Upregulation of α-synuclein during localized radiation therapy signals the association of cancer-related fatigue with the activation of inflammatory and neuroprotective pathways.

PURPOSE: Neuroinflammatory mechanisms are associated with fatigue in neurodegenerative conditions such as Parkinson's. The symptoms in Parkinson's including fatigue are thought to be related to α-synuclein overexpression. This study investigated genomic correlates of fatigue experienced by men with prostate cancer receiving external beam radiation therapy (EBRT). PATIENTS AND METHODS: Sixteen men with non-metastatic prostate cancer who were scheduled to receive EBRT were enrolled. Fatigue scores and blood were obtained at baseline (prior to EBRT, D0); one hour following initiation of EBRT (D1), day 7 (D7), day 14 (D14), midpoint (days 19-21, D21), completion (days 38-42, D42), and four weeks post-EBRT (days 68-72, D72). Gene expression profiling using microarray analysis was performed from peripheral blood and confirmatory qPCR and protein (ELISA) analyses verified the microarray results. Correlations between fatigue and gene/protein expressions were determined using a mixed model approach. RESULTS: Microarray data showed significant, differential expression of 463 probesets following EBRT. SNCA had a 2.95-fold change at D21 from baseline. SNCA expression was confirmed by qPCR (p<0.001) and ELISA (p<0.001) over time during EBRT. Fatigue scores were significantly correlated with SNCA gene expression on D14 (r=0.55, p<0.05) and plasma α-synuclein concentrations on D42 of EBRT (r=0.54, p=0.04). CONCLUSION: Fatigue experienced during EBRT may be mediated by α-synuclein overexpression. Alpha-synuclein may serve as a useful biomarker to understand the mechanisms and pathways related to the development of fatigue in this population.

Cooperative and redundant signaling of leukotriene B4 and leukotriene D4 in human monocytes.

BACKGROUND: Leukotriene B(4) (LTB(4)) and cysteinyl leukotrienes (cysLTs) are important immune mediators, often found concomitantly at sites of inflammation. Although some of the leukotriene-mediated actions are distinctive (e.g., bronchial constriction for cysLTs), many activities such as leukocyte recruitment to tissues and amplification of inflammatory responses are shared by both classes of leukotrienes. OBJECTIVE: We used human monocytes to characterize leukotriene-specific signaling, gene expression signatures, and functions and to identify interactions between LTB(4)- and cysLTs-induced pathways. METHODS: Responsiveness to leukotrienes was assessed using oligonucleotide microarrays, real-time PCR, calcium mobilization, kinase activation, and chemotaxis assays. RESULTS: Human monocytes were found to express mRNA for high- and low-affinity LTB(4) receptors, BLT(1) and BLT(2), but signal predominantly through BLT(1) in response to LTB(4) stimulation as shown using selective agonists, inhibitors, and gene knock down experiments. LTB(4) acting through BLT(1) coupled to G-protein α inhibitory subunit activated calcium signaling, p44/42 mitogen-activated protein kinase, gene expression, and chemotaxis. Twenty-seven genes, including immediate early genes (IEG), transcription factors, cytokines, and membrane receptors were significantly up-regulated by LTB(4). LTB(4) and LTD(4) had similar effects on signaling, gene expression, and chemotaxis indicating redundant cell activation pathways but costimulation with both lipid mediators was additive for many monocyte functions. CONCLUSION: Leukotriene B(4) and LTD(4) display both redundant and cooperative effects on intracellular signaling, gene expression, and chemotaxis in human monocytes. These findings suggest that therapies targeting either leukotriene alone may be less effective than approaches directed at both.

Distinct gene expression profiles in norepinephrine- and epinephrine-producing hereditary and sporadic pheochromocytomas: activation of hypoxia-driven angiogenic pathways in von Hippel-Lindau syndrome.

Pheochromocytomas in von Hippel-Lindau (VHL) syndrome produce exclusively norepinephrine, whereas those in multiple endocrine neoplasia type 2 (MEN 2) produce epinephrine. This study examined the pathways activated in VHL-associated pheochromocytomas by comparing gene expression profiles in VHL and MEN 2 tumors in relationship to profiles in sporadic norepinephrine- and epinephrine-producing tumors. Larger and more distinct differences in gene expression among hereditary than sporadic tumors indicated the importance of the underlying mutation to gene expression profiles. Many of the genes over-expressed in VHL compared with MEN 2 tumors were clearly linked to the hypoxia-driven angiogenic pathways that are activated in VHL-associated tumorigenesis. Such genes included those for the glucose transporter, vascular endothelial growth factor (VEGF), placental growth factor, angiopoietin 2, tie-1, VEGF receptor 2 and its coreceptor, neuropilin-1. Other up-regulated genes, such as connective tissue growth factor, cysteine-rich 61, matrix metalloproteinase 1, vascular endothelial cadherin, tenascin C, stanniocalcin 1, and cyclooxygenases 1 and 2 are known to be involved in VEGF-regulated angiogenesis. Shared differences in expression of subsets of genes in norepinephrine- versus epinephrine-producing hereditary and sporadic pheochromocytomas indicated other differences in gene expression that may underlie the biochemical phenotype. Over-expression of the hypoxia-inducible transcription factor, HIF-2alpha, in norepinephrine-predominant sporadic and VHL tumors compared with epinephrine-producing tumors indicates that expression of this gene depends on the noradrenergic biochemical phenotype. The findings fit with the known expression of HIF-2alpha in norepinephrine-producing cells of the sympathetic nervous system and might explain both the development and noradrenergic biochemical phenotype of pheochromocytomas in VHL syndrome.

Proteomic profiling of the cancer microenvironment by antibody arrays.

Critical changes in protein expression that enable tumors to initiate and progress originate in the local tissue microenvironment, and there are increasing indications that these microenvironmental alterations in protein expression play critical roles in shaping and directing this process. As a model to better understand how patterns of protein expression shape the tissue microenvironment, we analyzed protein expression in tissue derived from squamous cell carcinoma of the oral cavity through an antibody microarray approach for high-throughput proteomic analysis. Utilizing laser capture microdissection to procure total protein from specific microscopic cellular populations, we demonstrate that quantitative, and potentially qualitative, differences in expression patterns of multiple proteins within epithelial cells reproducibly correlate with oral cavity tumor progression. Furthermore, differential expression of multiple proteins was also found in stromal cells surrounding and adjacent to regions of diseased epithelium that directly correlated with tumor progression of the epithelium. Most of the proteins identified in both cell types are involved in signal transduction pathways, thus we hypothesize that extensive molecular communication involving complex cellular signaling between epithelium and stroma play a key role in driving oral cavity cancer progression.

Augmentation in expression of activation-induced genes differentiates memory from naive CD4+ T cells and is a molecular mechanism for enhanced cellular response of memory CD4+ T cells.

In an attempt to understand the molecular basis for the immunological memory response, we have used cDNA microarrays to measure gene expression of human memory and naive CD4+ T cells at rest and after activation. Our analysis of 54,768 cDNA clones provides the first glimpse into gene expression patterns of memory and naive CD4+ T cells at the genome-scale and reveals several novel findings. First, memory and naive CD4+ T cells expressed similar numbers of genes at rest and after activation. Second, we have identified 14 cDNA clones that expressed higher levels of transcripts in memory cells than in naive cells. Third, we have identified 135 (130 known genes and 5 expressed sequence tags) up-regulated and 68 (42 known genes and 26 expressed sequence tags) down-regulated cDNA clones in memory CD4+ T after in vitro stimulation with anti-CD3 plus anti-CD28. Interestingly, the increase in mRNA levels of up-regulated genes was greater in memory than in naive CD4+ T cells after in vitro stimulation and was higher with anti-CD3 plus anti-CD28 than with anti-CD3 alone in both memory and naive CD4+ T cells. Finally, the changes in expression of actin and cytokine genes identified by cDNA microarrays were confirmed by Northern and protein analyses. Together, we have identified approximately 200 cDNA clones whose expression levels changed after activation and suggest that the level of expression of up-regulated genes is a molecular mechanism that differentiates the response of memory from naive CD4+ T cells.

Sequence databases and microarrays as tools for identifying prostate cancer biomarkers.

Identification, acquisition, and assessment of molecular markers that could be adopted as surrogate endpoints for evaluating a response to prostate cancer intervention strategies is highly desirable. Recent advances in the fields of genomics and biotechnology have dramatically increased the quantity and accessibility of molecular information that is relevant to the study of prostate carcinogenesis. One major advance involves the construction of comprehensive databases that archive gene sequences and gene expression data. This information is in a format suitable for virtual queries designed to distinguish the molecular differences between normal and cancer cells. A second major advance uses robotic tools to construct microarrays comprising thousands of distinct genes expressed in prostate tissues. Such arrays offer a powerful approach for monitoring the expression of thousands of genes simultaneously and provide access for techniques designed to assess patterns or "fingerprints" of gene expression that may ultimately be used as signatures of response to therapeutic intervention.

Development of a prostate cDNA microarray and statistical gene expression analysis package.

A cDNA microarray comprising 5184 different cDNAs spotted onto nylon membrane filters was developed for prostate gene expression studies. The clones used for arraying were identified by cluster analysis of > 35 000 prostate cDNA library-derived expressed sequence tags (ESTs) present in the dbEST database maintained by the National Center for Biotechnology Information. Total RNA from two cell lines, prostate line 8.4 and melanoma line UACC903, was used to make radiolabeled probe for filter hybridizations. The absolute intensity of each individual cDNA spot was determined by phosphorimager scanning and evaluated by a bioinformatics package developed specifically for analysis of cDNA microarray experimentation. Results indicated 89% of the genes showed intensity levels above background in prostate cells compared with only 28% in melanoma cells. Replicate probe preparations yielded results with correlation values ranging from r = 0.90 to 0.93 and coefficient of variation ranging from 16 to 28%. Findings indicate that among others, the keratin 5 and vimentin genes were differentially expressed between these two divergent cell lines. Follow-up northern blot analysis verified these two expression changes, thereby demonstrating the reliability of this system. We report the development of a cDNA microarray system that is sensitive and reliable, demonstrates a low degree of variability, and is capable of determining verifiable gene expression differences between two distinct human cell lines. This system will prove useful for differential gene expression analysis in prostate-derived cells and tissue.

The diagnosis of low-grade peripheral B-cell neoplasms in bone marrow trephines.

The aim of this study was to establish how effective is the use of immunohistochemistry on formalin-fixed bone marrow in diagnosing low-grade B-cell neoplasms. We investigated a series of 41 consecutive patients with bone marrow involvement for whom no other diagnostic tissues were available. The sections were stained with the following antibodies: CD3, CD20, CD79a, CD5, CD10, CD23, anti-cyclin D1 and kappa and lambda light chains. Antigen retrieval was performed using either a microwave oven or a pressure cooker. Labelling was performed with an avidin-biotin-peroxidase labelling system. A final diagnosis was reached in 37 out of 41 cases (90%): B-chronic lymphocytic leukaemia (15 cases), follicular lymphoma (10 cases), mantle-cell lymphoma (eight cases) and lymphoplasmacytoid lymphoma/immunocytoma (four cases). In the remaining four cases, a generic diagnosis of low-grade B-cell neoplasm was made. The immunophenotyping of formalin-fixed marrow is a useful technique for diagnosing most of the low-grade B-cell neoplasms.

Analysis of gene expression in mutiple sclerosis lesions using cDNA microarrays.

In multiple sclerosis (MS) patients, a coordinated attack of the immune system against the primary constituents of oligodendrocytes and/or the myelin sheath of oligodendrocytes results in the formation of lesions in the brain and spinal cord. Thus far, however, a limited number of genes that potentially contribute to lesion pathology have been identified. Using cDNA microarray technology, we have performed experiments on MS tissue monitoring the expression pattern of over 5,000 genes and compared the gene expression profile of normal white matter with that found in acute lesions from the brain of a single MS patient. Sixty-two differentially expressed genes were identified, including the Duffy chemokine receptor, interferon regulatory factor-2, and tumor necrosis factor alpha receptor-2 among others. Thus, cDNA microarray technology represents a powerful new tool for the identification of genes not previously associated with the MS disease process.

FORESST: fold recognition from secondary structure predictions of proteins.

MOTIVATION: A method for recognizing the three-dimensional fold from the protein amino acid sequence based on a combination of hidden Markov models (HMMs) and secondary structure prediction was recently developed for proteins in the Mainly-Alpha structural class. Here, this methodology is extended to Mainly-Beta and Alpha-Beta class proteins. Compared to other fold recognition methods based on HMMs, this approach is novel in that only secondary structure information is used. Each HMM is trained from known secondary structure sequences of proteins having a similar fold. Secondary structure prediction is performed for the amino acid sequence of a query protein. The predicted fold of a query protein is the fold described by the model fitting the predicted sequence the best. RESULTS: After model cross-validation, the success rate on 44 test proteins covering the three structural classes was found to be 59%. On seven fold predictions performed prior to the publication of experimental structure, the success rate was 71%. In conclusion, this approach manages to capture important information about the fold of a protein embedded in the length and arrangement of the predicted helices, strands and coils along the polypeptide chain. When a more extensive library of HMMs representing the universe of known structural families is available (work in progress), the program will allow rapid screening of genomic databases and sequence annotation when fold similarity is not detectable from the amino acid sequence. AVAILABILITY: FORESST web server at http://absalpha.dcrt.nih.gov:8008/ for the library of HMMs of structural families used in this paper. FORESST web server at http://www.tigr.org/ for a more extensive library of HMMs (work in progress). CONTACT: valedf@tigr.org; munson@helix.nih.gov; garnier@helix.nih.gov

Protein topology recognition from secondary structure sequences: application of the hidden Markov models to the alpha class proteins.

The three-dimensional fold of a protein is described by the organization of its secondary structure elements in 3D space, i.e. its "topology". We find that the protein topology can be recognized from the ID sequence of secondary structure states of the residues alone. Automated recognition is facilitated by use of hidden Markov models (HMMs) to represent topology families of proteins. Such models can be trained on the experimentally observed secondary structure sequences of family members using well established algorithms. Here, we model various topology groups in the alpha class of proteins and identify, from a large database, those proteins having the topology described by each model. The correct topology family for protein secondary structure sequences could be recognized 12 out of 14 times. When the observed secondary structure sequences are replaced with predicted sequences recognition is still achievable 8 out of 14 times. The success rate for observed sequences indicates that our approach will become increasingly useful as the accuracy of secondary prediction algorithms is improved. Our study indicates that the HMMs are useful for protein topology recognition even when no detectable primary amino acid sequence similarity is present. To illustrate the potential utility of our method, protein topology recognition is attempted on leptin, the obese gene product, and the human interleukin-6 sequence, for which fold predictions have been previously published.

Multi-body interactions within the graph of protein structure.

We construct a graphical representation of protein structure based on the 3D C-alpha carbon point set, using the Delaunay tessellation to define interacting quadruples of amino acid residues. The tessellation is filtered by two criteria: interaction distance less than 9.5 angstroms and circumsphere radius less than 8.0 angstroms using dataset of 608 protein structures of low mutual sequence identity and a likelihood ratio test, we show that 3-body and 4-body interactions are indeed significant. We identify particular significant three-body interactions by first reducing the dataset to interacting triples, and classifying amino acid residues in a reduced alphabet. Although cystein was previously shown to be a dominant source of 3-body interactions, we now identify additional significant 3-body interactions of charged, hydrophobic and small residues.

Simplified representation of proteins.

The conventional methods for characterizing the secondary structures of proteins based on hydrogen bonding patterns and phi,rho torsions are not fully specific in determining the spatial arrangements of various secondary structural elements. This fact motivates a search for an efficient description of the various secondary structures and their interactions. The successive identical repeating units of a polypeptide chain, namely the atoms of the peptide plane may be superposed using a method based on the mathematical quaternion. The superposition angle then characterizes different secondary structures. The distortions in protein alpha-helices such as kinks and bends are also precisely determined. The twist in beta-sheets is quantified and reverse turns are found to have characteristic variations. This new representation might pave the way for a better understanding of the final folding conformation of the polypeptide chain.

The maternal hypothalamic-pituitary-adrenal axis in the third trimester of human pregnancy.

OBJECTIVE: The third trimester of pregnancy is characterized by a mildly hyperactive hypothalamic-pituitary-adrenal (HPA) axis, possibly driven by elevated circulating levels of corticotrophin releasing hormone (CRH) of placental origin. In-vitro studies have demonstrated that glucocorticoids and oestrogen stimulate while progesterone inhibits the expression of CRH mRNA and/or protein, suggesting that several potential interactions between the placenta and the HPA axis may exist. DESIGN AND PATIENTS: To investigate the detailed pattern of circulating immunoreactive (ir) CRH, ACTH, cortisol, oestradiol and progesterone during the third trimester of pregnancy, plasma samples were drawn serially every 30 minutes from 22 healthy pregnant women (age 32.0 +/- 1.1 years, mean +/- SE) between the 34th and 36th week of gestation. Ten women had plasma samples drawn between 0800 h and 2000 h (daytime group), and 12 between 2000 h and 0800 h (night-time group). The hormone concentrations obtained were analysed for pulsatility by the Detect program, for detection of circadian rhythmicity by comparison between the first and second 6-hour periods within each group by Student's t-test, and for time-dependent correlations by cross-correlation analysis. RESULTS: All five hormones were secreted in a pulsatile fashion. There was no apparent circadian rhythm of CRH or oestradiol secretion, whereas there was a clear circadian rhythm in plasma ACTH, cortisol and progesterone secretion, with the latter in reverse phase (P < 0.05). No significant correlations were observed between CRH and ACTH, whereas, as expected, ACTH and cortisol concentrations were strongly correlated with each other over time (r = 0.32 and 0.70 at lag time 30 minutes for the daytime and night-time groups, respectively), with ACTH leading cortisol. A weak positive correlation was observed between CRH and cortisol concentrations for the night-time group at lag time 0 minute, suggesting that the latter may have a positive effect on the former in vivo. CONCLUSIONS: These data suggest that placental CRH, although pulsatile, drives quantitatively the maternal HPA axis in the third trimester of pregnancy in a non-circadian, non-pulsatile fashion. The maternal HPA axis is probably driven in a circadian and pulsatile fashion by another major ACTH secretagogue, most likely AVP of parvocellular paraventricular nucleus origin.

A model for the effect of estrogen antagonists on cooperative estradiol binding.

Partial agonists such as estriol and estrone have been reported to diminish or even eliminate the upward convexity of the Scatchard plot of the binding of labeled estradiol to estrogen receptor. This has been interpreted as agonist interference with the receptor dimerization induced by estradiol. In order to investigate how a partial agonist or antagonist might interfere with dimerization we have developed a theoretical mass-action law model, where soluble receptors can dimerize and bind to two different ligands. Special attention was devoted to manifestations of positive cooperativity to determine whether they could be modified by competition with a second ligand. This was done using a computer program that evaluated a large set of combinations of affinity constants in an effort to explore all possible situations. The model could reproduce the effect of a second ligand on the cooperative binding of estradiol to the estrogen receptor but only if the second ligand was anticooperative, which is not the case of estriol, estrone and tamoxifen. Furthermore, even when the Scatchard plot was linear, the model still required dimerization of the receptor in most of the cases, showing that the addition of an antagonist may eliminate the upward curvature of the Scatchard without truly eliminating dimerization or cooperativity. We conclude that the effect of a second ligand on the binding of labeled estradiol to estrogen receptor is not necessarily due to interference with dimerization and/or cooperativity. The inability of this model to fully explain the published data for estriol, estrone, clomiphene, and tamoxifen suggests that a more complex mechanism is involved.

Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand.

The mutual effects that a hormonal ligand (H) and a guanine nucleotide regulatory protein (G protein) exert on each other when simultaneously occupying distinct sites of the receptor molecule (R) can be viewed as the molecular mechanism of drug efficacy. These effects are predictable on the basis of a model assuming that the ternary complex between the three partners (HRG) reaches equilibrium in the membrane [J. Biol. Chem. 255:7108-7117 (1980)]. Ligands can be classified as agonists, neutral antagonists, or negative antagonists, depending on whether they enhance, leave unchanged, or reduce, respectively, the spontaneous tendency of R to interact with G. Using this model and the assumption that the G protein response observed in membranes reflects the sum of ligand-independent (RG) and ligand-dependent (HRG) receptor-G protein complexes, we can explain virtually all the phenomenology reported earlier for opioid receptor-mediated stimulation of GTPase, i.e., 1) existence of ligands with both "positive" and "negative" intrinsic activity (the latter termed negative antagonists), 2) equipotency of neutral antagonists for the competitive blockade of the responses elicited both by agonists and by negative antagonists, and 3) apparent heterogeneity of binding sites for the binding isotherms of negative antagonists. The ternary complex model can also explain the differential effects of sodium on ligand binding and ligand-dependent GTPase activity, if we assume that this ion reduces the stability constant between receptor and G protein in membranes. Computer simulations predict that a negative antagonist exhibits a discrepancy between "biological" Ki (obtained by Schild plots) and true dissociation constant for the receptor, which increases as the fraction of "precoupled" receptors in the membrane increases. The demonstration of negative antagonism is definitive evidence for the existence of receptor coupling (hence activity) in the absence of ligand. Using this experimental paradigm, we show here that spontaneous receptor activity occurs in isolated membranes but not in intact NG108-15 cells.

Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells.

When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells.

Vasopressin and oxytocin receptors in vagina, myometrium, and oviduct of rabbits.

In view of the presence of distinct oxytocin (OT) and vasopressin (VP) receptors in the male genital tract (porcine) we have reexamined the receptors for OT and AVP in the classical OT target tissue, female genital tract (rabbit). Neurohypophysial hormone receptors have been investigated in vagina, myometrium, and oviduct using quantitative ligand binding, adenylate cyclase, and contractility studies. Our results clearly indicate the presence of distinct OT and V1 VP receptors in the myometrium, while only the latter was detected in vagina and oviduct. In myometrium, estrogen treatment increases the density of OT and AVP receptors, while progesterone administration inhibits the estrogen effect. At the time of spontaneous delivery a dramatic (17-fold) increase was observed for the OT sites, while the AVP sites were unchanged. AVP receptors in vagina were sensitive to sex steroid administration and were reduced during pregnancy and delivery. Isometric contractility studies suggest that not just OT, but AVP can stimulate uterine strips, an effect that is partially reversible by the V1 antagonist d(CH2)5TyrMeAVP. In vagina only AVP is effective in inducing contractions at nanomolar concentrations. These results suggest a role for AVP as well as OT in regulation of the motility of female genital tract.