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L Vanhamme

Publications and source records attributed to L Vanhamme.

At least 37 records · Page 2Linked to original sources
PubMed1999

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.

Animals↗
PubMed1999

Accurate quantification of (1)H spectra: from finite impulse response filter design for solvent suppression to parameter estimation.

A scheme for accurate quantification of (1)H spectra is presented. The method uses maximum-phase finite impulse response (FIR) filters for solvent suppression and an iterative nonlinear least-squares (NLLS) algorithm for parameter estimation. The estimation algorithm takes the filter influence on the metabolites of interest into account and can thereby correctly incorporate a large variety of prior knowledge into the estimation phase. The FIR filter is designed in such a way that no distortion of the important initial samples is introduced. The FIR filter method is compared numerically with the HSVD method for water signal removal in a number of examples. The results show that the FIR method, using an automatic filter design scheme, slightly outperforms the HSVD method in most cases. The good performance and ease of use of the FIR filter method combined with its low computational complexity motivate the use of the proposed method.

Algorithms↗
PubMed1999

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.

Adenosine Triphosphate↗
PubMed1998

A VSG expression site-associated gene confers resistance to human serum in Trypanosoma rhodesiense.

Infectivity of Trypanosoma brucei rhodesiense to humans is due to its resistance to a lytic factor present in human serum. In the ETat 1 strain this character was associated with antigenic variation, since expression of the ETat 1.10 variant surface glycoprotein was required to generate resistant (R) clones. In addition, in this strain transcription of a gene termed SRA was detected in R clones only. We show that the ETat 1.10 expression site is the one selectively transcribed in R variants. This expression site contains SRA as an expression site-associated gene (ESAG) and is characterized by the deletion of several ESAGs. Transfection of SRA into T.b. brucei was sufficient to confer resistance to human serum, identifying this gene as one of those responsible for T.b. rhodesiense adaptation to humans.

Animals↗
PubMed1998

Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity.

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem. 227, 780-786).

Alternative Splicing↗
PubMed1998

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.

Animals↗
PubMed1998

New approach for quantitation of short echo time in vivo 1H MR spectra of brain using AMARES.

Short echo time in vivo STEAM 1H MR spectra (4.7 T, TE = 16 ms) of normal rat brain were fitted in the time domain using a VARPRO-like algorithm called AMARES which allows an inclusion of a large amount of prior knowledge. The prior knowledge was derived from phantom spectra of pure metabolite solutions measured under the same experimental conditions as the in vivo spectra. The prior knowledge for the in vivo spectra was constructed as follows: for each VARPRO-fitted phantom spectrum one peak (the most prominent one in the in vivo spectrum) was chosen and left unconstrained in the AMARES fitting while all the other peaks in the metabolite spectrum (i.e. their corresponding parameters--amplitudes, damping factors, frequencies and phases) were fixed to the parameter values of the unconstrained peak via amplitude and damping ratios and frequency and phase shifts. Including N-acetyl-aspartate, glutamate, total creatine, cholines, glucose and myo-inositol into the fits provided results which were in agreement with published data. An inclusion of glutamine into the set of fitted metabolites was also investigated.

Algorithms↗
PubMed1998

Fast Removal of Residual Water in Proton Spectra.

Quantification of metabolites in 1H spectra is difficult because of the presence of an unwanted water signal. Preprocessing, or removing the water contribution of a 1H spectrum, in the time domain is usually done using the state-space approach HSVD. HSVD removes the residual water and its side lobes, thereby reducing the baseline for the metabolites of interest and allowing subsequent data analysis using more sophisticated nonlinear least squares algorithms. However, the HSVD algorithm is computationally expensive because it estimates the signal subspace using the singular value decomposition (SVD). We show here that replacing the SVD by a low-rank revealing decomposition speeds up the computations without affecting the accuracy of the wanted parameter estimates. Copyright 1998 Academic Press.

Journal Article↗
PubMed1997

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.

Animals↗
PubMed1997

Expression of a variant surface glycoprotein of Trypanosoma gambiense in procyclic forms of Trypanosoma brucei shows that the cell type dictates the nature of the glycosylphosphatidylinositol membrane anchor attached to the glycoprotein.

Procyclic forms of Trypanosoma brucei have been genetically modified to express the major metacyclic variant surface glycoprotein (VSG variant AnTat 11.17) of Trypanosoma gambiense. The VSG is expressed in an intact membrane-bound form that can be detected over the entire plasma membrane, together with procyclin, and as a series of lower-molecular-mass fragments that are mostly soluble degradation products. The presence of degraded VSG in the cells and the culture medium suggests that VSG is not efficiently processed and/or efficiently folded when expressed in procyclic cells. The level of procyclin expressed on the surface of these cells is slightly reduced, although there is no difference in procyclin mRNA levels. The intact membrane-bound form of the VSG is N-glycosylated with oligomannose structures and contains a glycosylphosphatidylinositol (GPI) membrane anchor that can be biosynthetically labelled with [3H]ethanolamine. The anchor is sensitive to mammalian GPI-specific phospholipase D but, like the anchor of procyclin, it is resistant to the action of bacterial phosphatidylinositol-specific phospholipase C. This pattern of phospholipase sensitivity suggests that the GPI anchor acquired by VSG when expressed in procyclics is acylated on the inositol ring and therefore resembles a procyclic procyclin-type anchor rather than a trypomastigote VSG-type anchor with respect to the lipid structure. The VSG expressed in procyclics was sensitive to the action of a mixture of sialidase, beta-galactosidase and beta-hexosaminidase, suggesting that the VSG GPI anchor also contains a sialylated polylactosamine side-chain modification similar to that described for procyclin. These results indicate that the nature of the protein expressed has little influence on the post-translational modifications performed in the secretory pathway of procyclic trypanosomes.

Animals↗
PubMed1997

Improved method for accurate and efficient quantification of MRS data with use of prior knowledge

We introduce AMARES (advanced method for accurate, robust, and efficient spectral fitting), an improved method for accurately and efficiently estimating the parameters of noisy magnetic resonance spectroscopy (MRS) signals in the time domain. As a reference time domain method we take VARPRO. VARPRO uses a simple Levenberg-Marquardt algorithm to minimize the variable projection functional. This variable projection functional is derived from a general functional, which minimizes the sum of squared differences between the data and the model function. AMARES minimizes the general functional which improves the robustness of MRS data quantification. The newly developed method uses a version of NL2SOL, a sophisticated nonlinear least-squares algorithm, to minimize the general functional. In addition, AMARES uses a singlet approach for imposition of prior knowledge instead of the multiplet approach of VARPRO because this greatly extends the possibilities of the kind of prior knowledge that can be invoked. Other new features of AMARES are the possibility of fitting echo signals, choosing a Lorentzian as well as a Gaussian lineshape for each peak, and imposing lower and upper bounds on the parameters. Simulations, as well as in vivo experiments, confirm the better performance of AMARES compared to VARPRO in terms of accuracy, robustness, and flexibility. Copyright 1997 Academic Press. Copyright 1997Academic Press

Journal Article↗
PubMed1995

The 3'-terminal region of the mRNAs for VSG and procyclin can confer stage specificity to gene expression in Trypanosoma brucei.

The variant surface glycoprotein (VSG) and procyclin are the respective major surface antigens of the bloodstream and the procyclic forms of Trypanosoma brucei. These proteins and their mRNAs are both the most abundant and absolutely characteristic of their respective life cycle stages. We show that the 3'-terminal region of these mRNAs regulates expression of a reporter gene in an inverse manner, depending on the developmental form of the parasite. In the case of VSG mRNA, the 97 nt sequence upstream from the polyadenylation site is responsible for these effects. The regulation occurs through a variation of mRNA abundance which is not due to a change in primary transcription. In the bloodstream form this effect is manifested by an increase in RNA stability, whereas in the procyclic form it seems to be related to a reduction in the efficiency of mRNA maturation. The 3'-end of VSG mRNA can obviate the 5- to 10-fold stimulation of transcription driven by the procyclin promoter during differentiation from the bloodstream to the procyclic form. The predominance of posttranscriptional over transcriptional controls is probably linked to the organization of the trypanosome genome in polycistronic transcription units.

Animals↗
PubMed1995

Stimuli of differentiation regulate RNA elongation in the transcription units for the major stage-specific antigens of Trypanosoma brucei.

In Trypanosoma brucei, the mutually exclusive expression of the major surface antigens, the variant surface glycoprotein (VSG) of the bloodstream form and procyclin of the procyclic form, is due to a stage-specific accumulation of the respective mRNAs. Through the targeting of a reporter construct in the procyclin promoter region, we show that independently of any selection pressure, a relatively high level of transcription (approximately 10%) occurs from the procyclin promoter in the bloodstream form. This transcription leads to the production of detectable amounts of polyadenylated mRNAs. However, RNA elongation in the procyclin transcription unit is down-regulated at this stage. Transcription elongation in the procyclin and VSG units is inversely controlled by the combination of factors which cause the differentiation of bloodstream into procyclic forms in vitro. These factors include temperature, citrate/cis-aconitate and the incubation medium. Our results suggest that inverse regulations of primary transcription in the VSG and procyclin units are early events that underly the differentiation of the parasite.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Control of gene expression in trypanosomes.

Trypanosomes are protozoan agents of major parasitic diseases such as Chagas' disease in South America and sleeping sickness of humans and nagana disease of cattle in Africa. They are transmitted to mammalian hosts by specific insect vectors. Their life cycle consists of a succession of differentiation and growth phases requiring regulated gene expression to adapt to the changing extracellular environment. Typical of such stage-specific expression is that of the major surface antigens of Trypanosoma brucei, procyclin in the procyclic (insect) form and the variant surface glycoprotein (VSG) in the bloodstream (mammalian) form. In trypanosomes, the regulation of gene expression is effected mainly at posttranscriptional levels, since primary transcription of most of the genes occurs in long polycistronic units and is constitutive. The transcripts are processed by transsplicing and polyadenylation under the influence of intergenic polypyrimidine tracts. These events show some developmental regulation. Untranslated sequences of the mRNAs seem to play a prominent role in the stage-specific control of individual gene expression, through a modulation of mRNA abundance. The VSG and procyclin transcription units exhibit particular features that are probably related to the need for a high level of expression. The promoters and RNA polymerase driving the expression of these units resemble those of the ribosomal genes. Their mutually exclusive expression is ensured by controls operating at several levels, including RNA elongation. Antigenic variation in the bloodstream is achieved through DNA rearrangements or alternative activation of the telomeric VSG gene expression sites. Recent discoveries, such as the existence of a novel nucleotide in telomeric DNA and the generation of point mutations in VSG genes, have shed new light on the mechanisms and consequences of antigenic variation.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗