands. Similarly, plasma and mammary ALP activities increased during carcinogenesis (95% and 43%, respectively), were significantly decreased in TRF-treated rats, whereas TRF mediated a further increase of 51% in hepatic ALP activity. TRF treatment to rats maintained low levels of GST activities in liver ( approximately 32%) and mammary glands ( approximately 21%), which is consistent with anti-carcinogenic properties of TRF. Administration of DMBA also caused a significant increase of 30% in plasma total cholesterol and 111% in LDL-cholesterol levels compared with normal control levels. Feeding of TRF to rats caused a significant decline of 30% in total cholesterol and 67% in LDL-cholesterol levels compared with the DMBA-administered rats. The experimental hypercholesterolaemia caused a significant increase in enzymatic activity (23%) and protein mass (28%) of hepatic 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase. Consistent with TRF-mediated reduction in plasma lipid levels, enzymatic activity and protein mass of HMG-CoA reductase was significantly reduced. These results indicate that TRF has potent anti-cancer and anti-cholesterol effects in rats.