Obesity and overfeeding affecting both tumor and systemic metabolism activates the progesterone receptor to contribute to postmenopausal breast cancer.

Obese postmenopausal women have increased risk of breast cancers with poorer clinical outcomes than their lean counterparts. However, the mechanisms underlying these associations are poorly understood. Rodent model studies have recently identified a period of vulnerability for mammary cancer promotion, which emerges during weight gain after the loss of ovarian function (surgical ovariectomy; OVX). Thus, a period of transient weight gain may provide a life cycle-specific opportunity to prevent or treat postmenopausal breast cancer. We hypothesized that a combination of impaired metabolic regulation in obese animals prior to OVX plus an OVX-induced positive energy imbalance might cooperate to drive tumor growth and progression. To determine if lean and obese rodents differ in their metabolic response to OVX-induced weight gain, and whether this difference affects later mammary tumor metabolism, we performed a nutrient tracer study during the menopausal window of vulnerability. Lean animals preferentially deposited excess nutrients to mammary and peripheral tissues rather than to the adjacent tumors. Conversely, obese animals deposited excess nutrients into the tumors themselves. Notably, tumors from obese animals also displayed increased expression of the progesterone receptor (PR). Elevated PR expression positively correlated with tumor expression of glycolytic and lipogenic enzymes, glucose uptake, and proliferation markers. Treatment with the antidiabetic drug metformin during ovariectomy-induced weight gain caused tumor regression and downregulation of PR expression in tumors. Clinically, expression array analysis of breast tumors from postmenopausal women revealed that PR expression correlated with a similar pattern of metabolic upregulation, supporting the notion that PR+ tumors have enhanced metabolic capacity after menopause. Our findings have potential explanative power in understanding why obese, postmenopausal women display an increased risk of breast cancer.

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples.

BACKGROUND: Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples. METHODS: Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10-16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT-PCR. RESULTS: In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC. CONCLUSION: FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.

Determination of ferrous and ferric iron in aqueous biological solutions.

A solvent extraction method was employed to determine ferrous and ferric iron in aqueous samples. Fe(3+) is selectively extracted into the organic phase (n-heptane) using HDEHP (bis(2-ethylhexyl) hydrogen phosphate) and is then stripped using a strong acid. After separation, both oxidation states and the total iron content were determined directly by ICP-MS analysis. This extraction method was refined to allow determination of both iron oxidation states in the presence of strong complexing ligands, such as citrate, NTA and EDTA. The accuracy of the method was verified by crosschecking using a refinement of the ferrozine assay. Presented results demonstrate the ability of the extraction method to work in a microbiological system in the presence of strong chelating agents following the bioreduction of Fe(3+) by the Shewanella alga BrY. Based on the results we report, a robust approach was defined to separately analyze Fe(3+) and Fe(2+) under a wide range of potential scenarios in subsurface environments where radionuclide/metal contamination may coexist with strongly complexing organic contaminants.

Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (MHV-68) is a relatively recently discovered pathogen of wild rodents and provides a unique opportunity to explore in detail the interactions of a gammaherpesvirus with its natural host. It may also provide a much needed small animal model for human gammaherpesviruses. As a step in the detailed analysis of virus gene structure and expression we have sequenced over 20 kb of the MHV-68 genome and mapped gene transcripts by Northern blot hybridization. The region we chose to analyse contains several conserved gene blocks as well as some less well conserved genes and allowed us to estimate the relationship of this virus to other herpesvirus family members. Of particular interest is the fact that none of the characteristic Epstein-Barr virus (EBV) genes is present at this genomic locus although MHV-68 does have one gene encoding a membrane glycoprotein, 9p150, which shows similarities to the major membrane glycoprotein of EBV. Our results further confirm that MHV-68 is a gammaherpesvirus marginally more closely related to a cluster of gammaherpesviruses including herpesvirus salmiri than to EBV. Northern analysis shows that the temporal regulation of expression is broadly similar to that of other herpesviruses in this region of the genome. We also show that like other gammaherpesviruses, MHV-68 splices its homologue of the EBV transcriptional activator gene BMRF1.

Differences in serological IgA responses to recombinant baculovirus-derived human papillomavirus E2 protein in the natural history of cervical neoplasia.

Infection with certain types of human papillomavirus (HPV) presents a high risk for the subsequent development of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. Immunological mechanisms are likely to play a role in control of cervical HPV lesions. The HPV E2 protein has roles in virus replication and transcription, and loss of E2 functions may be associated with progression of cervical neoplasia. Accordingly, it is of interest to monitor immune responses to the E2 protein, and previous studies have reported associations between serological reactivity to E2 peptide antigens and cervical neoplasia. In order to investigate serological responses to native, full-length E2 protein, we expressed HPV-16 E2 proteins with and without an N-terminal polyhistidine tag using the baculovirus system. Purified HPV-16 E2 protein was used to develop enzyme-linked immunosorbent assays to detect serological IgG and IgA responses in cervical neoplasia patients and controls. We found that serum IgA levels against the E2 protein were elevated in CIN patients relative to normal control subjects but were not elevated in cervical cancer patients. Moreover, there appeared to be a gradient of response within cervical neoplasia such that the highest antibody levels were seen in lower grades of neoplasia up to CIN 2, whereas lower levels were observed in CIN 3 and still lower levels in cervical carcinoma. These findings suggest that the IgA antibody response to E2 may associate with stage and progression in cervical neoplasia.

Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV.

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a variety of life-threatening diseases in humans. Therefore the development of an effective vaccine is an important objective. Many of the initial studies of vaccine efficacy analyse the ability of vaccine preparations to prevent the induction of lymphomas in cottontop tamarins by the B95-8 strain of EBV. We used a vaccinia virus recombinant expressing gp340, vMA1, tested previously in the cotton-top tamarin, to evaluate a common marmoset model in which the challenge virus, M81, resembles more closely the wild-type strains of EBV in the general population than does the standard B95-8 strain. We characterised the M81 strain of EBV with respect to the sequence of its gp340/220 gene and in regard to the presence of a region deleted in B95-8. Replication of the challenge virus in the group vaccinated with vMA1 was decreased when compared to control groups.

Effects of friendship and gender on peer group entry.

Effects of hosts' conflicting motives (to win a game vs. to be a good friend) on peer group entry processes and outcomes were examined. Subjects were 68 triads (35 female) of 10-12-year-old predominantly White children. Two host friends played a game for a large prize that was forfeited for a smaller prize if the guest (a friend or nonfriend of both hosts) was included. Hosts admitted guest friends more often than nonfriends (44% vs. 26%), suggesting that friendship norms prescribe self-sacrifice. Hosts behaved similarly with guest friends and nonfriends, but guest friends were more active than nonfriends, reflecting freedom derived from friendship security. Female hosts admitted guests more often than male hosts (51% vs. 21%), consistent with communal and agentic gender role prescriptions for girls and boys, respectively. Results suggest that hosts' friendship obligations and psychological orientation affect their response to a newcomer in a group entry situation.

Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein.

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.

Murine gammaherpesvirus-68 encodes homologues of thymidine kinase and glycoprotein H: sequence, expression, and characterization of pyrimidine kinase activity.

We have sequenced a 4.5-kb fragment of DNA spanning the junction of the BamHI D and E fragments of murine gammaherpesvirus-68 (MHV-68). This sequence was found to code for two major open reading frames (orfs) of 1934 and 2192 bp which showed significant homology to the thymidine kinase (TK) and glycoprotein H (gH) sequences of other gammaherpesviruses. Upstream from the TK gene another orf was found which showed amino acid sequence homology to the HSV1 UL24 gene. Analysis of the 1934-bp orf revealed the presence of all six of the recognized sites that are conserved between herpesvirus TKs although, uniquely among sequenced herpesvirus TK enzymes, MHV-68 lacks the consensus nucleotide binding site GXXGXGK, the second glycine being replaced by alanine. The MHV-68 TK has a predicted M(r) of 68,443, while the gH is predicted to have a M(r) of 82,890. Northern blot analysis showed an early TK message of 2.6 kb and a late gH-specific message of 2.5 kb. Both TK and gH probes detected a 4.3-kb late message, implying that this late message spans gH and TK. The TK coding sequence was expressed using an in vitro transcription translation system and was shown to encode functional TK activity.

The level and tempo of children's physical activities: an observational study.

We develop an observation system that quantifies the duration, intensity, and frequency of children's physical activities. We use this system to assess the level and tempo of energy expenditure under free-ranging, natural conditions experienced by 15 children aged 6-10 yr in southern California. Observations were recorded every 3 s during 4-h time blocks from 8:00 a.m.-8:00 p.m. Agreement among observers using the coding system was 91%. Using indirect calorimetry, calibration studies in the laboratory determined VO2 (ml.min-1.min-1) during each coded activity, and activities were categorized by intensity (low, medium, or high). Subjects were found to engage in activities of low intensity 77.1% of time and activities of high intensity 3.1% of time. The median duration of low and medium intensity activities was 6 s, of high intensity activities only 3 s with 95% lasting less than 15 s. Children engaged in very short bursts of intense physical activity interspersed with varying intervals of low and moderate intensity. These findings may be important for discovering how children's activity patterns under natural conditions influence physiological processes leading to growth and development. This study demonstrates the advantages of using an observational system that captures more than the intensity and frequency of children's activities to include duration and the length of intervals between activities of varying intensity.

Antigenic and sequence variation in the C-terminal unique domain of the Epstein-Barr virus nuclear antigen EBNA-1.

The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 is essential for viral genome maintenance in vitro and may be the only EBV protein expressed by the majority of latently infected cells in vivo. EBNA-1 may therefore be critical to the evasion of host immunity which allows persistent infection. EBNA-1 includes a polymorphic internal repeat domain of unknown significance and unique regions which mediate all known functional activities and which have hitherto been assumed to be conserved between strains. Monoclonal antibodies were generated using a construct based on EBNA-1 of the prototype B95-8 strain, deleted for the repeat domain. These antibodies showed a limited profile of recognition of EBNA-1 in common laboratory EBV+ cell lines by immunoprecipitation and immunostaining. The observed antigenic heterogeneity also extended to spontaneously transformed B lymphoblastoid cell lines (LCLs) representing viral isolates circulating within US and UK populations. DNA fragments spanning the C-terminal unique domain of EBNA-1 from eleven spontaneous LCLs were amplified by polymerase chain reaction for sequencing, which directly demonstrated extensive and unexpected variability between diverse type 1 EBV isolates. The resulting polymorphism affects most of the putative MHC Class I binding epitopes which could be identified within this region using published sequence motifs, and influences MHC binding by variants of at least one such peptide in the processing mutant cell line T2. These findings could be related to the apparent lack of recognition of EBNA-1 by cytotoxic T lymphocytes.

The Epstein-Barr virus candidate vaccine antigen gp340/220 is highly conserved between virus types A and B.

Anti-Epstein-Barr Virus (EBV) vaccines are being developed which are based on the gp340/220 membrane antigen (MA) gene products from the B95-8 strain. Some proteins are known to be immunologically quite different between type-A (1) and type-B (2) strains of EBV and therefore from a vaccine point of view it was critical to evaluate the degree of conservation of gp340/220. The complete MA coding sequence was determined for two B-type viruses, AG876 and P3HR-1, for comparison with the A-type B95-8. A variable region within MA was sequenced from several other strains. In addition the other open reading frames within the MA-containing BamHI-L fragment of AG876 were sequenced and compared. The results show that there is a high degree of homology between all strains examined. Although some differences were found within the MA coding sequence the only major site of variation was within the repeat region and no consistent A/B changes were found. Monoclonal antibodies generated against A-type MA and representing six epitope groups along the length of the gp340 molecule were found to recognize B-type gp340, thereby demonstrating functional homology. We conclude that, as a vaccine antigen, B95-8 gp340/220 should be equally effective against both types of EBV.

Effect of dose on the ability of caffeine to serve as a reinforcer in humans.

This study tested the effects of dose on the reinforcing effects of caffeine in humans. Eight moderate coffee drinkers were given concurrent access to decaffeinated coffee vs. decaffeinated coffee to which different doses of caffeine (25, 50, 150 and 200mg/cup) were added. Subjects were tested across several independent double-blind trials. The coffees with 25mg of caffeine were repeatedly self-administered at a rate greater than that of decaffeinated coffee in two of six subjects, the 50mg dose in four of eight subjects, the 150mg dose in three of six subjects, and the 200mg dose in none of the three subjects tested. Headaches, drowsiness and fatigue occurred with use of decaffeinated coffee in five subjects. When these symptoms occurred, there was a greater probability of self-administration of the caffeinated coffee. We conclude that doses of caffeine similar to those in tea or soda can serve as reinforcers.

Prevalence of the A and B types of Epstein-Barr virus DNA in nasopharyngeal carcinoma biopsies from southern China.

Epstein-Barr virus (EBV) exists in the human population in two genetic forms, usually referred to as type A and type B. Although many earlier studies had indicated that the A type was generally predominant, there were suggestions that the B type may exhibit a preferential tropism for nasopharyngeal epithelial cells. This study examines the prevalence of the two forms of EBV DNA present in nasopharyngeal carcinoma biopsies obtained from the high incidence area of Southern China. The results obtained by Southern blot or polymerase chain reaction analyses show that in this patient group the A type of EBV is predominant.

Smoking cessation among self-quitters.

We examined cessation among 630 smokers who quit abruptly on their own. Continuous, complete abstinence rates were 33% at 2 days, 24% at 7 days, 22% at 14 days, 19% at 1 month, 11% at 3 months, 8% at 6 months postcessation, and 3% at 6 months with biochemical verification. Slipping (smoking an average of less than 1 cigarette/day) was common (9% to 15% of subjects) and was a strong predictor of relapse; however, 23% of long-term abstainers slipped at some point. These results challenge beliefs that most smokers can initially stop smoking and that most relapse occurs later on postcessation.

Forced-choice versus free-choice procedures: caffeine self-administration in humans.

Methodological comparisons of procedures for drug self-administration are rare. In studies examining the reinforcing effect of caffeine in humans, caffeine self-administration usually has been inferred from performance under forced-choice procedures. In the present experiment, caffeine self-administration via coffee was compared under forced-choice and free-choice conditions; i.e., when subjects were and were not required to use a minimum number of coffees. Ten moderate coffee drinkers (2-7 cups/day) were assigned to forced- and free-choice conditions using a randomized cross-over design. Under each choice condition, subjects completed six independent, double-blind trials, consisting of a 2-day exposure period followed by a 2-day test period. During exposure, subjects consumed either decaffeinated or caffeinated (100 mg/serving) coffee on day 1 and the other coffee on day 2. During the test period, subjects had concurrent access to the same decaffeinated and caffeinated coffees. Under the forced-choice condition, subjects were required to drink at least four cups of coffee per day during the test period. Under the free-choice condition, subjects did not have a minimum-cup requirement. In general, the relative rate at which subjects self-administered caffeinated versus decaffeinated coffee was similar across choice conditions, even though subjects self-administered significantly fewer cups of both coffee types under the free-choice than the forced-choice condition. These results suggest that, at least for caffeine, forced-choice and free-choice procedures produce comparable results. Whether this finding generalizes to a context in which caffeine or another drug is more robustly self-administered, remains to be determined.

Effects of caffeine on tobacco withdrawal.

Smoking cessation increases caffeine blood levels, and this has been hypothesized to cause some of the symptoms of tobacco withdrawal (e.g., anxiety and insomnia). To test this hypothesis, 10 coffee drinkers who smoked cigarettes were entered into a completely within-subjects experimental design in which the effects of caffeine dose (0, 50, and 100 mg/coffee serving) and smoking status (smoking versus abstinence) were examined over a 4-day period. Self-reported and observed measures of tobacco withdrawal, caffeine withdrawal, and intoxication, as well as psychomotor tasks and vital signs, were completed daily; blood was drawn at the end of each period. Temporary abstinence produced typical withdrawal symptoms but did not significantly increase caffeine blood levels. Caffeine did not increase the severity of symptoms but did decrease the severity of withdrawal-induced hunger. These findings suggest that, in the absence of increased blood levels, caffeine does not increase the severity of tobacco withdrawal.

Oxygen saturation of brachial venous blood correlates with fingertip temperatures between 11 and 39 degrees C.

The relationships between fingertip skin temperature (Tfin), brachial venous hemoglobin oxygen saturation (SVO2), and plasma catecholamines were studied by exposing 15 semi-nude men to 30 min of 5 degrees C and 48 degrees C ambient air temperature extremes. After 30 min of cold air, norepinephrine (NE) increases from 2.01 +/- 0.26 to 7.15 +/- 0.64 nmol/L, and epinephrine (Epi) increases from 201 +/- 25 to 295 +/- 34 pmol/L. In contrast, after 30 min of hot air exposure, Epi rises from 161 +/- 32 to 348 +/- 43 pmol/L, while NE rises slightly from 2.07 +/- 0.26 to 2.57 +/- 0.64 nmol/L. Heat elevates SVO2 from 68.8 +/- 0.72 to 93.3 +/- 0.66% and Tfin from 29.5 +/- 1.09 to 39.4 +/- 0.56 degrees C. Cold lowers SVO2 to 32.1 +/- 0.70% and Tfin to 11.5 +/- 0.55 degrees C. SVO2 correlates with Tfin (r = 0.895, p less than 0.0001). We conclude that exposure to an environmental temperature of 5 degrees C decreases Tfin with concurrent decreases in blood flow and SVO2 in the extremity by the vasoconstrictive action of NE. Exposure to 48 degrees C increases Tfin with increases in blood flow and SVO2 in the extremity by vasodilation of peripheral vascular beds, which may be related to the rise in the plasma concentration of Epi.