Myeloperoxidase and Prostate volume: A preliminary study.

OBJECTIVES: Oxidative stress is associated with the development of BPH and might be modulated by several factors. Myeloperoxidase (MPO) has recently been observed in prostate tissue. Our goal was to investigate the correlation between MPO and the prostate volume. MATERIAL AND METHODS: Hundred and twenty-one patients (48-70 years) with a filled IPSS were prospectively included. Blood sampling (PSA, testosterone, Angiotensin II (AngII), MPO, Mox-LDL) and transrectal ultrasound of the prostate were performed with total volume (TV) and transitional zone volume (TZ) measurements. For correlation, univariate analyses were depicted by Pearson's coefficient. Multilinear regression analysis used a stepwise backward selection of the explicative variables. RESULTS: In multivariate analysis, the TV was positively correlated to the combination of age and Ang II but negatively to MPO specific activity (Std Coef=-0.272, P=0.004). Significant correlations were confirmed between TZ, age and MPO specific activity but not with Ang II. A negative correlation between TZ and MPO specific activity was also observed (Std Coef=-0.21, P=0.016). No correlation was found with Mox-LDL. CONCLUSIONS: Negative correlation between MPO and prostate volume was observed but careful interpretations may be endorsed and longitudinal study is necessary. It seems relevant to focus on the potential contribution of MPO in the development of prostatic diseases as this enzyme can also promote DNA oxidation. LEVEL OF EVIDENCE: 4.

Intriguing location of myeloperoxidase in the prostate: a preliminary immunohistochemical study.

BACKGROUND: Myeloperoxidase (MPO) is a member of the peroxidase-cyclooxygenase superfamily, which is secreted from stimulated leucocytes at inflammatory sites. It is well known that MPO catalyses oxidation reactions via the release of reactive halogenating and nitrating species and thus induces tissue damage. Several studies have already implicated MPO in the development of neoplasia. Chronic or recurrent prostatic inflammation has long been recognized as having the potential to initiate and promote the development of prostate cancer. The objective was to investigate whether MPO is present in the prostate. METHODS: Human prostate material was obtained from biopsies, transurethral resections of the prostate (TURP), prostatic adenomectomies, and retropubic radical prostatectomies. Twenty-nine slides of normal prostate tissue, benign prostatic hyperplasia (BPH), and prostate cancer were reviewed by a pathologist. Immunohistochemical analysis using MPO-specific human antibody was performed to detect MPO in the prostate tissue. RESULTS: Immunocytohistochemistry showed cellular colocalization of MPO in the secretory epithelial cells of the prostate with staining varying from light to strong intensity. Staining in the glandular apical snouts was often reinforced although staining of basal as well as of luminal glandular cells was also present. CONCLUSIONS: We identified, for the first time, the presence of MPO at the surface of prostatic epithelial cells. In view of the pro-oxidant properties of this enzyme, further research is needed to define whether MPO contributes to the development of prostatic lesions.

The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.

Specific binding of proteins to the noncoding strand of a crucial element of the variant surface glycoprotein, procyclin, and ribosomal promoters of trypanosoma brucei.

The variant surface glycoprotein (VSG) and procyclin promoters of Trypanosoma brucei recruit an RNA polymerase sharing characteristic with polymerase I, but there is no sequence homology between them nor between these promoters and ribosomal promoters. We report the detailed characterization of the VSG promoter. The 70-bp region upstream of the transcription start site was sufficient for full promoter activity. Mutational analysis revealed three short critical stretches at positions -61 to -59 (box 1), -38 to -35 (box 2), and -1 to +1 (start site), the spacing of which was essential. These elements were conserved in the promoter for a metacyclic VSG gene. Hybrid sequences containing box 1 of the VSG promoter and box 2 of the ribosomal promoter were active. A specific binding of proteins to the noncoding strand of box 2, but not to double-stranded DNA, occurred. Competition experiments indicated that these proteins also bind to the corresponding region of the metacyclic VSG, procyclin, and ribosomal promoters. Binding of such a protein, of 40 kDa, appeared to be shared by these promoters.

Java-based framework for processing and displaying short-echo-time magnetic resonance spectroscopy signals.

Magnetic resonance spectroscopy (MRS) can be used to determine in a non-invasive way the concentrations of certain chemical substances, also called metabolites. The spectra of MRS signals contain peaks that correspond to the metabolites of interest. Short-echo-time signals are characterized by heavily overlapping metabolite peaks and require sophisticated processing methods. To be useful in a clinical environment tools are needed that can process those signals in an accurate and fast way. Therefore, we developed novel processing methods and we designed a freely available and open-source framework (http://www.esat.kuleuven.ac.be/sista/members/biomed) in which the processing methods can be integrated. The framework has a set of abstract classes, called hot spots, and its goal is to provide a general structure and determine the control flow of the program. It provides building blocks or components in order to help developers with integrating their methods in the framework via a plug-in system. The framework is designed with the unified modeling language (UML) and implemented in Java. When a developer implements the framework he gets an application that acts like a simple and user-friendly graphical user interface (GUI) for processing MRS data. This article describes in detail the structure and implementation of the framework and the integration of our processing methods in it.

A single-stranded DNA-binding protein shared by telomeric repeats, the variant surface glycoprotein transcription promoter and the procyclin transcription terminator of Trypanosoma brucei.

In Trypanosoma brucei the genes are organised into long polycistronic transcription units and only three promoters for protein-encoding genes and a single terminator have been characterised. These promoters recruit a polI-like RNA polymerase for the transcription units encoding the two major stage-specific antigens of the parasite, the variant surface glycoprotein (VSG) of the bloodstream form and procyclin of the insect-specific procyclic form, while the terminator is that of a procyclin transcription unit. By deletional and mutational analysis we defined the two DNA sequences essential for the activity of the VSG promoter from a bloodstream form transcription unit and one of the functional elements of the procyclin terminator. These three short sequences are similar, and their C-rich strand binds the same protein of 40 kDa. In addition, this factor also binds to the C-rich strand of the telomeric repeats, the consensus target sequence being 5'-CCCTNN-3'. The factor-binding sequences are functionally interchangeable in chimeric promoter or terminator constructs, although additional elements are required for full activity.

Characterization of sarcoma cell lines from v-jun transgenic mice.

Wounding is a prerequisite for tumor formation in v-jun transgenic mice. The progression from wound to dermal sarcoma is a multistep process which, at some stage, results in an increase in transgene mRNA expression in tumor tissue. However, transgene expression in individual sarcoma cells stained for Jun protein cultures is heterogeneous. We cloned several cell lines from wound-related v-jun transgenic tumors to determine whether a relationship existed between the cellular growth properties and structure, expression, or function of the transgene. Cell lines with very high v-jun expression had a high cloning efficiency in soft agar and tumorigenicity in nude mice. However, for cell lines with an intermediate or low level of transgene expression there was no correlation between transgene expression and the transformed phenotype. There was also no correlation between transgene expression and individual cell line morphologies, growth rates, transgene genomic DNA copy number, or mRNA expression of jun-related genes. The tumor cell subclones (1-20.2, 3-24.3) with very low transgene expression, very poor cloning efficiency, and low tumorigenicity also showed reduced activator protein 1 DNA binding activity and had an increased expression of endogenous c-jun when compared to other tumor cell lines. Transfection of a v-jun expression vector into cell lines with poor cloning efficiency and low tumorigenicity enhanced both in vitro cloning and in vivo tumor formation. However, such overexpressed v-jun had no effect on NIH3T3 cells. Our studies show that expression of the v-jun transgene contributes to the transformed phenotype of tumor cell lines but that there are additional factors that determine growth properties in culture and in the animal.

Tumor necrosis factor alpha and interleukin 1 alpha induce anchorage independence in v-jun transgenic murine cells.

The oncogene jun encodes a transcription factor of the AP-1 family. In mice carrying viral jun (v-jun) as a transgene, wounding is a prerequisite for tumorigenesis, suggesting collaboration between the transgene and a wound-related event. To define possible candidates for this collaborative process, we examined the effect of several wound-related polypeptide growth factors on cells from transgenic mice. Tumor necrosis factor alpha and interleukin 1 alpha induce anchorage independence in embryo fibroblasts and tumor cell revertants from these mice. This effect was specific for the two cytokines and was restricted to cells from v-jun transgenic mice. Anchorage independence required the continued presence of the cytokines. Transfection of transgenic cells with a v-jun expression plasmid also induced anchorage independence and a tumorigenic phenotype in transgenic tumor cell revertants. However, there was no correlation between anchorage independence, expression of Jun, and AP-1 activity. These results suggest that while increased transgene expression can enhance the growth properties of v-jun transgenic cells, there exist other cytokine-dependent mechanisms that have a similar effect. Retinoic acid, dexamethasone, or forskolin inhibits induction of anchorage independence by tumor necrosis factor alpha, interleukin 1 alpha, and transfected v-jun. Although these agents affect both AP-1 transactivation potential and DNA binding in the transgenic cells, the changes are not correlated with the inhibition of growth.

The use of multivariate MR imaging intensities versus metabolic data from MR spectroscopic imaging for brain tumour classification.

This study investigated the value of information from both magnetic resonance imaging and magnetic resonance spectroscopic imaging (MRSI) to automated discrimination of brain tumours. The influence of imaging intensities and metabolic data was tested by comparing the use of MR spectra from MRSI, MR imaging intensities, peak integration values obtained from the MR spectra and a combination of the latter two. Three classification techniques were objectively compared: linear discriminant analysis, least squares support vector machines (LS-SVM) with a linear kernel as linear techniques and LS-SVM with radial basis function kernel as a nonlinear technique. Classifiers were evaluated over 100 stratified random splittings of the dataset into training and test sets. The area under the receiver operating characteristic (ROC) curve (AUC) was used as a global performance measure on test data. In general, all techniques obtained a high performance when using peak integration values with or without MR imaging intensities. For example for low- versus high-grade tumours, low- versus high-grade gliomas and gliomas versus meningiomas, the mean test AUC was higher than 0.91, 0.94, and 0.99, respectively, when both MR imaging intensities and peak integration values were used. The use of metabolic data from MRSI significantly improved automated classification of brain tumour types compared to the use of MR imaging intensities solely.

A binding site for the cyclic adenosine 3',5'-monophosphate-response element-binding protein as a regulatory element in the grp78 promoter.

The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.

An update on antigenic variation in African trypanosomes.

African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?

A transcript encoding a proteasome beta-subunit and a zinc finger protein in Trypanosoma brucei brucei.

During the screening of a Trypanosoma brucei brucei (T. b. brucei) cDNA library constructed from bloodstream form mRNA, we identified a 2.3kb cDNA encoding a proteasome beta subunit (ORF1) and a putative zinc finger protein (ORF2). Northern blot analysis indicated the presence of a digenic transcript as well as the two individual messengers in both procyclic and bloodstream forms of the parasite. Southern blot analysis showed the relevant locus to be unique. ORF1 encoded a 22.7kDa protein sharing over 50% identity with the eukaryotic PRCE (aka beta5) proteasome beta subunit. This protein contained a beta amino acid signature and residues involved in the catalytic activity. Further phylogenetic analysis indicated that this subunit as well as those from other kinetoplastids could be confidentially assigned to extant eukaryotic subfamilies such as beta1, beta2, and beta5. ORF2 encoded a 14.6kDa putative zinc finger protein containing five repeats of a CCHC motif commonly present in retroviral nucleocapsid proteins as well as proteins involved in vertebrate embryogenesis.

A simple method for the immunocytochemical processing of large numbers of floating sections and its application in screening for monoclonal antibodies.

A technically simple modification of routine (non-adsorbent) multi-well plates, permitting the simultaneous immunocytochemical processing of hundreds of free-floating sections is described. The adaptations consist of (1) making a 1.5 mm wide perforation in the bottom of each well of the multi-well plate, (2) placing a 6 mm wide 50 microns mesh nylon filter on the bottom of each well and (3) preincubating the plate with excess inert protein in order to prevent adsorption of protein reagents. During the incubation of the floating sections with the immunocytochemical reagents, the fluid is retained in the well by capillarity, provided the detergent concentrations within the well do not exceed 0.005% (v/v). The wells can be emptied simply and quickly by blotting the plate bottom with a piece of laboratory paper toweling: the fragile sections are gently caught on the filter, without the risk of loss or damage. Sections start floating again as soon as the next reagent is added to the well. The present method drastically reduces the time needed for rinsing and reagent exchange, making immunocytochemistry on free-floating sections feasible as a primary screening method during hybridoma production.

Controls of the expression of the Vsg in Trypanosoma brucei.

We present an overview of the regulation of vsg expression, focusing on initiation and elongation of transcription as well as processing and stabilization of the transcripts. We propose a model where common factors are involved in the reverse controls of the genes for the two main stage-specific antigens, the Vsg and procyclin: a cross-talk between the two transcription units would allow a fast rerouting of limiting factors at differentiation, thereby allowing the expression of only one type of antigen at a time. A similar mechanism would ensure that only one vsg ES is fully expressed at a time in bloodstream forms.

Differential regulation of ESAG transcripts in Trypanosoma brucei.

In Trypanosoma brucei, several genes termed ESAGs for expression site-associated genes are contained within the polycistronic transcription units of the VSG gene, and their transcription is coordinately regulated during the parasite life-cycle. Since the VSG mRNA is characterized by a drastic destabilization under conditions where translation is inhibited, we examined if this post-transcriptional control also applies to the ESAG mRNAs. While the ESAG 7/6 mRNA behaved like the VSG mRNA, the ESAG 8 and ESAG 3 mRNAs did not. We ascribe this differential behaviour to the residual transcription that still occurs only in the ESAG 7/6 region of the VSG unit under conditions where this unit is down-regulated.

The VSG expression sites of Trypanosoma brucei: multipurpose tools for the adaptation of the parasite to mammalian hosts.

The variant surface glycoprotein (VSG) genes of Trypanosoma brucei are transcribed in telomeric loci termed VSG expression sites (ESs). Despite permanent initiation of transcription in most if not all of these multiple loci, RNA elongation is abortive except in bloodstream forms where full transcription up to the VSG occurs only in a single ES at a time. The ESs active in bloodstream forms are polycistronic and contain several genes in addition to the VSG, named ES-associated genes (ESAGs). So far 12 ESAGs have been identified, some of which are present only in some ESs. Most of these genes encode surface proteins and this list includes different glycosyl phosphatidyl inositol (GPI)-anchored proteins such as the heterodimeric receptor for the host transferrin (ESAG7/6), integral membrane proteins such as the receptor-like transmembrane adenylyl cyclase (ESAG4) and a surface transporter (ESAG10). An interesting exception is ESAG8, which may encode a cell cycle regulator involved in the differentiation of long slender into short stumpy bloodstream forms. Several ESAGs belong to multigene families including pseudogenes and members transcribed out of the ESs, named genes related to ESAGs (GRESAGs). However, some ESAGs (7, 6 and 8) appear to be restricted to the ESs. Most of these genes can be deleted from the active ES without apparently affecting the phenotype of bloodstream form trypanosomes, probably either due to the expression of ESAGs from 'inactive' ESs (ESAG7/6) or due to the expression of GRESAGs (in particular, GRESAGs4 and GRESAGs1). At least three ESAGs (ESAG7, ESAG6 and SRA) share the evolutionary origin of VSGs. The presence of these latter genes in ESs may confer an increased capacity of the parasite for adaptation to various mammalian hosts, as suggested in the case of ESAG7/6 and proven for SRA, which allows T. brucei to infect humans. Similarly, the existence of a collection of slightly different ESAG4s in the multiple ESs might provide the parasite with adenylyl cyclase isoforms that may regulate growth in response to different environmental conditions. The high transcription rate and high recombination level that prevail in VSG ESs may have favored the generation and/or recruitment in these sites of genes whose hyper-evolution allows adaptation to a larger variety of hosts.

Control and function of the bloodstream variant surface glycoprotein expression sites in Trypanosoma brucei.

African trypanosomes escape the host immune response through a periodical change of their surface coat made of one major type of protein, the variant surface glycoprotein. From a repertoire of a thousand variant surface glycoprotein genes available, only one is expressed at a time, and this takes place in a specialised expression site itself selected from a collection of an estimated 20-30 sites. As the specialised expression sites are long polycistronic transcription units, the variant surface glycoprotein is co-transcribed with several other genes termed expression site-associated genes. How do the trypanosomes only use a single specialised expression site at a time? Why are there two dozen specialised expression sites? What are the functions of the other genes of these transcription units? We review the currently available answers to these questions.

Time-domain quantification of series of biomedical magnetic resonance spectroscopy signals.

Quantification of individual magnetic resonance spectroscopy (MRS) signals is possible in the time domain using interactive nonlinear least-squares fitting methods which provide maximum likelihood parameter estimates under certain assumptions or using fully automatic, but statistically suboptimal, black-box methods. In kinetic experiments time series of consecutive MRS spectra are measured in which information concerning the time evolution of some of the signal parameters is often present. The purpose of this paper is to show how AMARES, a representative example of the interactive methods, can be extended to the simultaneous processing of all spectra in the time series using the common information present in the spectra. We show that this approach yields statistically better results than processing the individual signals separately.

Improved Lanczos algorithms for blackbox MRS data quantitation.

Magnetic resonance spectroscopy (MRS) has been shown to be a potentially important medical diagnostic tool. The success of MRS depends on the quantitative data analysis, i.e., the interpretation of the signal in terms of relevant physical parameters, such as frequencies, decay constants, and amplitudes. A variety of time-domain algorithms to extract parameters have been developed. On the one hand, there are so-called blackbox methods. Minimal user interaction and limited incorporation of prior knowledge are inherent to this type of method. On the other hand, interactive methods exist that are iterative, require user involvement, and allow inclusion of prior knowledge. We focus on blackbox methods. The computationally most intensive part of these blackbox methods is the computation of the singular value decomposition (SVD) of a Hankel matrix. Our goal is to reduce the needed computational time without affecting the accuracy of the parameters of interest. To this end, algorithms based on the Lanczos method are suitable because the main computation at each step, a matrix-vector product, can be efficiently performed by means of the fast Fourier transform exploiting the structure of the involved matrix. We compare the performance in terms of accuracy and efficiency of four algorithms: the classical SVD algorithm based on the QR decomposition, the Lanczos algorithm, the Lanczos algorithm with partial reorthogonalization, and the implicitly restarted Lanczos algorithm. Extensive simulation studies show that the latter two algorithms perform best.

The filtering approach to solvent peak suppression in MRS: a critical review.

Suppressing the solvent peak is important in many applications of biomedical NMR spectroscopy in order to quantify the metabolites with a great accuracy. Among the postprocessing methods proposed in the literature, many deal with the concept of filtering. However, several proposals lack a theoretical perspective and some have not been explicitly applied to quantification problems. The present article is intended to bridge this gap: five methods are analyzed from a theoretical perspective. Subsequently the different methods are applied to the same set of data, and then the latter are quantified using the model fitting method AMARES. With our set, the scheme proposed by T. Sundin et al. (J. Magn. Reson. 139(2), 189-204 (1999)) proved to be the most reliable method.