extfieldsofstudy: Biology Medicine

text:
Identifying the most suitable endogenous control for determining gene expression in hearts from organ donors

Background Quantitative real-time reverse transcription PCR (qRT-PCR) is a useful tool for assessing gene expression in different tissues, but the choice of adequate controls is critical to normalise the results, thereby avoiding differences and maximizing sensitivity and accuracy. So far, many genes have been used as a single reference gene, without having previously verified their value as controls. This practice can lead to incorrect conclusions and recent evidence indicates a need to use the geometric mean of data from several control genes. Here, we identified an appropriate set of genes to be used as an endogenous reference for quantifying gene expression in human heart tissue. Results Our findings indicate that out of ten commonly used reference genes (GADPH, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP and HPRT), PPIA, RPLP and GADPH show the most stable gene transcription levels in left ventricle specimens obtained from organ donors, as assessed using geNorm and Normfinder software. The expression of TBP was found to be highly regulated. Conclusion We propose the use of PPIA, RPLP and GADPH as reference genes for the accurate normalisation of qRT-PCR performed on heart tissue. TBP should not be used as a control in this type of tissue.

Background
Gene expression analysis is a useful technique for determining and comparing gene expression levels in healthy and diseased tissues. One of the most commonly used tools in the area of gene expression quantification is quantitative real-time reverse transcription PCR (qRT-PCR). When small amounts of nucleic acids are available, qRT-PCR is especially suitable and provides simultaneous measurement of gene expression in many different samples. If we compare this technique with others such as in situ hybridisation, qRT-PCR offers several advantages: it is not time-consuming, only a small amount of tissue is required, it can be used in high throughput systems and no post-reaction manipulation is needed. However, the use of qRT-PCR requires compensation for differences between samples, arising from the varying quality and quantity of the starting material, especially when starting with solid tissue, due to the method of RNA extraction and cDNA synthesis [1]. Normalisation should include endogenous control genes (reference genes), and some of the most frequently used reference genes are housekeeping genes. The ideal endogenous control should be expressed at a constant level in the different tissues of an organism at all stages of development and should be unaffected by experimental treatments. It should also be constitutively expressed in the same tissue under different circumstances. There is, however, no universal control gene that is expressed at a constant level under all conditions and in all tissues. Hence, experimental treatments [2] and hormonal stimulation [3], as well as the different methods used to process tissue specimens [4] can induce changes in the expression of typical housekeeping genes.
Consequently, the success of the technique used depends on the adequate choice of the appropriate reference genes. Despite many qRT-PCR studies having reported the use of a single endogenous control [5], a normalisation strategy based on a single housekeeping gene can lead to erroneous results [6,7]. Vandesompele et al. [6] propose the use of a panel of putative reference genes on a representative number of samples, to identify the most stable of these and then establish the optimal number of genes required for the reliable normalisation of RT-PCR data. In the present study, we tested ten commonly used reference genes (GADPH, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP and HPRT) of different functional classes (significantly reducing the chance that the genes will be co-regulated) in heart tissue obtained from organ donors. Using geNorm software [6], we were able to assess gene expression stability under our experimental conditions and determine how many reference genes were needed for accurate normalisation. Then, by comparing these results with those generated by a similar programme, Normfinder [8,9], we identified a set of reference genes that offers reliable results for qRT-PCR data normalisation for use in gene expression studies involving heart tissue from brain-dead multiorgan donors.

Results
Thirty five samples of left ventricular tissue were obtained from 35 multiorgan donors. RNA was successfully isolated and cDNA synthesised from all these specimens. All the samples analysed showed a single β-actin band in the 2% agarose gel stained with ethidium bromide at the expected size (data not shown), confirming the total absence of residual DNA.
In each sample, qRT-PCR using Sybr Green was performed for ten frequently-used reference genes (GADPH, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP and HPRT). The accuracy of the qRT-PCR was assessed by melt curve analysis and gel electrophoresis. Gene-specific amplification was verified, by both a single peak in the melt curve and a single band in the agarose gel, for the 10 genes analysed in the 35 cDNA samples. Correlation coefficients (R 2 ) ranged from 0.995 to 0.999 and PCR efficiencies from 89.7% to 104%. Using the Proc VARCOMP in the SAS/STAT™ software [10], the reproducibility of the assay was assessed using as control material samples obtained by pooling the whole set of samples analysed in the present study.  (Figure 1).
The GeNorm software also provided a rank order of the ten candidate reference genes according to their M values ( Figure 2). PPIA, RPLP and GADPH were the three most stable genes. M values increased moderately for all genes, while TBP abruptly attained the highest M value. The pairwise variation (V) upon normalisation with the two most stable genes and introduction of the third one was 0.236. This value decreased gradually until the addition of the fifth gene, when the trend became more or less stable but increased considerably upon the incorporation of the tenth gene ( Figure 3). Table 1 provides the results of the Normfinder analysis performed on our data. The reference genes tested were ordered according to their stability values. Thus, the most stable single gene was GADPH with a stability value of 0.345 and the least stable was TBP with a value of 2.287.

Discussion
Human heart tissue specimens are very difficult to acquire. However, we managed to obtain 35 samples from the left ventricles of 35 organ donors of different age, weights and sex, whose hearts could not be used for transplant purposes for various reasons (see Methods). Thus, we can assume that the number and type of samples used was sufficiently high and diverse to ensure their representativity and randomness. Reports of the use of qRT-PCR in human heart tissue are scarce because of the technical difficulties involved in obtaining such samples. However, given that qRT-PCR is especially suitable for determining gene expression in small pieces of tissue, we tried to establish guidelines for accurate data normalisation intended for human heart studies based on qRT-PCR. Our technical procedure proved to be a valid method of quantifying gene expression, rendering high correlation coefficients (R 2 ) and robust PCR efficiencies.

Expression levels of candidate housekeeping genes
Through geNorm analysis, PPIA, RPLP and GADPH appeared as the most stable genes and TBP the least stable ( Figure 2). It has been reported that in over 90% of cases, gene expression data are normalised using GADPH, ACTB, 18S rRNA or 28S rRNA as single control genes [5]. However, several studies have shown that these reference genes undergo variation according to the experimental conditions, treatment and cell cycle stage [11,12].
In human adipocytes and preadipocytes, ACTB and 18S rRNA gene expression levels change under hormonal stimulation [3]. Moreover, it has been reported that ACTB is unsuitable as a control for gene expression analysis in interstitial cells derived from sheep heart valves [13]. The 18S rRNA gene has been considered an ideal internal control in qRT-PCR analysis. However, ribosomal RNA accounts for up to 80-90% of total cellular RNA, and several studies have shown that rRNA varies less under conditions that affect the expression of mRNAs [14] but that possible imbalances in rRNA and mRNA fractions between different samples makes genes encoding ribosomal RNAs unsuitable as references [6]. As far as we are aware, no previous study has tried to identify adequate housekeeping genes for use in human heart tissue. Morgan et al. [15] examined the expression of genes related to the renin-angiotensin system in human atrial tissue using GADPH as an endogenous control. Other authors [16] have used RNA extracted from human endomyocardial biopsies and isolated cardiomyocytes for real-time RT-PCR using GADPH, HPRT and the oncogene ABL as housekeeping genes. These endogenous controls showed very low variation in individual gene expression levels Pairwise variation analysis between normalisation factors to determine the optimal number of control genes for normalisation Figure 3 Pairwise variation analysis between normalisation factors to determine the optimal number of control genes for normalisation. Optimal number of control genes for normalisation calculated on the basis of pairwise variation (V) analysis. The highest V9/10 value is due to the inclusion of a relative unstable gene and is in accordance with the average expression stability (M). across cardiac pathologies, suggesting their suitable use as reference genes for quantitative PCR studies in cardiac tissue. In these previous studies, the specific testing of several candidate reference genes to determine the most suitable reference for use in cardiac tissue was not reported [15,16]. In contrast, Radonic et al. [17] determined transcription levels of several housekeeping genes in different human tissues, including heart, and identified TBP as the gene with the lowest range of RNA transcription across tissues. This finding is in agreement with the present results.
We found PPIA, RPLP plus GADPH to be a reliable set of genes for normalising data (Figure 3) according to the geNorm programme. As reported by Vandesompele et al. [6], geNorm proposes a pairwise variation of 0.15 as the cut-off under which the inclusion of an additional control gene is not required. Using our set of candidate genes, this would mean that adding a fifth gene to the four most stable genes identified would really provide the best results. Notwithstanding, this cut-off of 0.15 should not be considered in a strict sense, but rather as a reference to determine the optimal number of housekeeping genes. Sometimes the observed trend can be equally informative, and using the three best reference genes is, in most cases, a valid strategy for much more accurate and reliable normalisation compared to the use of a single housekeeping gene.
Using NormFinder software [8] as another tool to validate the expression stability of the ten candidate reference genes; GADPH, PPIA and RPLP also achieved the best stability values. While geNorm detected the two genes whose expression ratios showed least variation from those of the other genes tested, NormFinder was able to identify the single gene with the most stable expression. Hence, the most stable candidate gene was found to be GADPH, and the least stable TBP. Using this programme, we obtained the same results as with GeNorm except for the rank position ascribed to GADPH as the most stable single gene. However, the least stable genes and most stable ones identified by GeNorm and Normfinder were generally wellmatched.
As a limitation to our study, we should mention that although we established the purity of our RNA samples, due to the amount of RNA available, we could not run electrophoretic tests to check RNA integrity.
Finally, the set of reference genes determined here as the best endogenous controls to be used in qRT-PCR studies in heart tissue has applications in developing cardiovascular diagnostic tests and therapeutic strategies that will substantially improve human health [18].

Conclusion
It is commonly accepted that gene expression studies should be normalised using more than one reference gene. Based on our results, we propose the use of the mean result rendered by PPIA, RPLP and GADPH as housekeeping genes to normalise gene expression values obtained by qRT-PCR in heart tissue.

Sample collection
Tissue samples were obtained from the left ventricle of 35 organ donors (19 male, 16 female; mean age 59 years, range 40 -80 years). The organ donors had died in a multidisciplinary ICU of a tertiary university hospital. In 85% of the organ donors, brain death was attributed to a non-traumatic cause and no donor had previous heart pathology. Consent for organ donation for transplant and clinical investigation was obtained from the relatives of each donor.
The hearts had been rejected for transplant because of donor age or weight, or recipient blood group incompatibility. The cardiac tissues were freshly harvested, stored immediately in O.C.T™ Compound (Tissue Tek, Sakura, Netherlands) and then frozen at -80°C.

RNA extraction and cDNA synthesis
Isolation of total RNA was performed using the Nucleo Spin RNA II Isolation Kit (Macherey-Nagel Gmb H & Co. KG) according to the manufacturer's instructions.
The quality of the RNA was assessed through absorbance measurements made using a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDropTechnologies, Wilmington, DE, USA). All the samples used in the PCR procedures showed a 260/280 nm absorbance ratio between 1.8 and 2.2. A ratio of ≈2 is generally accepted as pure for RNA.
Each RNA sample was reverse transcribed using the Oligo-pdT and Random primers provided in the first-strand complementary DNA (cDNA) synthesis kit for RT-PCR (AMV; Roche Diagnostic, Switzerland) following the manufacturer's instructions. The tubes were incubated at 25°C for 10 minutes to allow annealing. Reverse transcription was conducted at 42°C for 60 minutes, followed by 5 minutes incubation at 99°C to denature the enzyme. The cDNA samples were then cooled at 4°C and stored at -20°C until use.
To confirm the total absence of residual DNA, we per- β-actin down 5'-CGGATGTCCACGTCACACTTC-3'). The PCR protocol consisted of an initial denaturation step at 94°C for 5 minutes, followed by 35 amplification cycles. Each cycle involved a denaturation step of 30 seconds at 94°C, followed by 30 seconds of primer annealing at 55°C and 30 seconds of primer extension at 72°C followed by a final extension of 10 minutes. Amplicon size was confirmed by electrophoresis using an ethidium bromide-stained 2% agarose gel in 1XTBE buffer.

Gene expression
We analysed the expression of the genes GADPH, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP and HPRT included in the Human Endogenous Control Gene Panel supplied by TATAA Biocenter (Goteborg, Sweden) (see Table 2). For this panel, the genes are carefully selected to be of different functional classes, thus significantly reducing the likelihood that they might be co-regulated. The genes selected here were also evaluated by Vandesompele et al. [6].
Quantitative PCR was performed using an i-Cycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). SYBR Green PCR Supermix (2X) (Bio-Rad), which detects amplification using Sybr Green as a doublestranded DNA-specific fluorescent dye, was used as a mastermix. Assays were performed in duplicate and a blank included in every assay. The reaction mixture for amplification consisted of 2 µl of cDNA in a final reaction volume of 25 µl, as indicated in the instructions for the Human Endogenous Control Gene Panel. The RT-PCR protocol included an initial step of 95°C (3 minutes), followed by 40 cycles of 30 seconds at 95°C for DNA denaturation, 20 seconds for primer annealing at 60°C and 20 seconds at 72°C for primer extension. Fluorescence data were acquired at 72°C. Melting-curve analysis to confirm product specificity was performed immediately after amplification, following 1 minute denaturation at 95°C, 1 minute annealing at 65°C and 60 cycles of 0.5°C increments (10 seconds each) beginning at 65°C while monitoring fluorescence. Product identity was confirmed by electrophoresis using an ethidium bromide-stained 2% agarose gel in 1XTBE buffer. PCR efficiencies were calculated from a relative standard curve derived from a pool of heart tissue specimens from the same organ donors and serially diluted (a ten-fold dilution series with at least six measuring points).

Reference gene expression stability
To determine the most stable housekeeping genes of the set of tested genes in our heart cDNA sample panel, we used firstable, the geNorm software package for Microsoft Excel ® developed by Vandesompele et al. [6]. The programme geNorm uses an algorithm to calculate M, a gene expression stability measure, defined as the mean pairwise variation for a given gene compared to the remaining tested control genes. Thus, the higher the M value of a gene, the greater the variation in its expression levels. The programme establishes a rank order of gene stability via stepwise exclusion. To determine how many reference genes should be used, normalisation factors (NF n ) based on the geometric mean of the expression levels of the n best reference genes were calculated by stepwise inclusion of an extra, less stable reference gene as described elsewhere [4]. The programme shows the pairwise variation Vn/V n+1 between two sequential normalisation factors, NFn and NF n+1 . A large variation means that the added gene has a significant effect and should probably be included for calculation of the normalisation factor.
To compare our results with those generated by a similar programme, we used Normfinder [9], another algorithmbased tool for identifying optimal reference genes among a set of candidates. The programme ranks the genes depending on their expression stability value in given samples derived from an experimental design. was designed by JO. All the authors have read and approved the final manuscript.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
VopA Inhibits ATP Binding by Acetylating the Catalytic Loop of MAPK Kinases*

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors. One of these effectors, Vibrio outer protein A (VopA), inhibits MAPK signaling via a novel mechanism, distinct from those described for other bacterial toxins, that disrupts this signaling pathway. VopA is an acetyltransferase that potently inhibits MAPK signaling pathways not only by preventing the activation of MAPK kinases (MKKs) but also by inhibiting the activity of activated MKKs. VopA acetylates a conserved lysine found in the catalytic loop of all kinases and blocks the binding of ATP, but not ADP, on the MKKs, resulting in an inactive phosphorylated kinase. Acetylation of this conserved lysine inhibits kinase activity by a new mechanism of regulation that has not been observed previously. Identifying the target of VopA reveals a way that the reversible post-translational modification of lysine acetylation can be used to regulate the activity of an enzyme.

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors. One of these effectors, Vibrio outer protein A (VopA), inhibits MAPK signaling via a novel mechanism, distinct from those described for other bacterial toxins, that disrupts this signaling pathway. VopA is an acetyltransferase that potently inhibits MAPK signaling pathways not only by preventing the activation of MAPK kinases (MKKs) but also by inhibiting the activity of activated MKKs. VopA acetylates a conserved lysine found in the catalytic loop of all kinases and blocks the binding of ATP, but not ADP, on the MKKs, resulting in an inactive phosphorylated kinase. Acetylation of this conserved lysine inhibits kinase activity by a new mechanism of regulation that has not been observed previously. Identifying the target of VopA reveals a way that the reversible post-translational modification of lysine acetylation can be used to regulate the activity of an enzyme.
Vibrio parahemeolyticus is a marine bacterium and causative agent of gastroenteritis associated with the consumption of contaminated seafood. It is endemic to Southeast Asia and is the leading cause of gastroenteritis in Japan (1). Sequencing of the V. parahemeolyticus genome revealed the presence of two type III secretion systems each encoded in separate pathogenicity islands (2). Type III secretion systems are used by bacterial pathogens to inject effector proteins into the cytoplasm of the host cells (3). Upon delivery into the host cytosol, the effectors manipulate host signaling to gain an advantage for the infecting pathogen. The bacterial effectors are proposed to usurp or mimic eukaryotic activity that is then used to target and deregulate normal functions in the host cell by either up-or downregulating signaling pathways (4). However, the bacterial effector proteins must initially be kept in a quiescent state within the bacterium. This is accomplished by a variety of methods, including their association with a chaperone in the bacterial cytosol, their specificity for a substrate, and/or their requirement for an activator or modification that is only provided by a eukaryotic source (4,5).
VopA (Vibrio outer protein A) is an effector found within one of the V. parahemeolyticus pathogenicity islands, and is a member of a family of type III effectors called YopJ-like proteins that are found in a wide variety of pathogens including other animal pathogens, plant pathogens, and the plant symbiont Rhizobium (5-7). The founding member of the family, YopJ from Yersinia spp., inhibits the mitogen-activated protein kinase (MAPK) 3 and NFB signaling cascades within the host cell, thereby inhibiting the host's innate immune response (7). All YopJ-like proteins contain a catalytic triad, and it was recently shown that YopJ functions as an acetyltransferase (8 -10). It has been speculated that other members of the family of YopJ-like proteins might also possess this activity (8). YopJ was demonstrated to inhibit the MAPK and NFB signaling pathways by acetylating the critical serine and threonine residues found on the activation loop of MAPK kinases (MKKs), thereby blocking their activation by phosphorylation (8 -10). VopA and YopJ share ϳ55% similarity at the amino acid level although they elicit different functions (11).
VopA inhibits MAPK signaling using a novel mechanism distinct from those described for other bacterial toxins that disrupt this signaling pathway. This effector potently inhibits MAPK signaling but shows no inhibitory effect on the NFB pathway (11). Herein, we show that VopA acetylates a conserved lysine located in the catalytic loop of all kinases that plays a critical role in the binding of the ␥-phosphate of ATP. Modification of this lysine by acetylation inhibits the binding of ATP, but not ADP, to the MAPK kinase, resulting in an inactivate kinase. These findings uncover a unique mechanism that targets a critical conserved lysine residue involved in coordinating nucleotide binding that could also be used by eukaryotic enzymes.

EXPERIMENTAL PROCEDURES
Cloning-pSSFV VopA-FLAG and pSFFV VopA-C167A constructs were generated as described previously (11). For bacterial overexpression, VopA and VopA-C167A were ampli-fied by PCR using pSFFV VopA or pSFFV VopA-C167A as template with 5Ј-EcoRI and 3Ј-stop-XhoI primers and cloned into pGEX-tobacco etch virus (a kind gift from Y. M. Chook). The resulting construct has a glutathione S-transferase (GST) tag on the N-terminus of VopA with both thrombin and tobacco etch virus cleavage sites in the linker. For expression in Yersinia pseudotuberculosis, VopA and VopA-C167A were amplified by PCR using pSFFV VopA or pSFFV VopA-C167A as template with 5Ј-HindIII and 3Ј-FLAG-stop-BamHI primers and cloned into pMMB67HE (a kind gift from J. B. Bliska).
Y. pseudotuberculosis Strains-YP126 (wild type) and YP26 (⌬YopJ) were kind gifts from J. B. Bliska. For expression of pMMB67HE VopA-FLAG and pMMB67HE VopA-C167A-FLAG in YP26, the constructs were transformed into SM10pir. Overnight cultures of SM10 strains were grown at 37°C, and overnight cultures of YP26 were grown at 26°C. 200 l of each strain was mixed and spotted onto an LB agar plate. Plates were incubated at 37°C right side up for several hours. Bacteria were scraped off plates, replated onto M9 agar plates supplemented with ampicillin (100 g/ml), and grown at 26°C. Colonies were picked, and VopA and VopA-C167A expression were verified by Western blot with anti-FLAG antibody (Sigma).
Macrophage Infections-Y. pseudotuberculosis infections were performed as described previously (12). In brief, J774A.1 murine macrophages were seeded into 6-well dishes at a density of 1.0 ϫ 10 6 cells/ml in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Hyclone), penicillin/streptomycin/glutamate (Invitrogen), and sodium pyruvate (Invitrogen). To induce type III secretion, overnight cultures of Yersinia strains were diluted back to an OD of 0.1 in LB supplemented with 20 mM sodium oxalate and 20 mM magnesium chloride and grown at 26°C for 1 h. Cultures were then shifted to 37°C for 2 h. Bacteria cells (1 ϫ 10 8 cells) were pelleted and resuspended in Dulbecco's modified Eagle's medium. Bacteria were overlaid onto macrophages and plates spun at 500 ϫ g for 5 min to bring bacteria into contact with the cells. Plates were kept at 37°C for 15 min and 45 min. Plates were put on ice and cells were washed once with cold phosphate-buffered saline. Cells were lysed for 10 min in radioimmunoprecipitation assay buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM Na 3 VO 4 , 10 mM NaF, 20 mM ␤-glycerophosphate, 1 M dithiothreitol, 0.5 mM EGTA, and Complete protease inhibitor tablet (Roche Applied Science)). Lysates were cleared by centrifugation at 10,000 rpm. 150 l of cleared lysate were boiled with 50 l of 5ϫ Laemmli sample buffer. Samples were separated by SDS-PAGE and analyzed by immunoblot with ␣-phospho-JNK and ␣-JNK or ␣-phospho-p38 and ␣-p38.
Epistasis Assays-293T cells were transfected with or without a MAPK (HA-ERK, 200 ng) in the presence or absence of pSFFV VopA or pSFFV VopA-C167A (50 ng) with vector RasV12 (100 ng), Raf BxB (gift from Mike White) (10 ng), or MKK1 ED (100 ng). After 36 h of growth, cells were starved for 3 h followed by treatment or no treatment with a stimulus (epidermal growth factor, 50 ng/ml). Cells lysates were analyzed by immunoblot with rabbit anti-phospho-ERK anti-body (Cell Signaling Technology, Inc.) and mouse anti-HA antibody (Babco).
Protein Purification-GST-VopA wild type and GST-VopA-C167A constructs were expressed in Escherichia coli BL21-DE3 cells and purified by GST affinity chromatography. In brief, cells were grown to an A 600 of 0.6 to 0.8 in 2ϫ yeast extract and tryptone medium and then induced with 400 M isopropyl-Dthiogalactopyranoside (Roche Applied Science) for 4 h at 25°C. The cells were lysed in PBS-T (phosphate-buffered saline (pH 8), 1% (v/v) Triton X-100 (Fisher), 0.1% (v/v) 2-mercaptoethanol (Bio-Rad), and 1 mM phenylmethylsulfonyl fluoride (Sigma)) using a cell disrupter (EmulsiFlex-C5, Avestin, Inc.). The lysates were incubated with glutathione-agarose beads, and bound protein was subjected to GST-tobacco etch virus cleavage overnight in PBS-T supplemented with 1 mM acetyl coenzyme A (acetyl-CoA) (Sigma) at room temperature. Flowthrough was collected and incubated with GST beads to remove any remaining GST-tobacco etch virus. Flow-through was collected and concentrated in a 30-kDa molecular mass cut-off Amicon-Ultra concentrator (Fisher).
rMKK6 construct was expressed in Rosetta Blue cells (Novagen) and purified by nickel affinity chromatography. In brief, cells were grown and lysed as above, except cells were induced with 200 M isopropyl-D-thiogalactopyranoside and grown at 25°C for 8 h. Lysates were bound to Ni 2ϩ -nitrilotriacetic acid beads (Sigma). Bound protein was washed and eluted per the manufacturer's protocol. His-MKK1 was expressed and purified as His-MKK6 except cells were induced with 400 M of isopropyl-D-thiogalactopyranoside and grown at 25°C for 4 h.
Molecular Weight Measurement-The protein molecular weight was measured on a Sciex QSTAR XL mass spectrometer (Applied Biosystems) with an off-line electrospray ionization source. The electrospray tips were purchased from Proxeon Biosystems. Sample solution was desalted using a Millipore C 18 ZipTip and eluted with 80% acetonitrile. The eluates were dried, and then resuspended in 1% formic acid, 50% acetonitrile.
Liquid Chromatography/Tandem Mass Spectrometry-The tryptic digests of the sample were fractionated using a Dionex LC-Packings HPLC. Peptides were first desalted on a 300-m ϫ 1-mm PepMap C 18 trap column with 0.1% formic acid in HPLC grade water at a flow rate of 20 l/min. After desalting for 5 min, peptides were flushed onto a LC Packings 75-m ϫ 15-cm C 18 nanocolumn (3 m, 100 Å) at a flow rate of 200 nl/min. A 45-min gradient was used for the HPLC separation with the acetonitrile concentration increased from 2 to 45%. Elutes were analyzed with a QSTAR XL mass spectrometer (Applied Biosystems). Data were acquired in information dependent acquisition mode where the top three precursor ions were selected per cycle for the tandem mass spectrometry experiment. Mass ranges for the mass spectrometry survey scan and tandem mass spectrometry were m/z 300 -1800 and m/z 50 -1800, respectively. Raw data were processed by Analyst QS to generate the peak list (PKL) file. Data were searched using the Mascot search engine (Matrix Science Ltd., London) against the SwissProt data base and the home-built data base that contains the sequence of the MEKK6 protein. Nucleotide

RESULTS
VopA Inhibits Mammalian MAPK Activation-We have shown previously that VopA is able to inhibit the ERK-MAPK signaling pathway in mammals and the high osmolarity growth pathway in yeast (11). To confirm that VopA inhibits other mammalian MAPK pathways, we analyzed the activation of the p38-MAPK and JNK-MAPK pathways by assessing the phosphorylation of p38 and JNK in the presence and absence of VopA or the catalytically inactive form of VopA (VopA-C167A) where the catalytic cysteine is mutated to an alanine (Fig. 1). For this analysis, VopA or VopA-C167A was cloned into the bacterial expression vector pMMB67HE and conjugated into a Y. pseudotuberculosis strain that is deleted for YopJ (12). These strains were used to infect J744A.1 murine macrophages to test the ability of VopA to rescue the deleted YopJ strain by restoring the inhibition of the p38 or JNK pathways. Infection with the wild type Yersinia strain that contains YopJ efficiently inhibits both the p38 and JNK-MAPK pathways (Fig. 1A, lanes  2 and 3, and B, lanes 2 and 3, respectively), whereas the Yersinia strain deleted for YopJ does not inhibit these signaling pathways (Fig. 1A, lanes 4 and 5, and B, lanes 4 and 5, respectively) (12). Inhibition of the p38 and JNK phosphorylation was restored in the YopJ deletion strain by induced expression of VopA (Fig. 1A, lanes 8 and 9, and B, lanes 8 and 9, respectively) but not the catalytically inactive VopA-C167A (Fig. 1A, lanes 12  and 13, and B, lanes 12 and 13, respectively).
VopA Inhibits Downstream of MKK Activation-To determine the point at which VopA blocks the MAPK pathway, we performed a series of epistasis experiments. Consistent with previous findings, when the MAPK pathway is stimulated with an external stimulus (such as epidermal growth factor) VopA, but not VopA-C167A, is able to inhibit the ERK-MAPK pathway as demonstrated by the lack of phosphorylated HA-ERK in treated cells ( Fig. 2A) (11). Phosphorylation of endogenous ERK is observed because of the background from nontransfected, stimulated cells ( Fig. 2A) (11). Constitutive activation of this pathway by transfected RasV12 or B-Raf BxB is also inhibited by wild type VopA, but not mutant VopA-C167A, as demonstrated by analysis of ERK phosphorylation (Fig. 2, B and C,  respectively). Surprisingly, when an activated form of MKK1, MKK1 ED, is used to activate the pathway just upstream of ERK, VopA is still able to inhibit phosphorylation of HA-ERK (Fig. 2D). This observation is in contrast to the inhibitory profile of YopJ, which is unable to inhibit signaling in the presence of an activated MKK (7). Based on these findings, we concluded that VopA inhibits MAPK signaling downstream of MKK1 activation and upstream of ERK activation.
VopA Acetylation of MKK6 Inhibits Activation-Given that VopA only affects the MAPK signaling pathways and that it also inhibits the activation of MAPKs, we predicted the molecular target of VopA would be MAPKs (11). In addition, because of the fact that VopA is a member of the family of YopJ-like proteins and that YopJ is an acetyltransferase, we hypothesized that VopA functions as an acetyltransferase that acetylates and inhibits MAPK activation (8). To test this hypothesis, we used purified p38 in an in vitro acetylation assay with purified wild type and mutant VopA. However, we observed that neither VopA nor the catalytically inactive VopA-C167A acetylates p38 (Fig. 3A). However, as a control we included rMKK6 as a substrate and discovered that VopA, but not mutant VopA-C167A, is able to acetylate rMKK6.
Previously, YopJ was observed to acetylate rMKK6 on its activation loop, thereby preventing phosphorylation by upstream kinases (9,10). To determine whether VopA modifies rMKK6 in a similar manner, we tested whether rMKK6 is phosphorylated by upstream kinases in the presence or absence of VopA using a previously established in vitro signaling assay (9). rMKK6 was preincubated with GST-VopA or GST-VopA-C167A in the presence or absence of acetyl-CoA followed by incubation with membrane-free lysates derived from serum- stimulated cells. In control samples without VopA, rMKK6 was phosphorylated and activated by upstream kinases as indicated by immunoblot analysis with an antiphospho-MKK6 antibody (Fig. 3B,  lanes 1 and 2). However, when rMKK6 is preincubated with wild type VopA in the presence of acetyl-CoA, rMKK6 is no longer activated by phosphorylation (Fig. 3B, lane 4). By contrast, incubation of the kinase with mutant VopA-C167A has no inhibitory effect on the phosphorylation of rMKK6 (Fig. 3B, lanes 5 and  6). Although these results demonstrate that VopA can inhibit activation of MKKs by acetylation, they do not explain how VopA is able to inhibit activated MKKs.
VopA Acetylates Four Conserved Residues in MKK6-To determine how VopA acetylates MKK6, we first assessed the total mass of rMKK6 isolated from bacterial cells expressed either alone (rMKK6) or in the presence of GST-VopA (rMKK6ϩV) for total mass spectrometric analysis. The mass of rMKK6 is observed as one strong peak at 39,655 kDa, but the mass of rMKK6ϩV is divided among five peaks, with mass differences in increments of 42 Da, equal to the mass of one acetyl group (Fig. 4A). These results indicate that VopA acetylates four residues on rMKK6.
To determine which four residues of rMKK6 are modified by GST-VopA, the samples were subjected to liquid chromatography/tandem mass spectrometry analyses. No acetylation was observed for rMKK6, whereas rMKK6ϩV was acetylated on three residues in the activation loop (Ser-207, Lys-210, and Thr-211) and on one residue in the catalytic loop (Lys-172) (Fig.  4B) (13). The three residues in the activation loop are the same three residues that are modified by YopJ, and as shown in Fig.  3A, the acetylation by VopA on the activation loop of the kinase directly competes with phosphorylation on the serine and threonine residues. The fourth residue is a conserved lysine that is involved in the coordination of the ␥-phosphate of ATP upon nucleotide binding.
To confirm that acetylation of the lysine residue on the catalytic loop can occur on an active kinase, we used the purified active phosphomimic, His 6 -MKK1(S218E,S222D), as a substrate in an acetylation assay (7). We were able to detect acetylation of His 6 -MKK1(S218E,S222D) by VopA but not VopA-C167A (Fig. 4C). We were also able to detect autoacetylation of VopA, which is not seen in the assay with rMKK6. This observed difference is likely because of the extended exposure required to see His 6 -MKK1(S218E,S222D) acetylation (1.5 weeks). The longer exposure time is required because only one acetylation site on His 6 -MKK1(S218E,S222D) is available for modification, due to the mutations of both serines on the acti-  Acetylation of Lys-172 by VopA Disrupts ATP Binding to MKK-Based on the location of the conserved lysine in the kinase (Lys-172 in MKK6) and its interaction with the ␥-phosphate of ATP, we hypothesized that acetylating this lysine would negatively affect the binding of ATP, but not ADP, to MKKs. Therefore, we analyzed the binding of the fluorescent nucleotide analogs MANT-ATP and MANT-ADP to constitutively activated His 6 -MKK1(S218E,S222D). When excited at 290 nm the MANT-labeled nucleotides, ATP and ADP, emit strong fluorescent peaks (Fig. 5, A and B, respectively) (14). When MANT-ATP or MANT-ADP is incubated with either His 6 -MKK1(S218E,S222D) or His 6 -MKK1(S218E,S222D) that is preincubated with VopA-C167A, an increase in the emission maximum at ϳ450 nm is observed (Fig. 5, A and B, respectively). This is similar to the emission profile observed for binding of these nucleotide analogs to other kinases (14). However, this increase in fluorescence in the presence of MANT-ATP is abrogated when His 6 -MKK1(S218E,S222D) is preincubated with wild type VopA (Fig. 5A). This inhibitory effect is not seen in the presence of MANT-ADP (Fig. 5B), although binding of both wild type VopA and VopA-C167A does increase the emission of MANT-ADP bound to His 6 -MKK1(S218E,S222D). We propose this difference be attributed to the disruption of the coordination of binding for the ␥-phosphate of ATP by the acetylated lysine residue (Lys-192) in MKK1.

VopA, a Multi-tasking Acetyltransferase-Vibrio
VopA is an acetyltransferase that modifies MKKs on serine, threonine, and lysine. The modification of the lysine residue on the catalytic loop of the kinase is a novel mechanism for inhibiting a kinase. Essentially, VopA uses an acetyl group to nullify the charge on the lysine and destabilize interactions with the ␥-phosphate of ATP. The modifications on serine and threonine residues in the active site loop prevent activation of the kinase by phosphorylation, similar to what has been observed before with Yersinia YopJ (9,10). The lysine in the activation loop of the MKK6 is not conserved and, therefore, not thought to be important for the inhibitory activity of the effector (9). Based on the architecture of their catalytic site, VopA and other YopJ-like proteins are predicted to transfer the acetyl group from acetyl-CoA to a protein substrate using a ping-pong mechanism (8). For this mechanism, a covalent acetyl-enzyme intermediate is formed that is attacked by the R group of an amino acid. Supported by the presented experiments, VopA must efficiently coordinate two distinct nucleophiles for attack on the acetylenzyme intermediate: an amine from a lysine residue or a hydroxyl from a serine or threonine. Although the structure/ function studies required to understand the biochemical mechanism used by these enzymes are beyond the scope of this study, from a molecular point of view the mechanism used by this group of enzymes is intriguing and will be interesting to decipher.
VopA, an Ideal Molecular Inhibitor-For decades, scientists have been, and still are, trying to design specific inhibitors that target the nucleotide binding site of kinases. Attempts to make selective inhibitors, such as imatinib, have been focused on targeting the inactive state of a kinase because of its unique fold (15). In their active states many kinases conform to a canonical structure, which makes it difficult to target a specific kinase or even a family of kinases. Natural selection has once again proven to be the champion chemist by creating VopA. This bacterial effector disrupts not only the inactive form of the kinase but the active form as well.
VopA efficiently inhibits the MKKs two ways by targeting different sites on the kinase. First, VopA targets the pharmacological "sweet spot" (i.e. the nucleotide binding site) of a kinase by masking the charge on a lysine residue with an acetyl moiety. This change in the charge and shape of the nucleotide binding pocket of a kinase drastically changes the activity of the enzyme.
The modified kinase is no longer able to bind ATP but is still able to bind ADP. Therefore, acetylated MKKs are covalently locked into an inactive state and are no longer able to modify their substrates. VopA also targets the activation of the MKKs by acetylation of the activation loop on the serine and threonine residues, directly competing with phosphorylation, resulting in a kinase that cannot be activated. Perhaps it is understandable that VopA appears to be a more potent inhibitor of MAPK signaling than YopJ because VopA is able to inactivate MKKs by two mechanisms, whereas YopJ only uses one (11).
Recently another effector, the Yersinia kinase YpkA, has been shown to block the binding of a GTP to G-protein G␣ q . In contrast to VopA that modifies the nucleotide binding site with a neutral acetyl group, YpkA phosphorylates a serine residue found in the proximity of the GTP binding site (16). Phosphorylation of Ser-47 on G␣ q is predicted to reduce its affinity for GTP, thereby inactivating the G-protein. The molecular mechanisms are quite distinct for these two effectors, but the global outcome is the same: they disrupt the binding of the nucleotide to a host enzyme.
Diversity of Acetyltransferase Effector Family-Bacterial effectors, much like viral oncoproteins, target and manipulate host signaling by usurping or mimicking a host activity (4, 5).
The bacterial effectors contain a variety of enzymatic activities that includes hydrolases, phosphatases, GTPase-activating proteins, guanine nucleotide exchange factors, and kinases (4,5,17). Recently, another enzyme, a serine/threonine acetyltransferase, was observed, and now we find lysine acetylation is used to manipulate host signaling 8 -10, 18. Some of these effectors, like VopA, belong to a large family of effectors that are expressed by a variety of pathogens (11,19). These families are arranged by the homology found in their primary amino acid sequence. As with other families of enzymes, the molecular activity of a bacterial effector can be predicted based on the biochemical activity of one or more of the family members. Proving an effector has the predicted activity requires direct biochemical experiments.
Even though the substrates of many eukaryotic enzymes can be predicted because of the conservative nature of signaling pathways throughout evolution, the identification of substrate for bacterial effectors is challenging to say the least. These enzymes have been mutated and molded over time to contain an activity that is custom designed by the pathogen that expresses it. For example, Yersinia YopJ cripples the innate immune response by inhibiting all MAPK pathways and the NFB pathway, whereas Vibrio VopA targets only MAPK pathways, albeit in a more potent manner (7,11). Homologues of these acetyltransferases are expressed by Rhizobium, a plant symbiont that may use this activity to attenuate host-signaling pathways to facilitate a commensal relationship between the bacteria and host (5,7). Plant pathogens seem to be the most aggressive with regard to this family of effectors because some of the bacteria that target plants contain over a half-dozen genes encoding YopJ-like effectors (20).
Modifying a protein with lysine acetylation is a reasonable way to regulate the nucleotide binding to a kinase because lysine acetylation, like phosphorylation, is reversible. We propose that VopA exemplifies an effector that usurped the eukaryotic activity of lysine acetylation. Consistent with this hypothesis is the observation that expression of VopA in yeast suspends cell growth, but the growth suppression can be reversed when expression of VopA is stopped. In addition, these observations support by inference that O-acetylation (serine and threonine acetylation), like N-acetylation (lysine acetylation), will be reversible. Therefore, serine, threonine, and lysine acetylation might be reversible post-translational modifications that are used to modulate the enzymatic activity of eukaryotic signaling machinery.


On a new species and genus in the Cypridini (Crustacea, Ostracoda, Cyprididae) from South Africa, with a phylogenetic analysis of the tribe and a discussion on the genus concept in this group

The present paper describes Mnementh brennei n. sp. n. gen., a new non‐marine ostracod species and genus from temporary wetlands in the Western Cape Province (South Africa). The new genus is the fifth in the subfamily Cypridinae and a phylogenetic analysis using morphological characters demonstrates that it is most closely related to its putative sister taxon Pseudocypris. Apart from this cluster, the phylogenies are not at all robust and this is to all probability due to the incongruent (mosaic) evolution amongst the different characters in valve and soft part modules. Implications of such incongruent evolution of characters for the concept of genera in this group are discussed.

Introduction
Ostracods are small (average length 51 mm), bivalved crustaceans which abound in most types of aquatic and (semi-) terrestrial environments. Up to 80% of all non-marine species are in the family Cyprididae. This family consists of more than 20 subfamilies, and their phylogenetic relationships are highly obscure. Some attempts have been made to deduce the phylogenetic positions of different genera within one subfamily, e.g. the Megalocypridinae (Martens and Coomans 1990) and Scottiinae ), but such analyses are hampered by the lack of a sufficient number of synapomorphic features. This is primarily due to the fact that ostracod morphology is most conservative, especially in soft parts (the limbs). Here, I describe a new genus within the Cypridinae and analyse the phylogenetic position of this new taxon within the subfamily. In addition, I test if the evolution in valve and soft part characters has been congruent, or if both groups of characters have had independent evolutions.

Material
Material for the present paper was collected in September 2001 from a series of temporary water bodies in the surroundings of Van Rhynsdorp (Figure 1), with the assistance of Liz Hoenson (South African Museum, Cape Town). The field trip was provoked by the analysis of ostracod material from that area, offered by Jenny Day and Genevieve Jones (University of Cape Town, Cape Town). Only nine water bodies were sampled during 2 days, but the collection turned out to be very diverse. Apart from the present new genus and species, several other new and rare taxa were found. The giant ostracod, Liocypris grandis, which was presumed extinct, was rediscovered for the first time since its original description by Sars (1924); the presence of additional appendages in the adult female necessitated the erection of a new subfamily, the Liocypridinae (Martens 2003;Matzke-Karasz and Martens 2005). The new genus and species of Cypridopsinae and the new species of Heterocypris and of Megalocypris will be described elsewhere. An overview of the different types of wetlands in the Van Rhynsdorp area is given in Jones and Day (2001).

Phylogenetic analysis
A generic phylogenetic analysis of the tribe Cypridini was performed based on 17 morphological characters (see Appendix 1), using the ostracod genus Chlamydotheca s.l. as outgroup. Chlamydotheca s.l. is thought to be the sister group to the Cypridini. The data matrix (see Appendix 2) was analysed using PAUP 4.06 (Swofford 1998). Input order of taxa was as in the matrix in Appendix 2. All characters were allocated the type ''ordered'' (Wagner parsimony). Trees were built using Maximum Parsimony (MP) with the branch-and-bound routine (furthest taxon input) and with Neighbour Joining (NJ) methods (mean character difference). Bootstrapping used the full heuristic method in MP; for both MP and NJ 10,000 replicates were performed. Testing the (in-)congruence of evolution in soft part and valve characters was done by allocating higher weights to either set of characters and assessing the effect on topology, robustness (bootstrap support) and various descriptive indices (Consistency Index, CI; Retention Index, RI; Homoplasy Index, HI) of the resulting trees.

Derivation of name
Named after the bronze dragon Mnementh, mate to Ramoth, last remaining golden queen dragon on the planet of Pern. I herewith renew my tribute to their creator, Mrs Anne McCaffrey.

Diagnosis
A cypridinid genus, globular and large. RV with frontal and caudal selvage marginally inwardly displaced, and with anterior valve margin not ventrally protruding; ventro-caudal part of calcified inner lamella linearly elevated, not running parallel to valve margin. LV without inwardly displaced selvage and without large inner list.
T1 with penultimate segment divided and with seta d 1 two to three times as long as seta d 2 . Hemipenis with ventral protuberance on the medial shield.

Differential diagnosis
Mnementh n. gen. clearly belongs in the Cypridini, as it has three to five additional ''8''-shaped loops of the post-labyrinthal spermiductus in the hemipenis. It can be distinguished from the other four genera in this tribe by a mixture of features: by the divided penultimate segment of the T2 from Cypris and Pseudocypris; by the sub-marginal selvages in both valves from Cypris, Ramotha, and Globocypris; by the absence of a large inner list on the LV from Pseudocypris; by the fact that seta d 1 is more than twice the length of d 2 from Cypris and Pseudocypris; and by the absence of the additional lateral setae on the T1-palp in the female from Globocypris. The new genus, Mnementh, shows morphological characteristics which classify it in between Pseudocypris and Ramotha, but it also has autapomorphic features, e.g. the additional ventral protuberance on the medial shield of the hemipenis which is thus far unique in the tribe. This ventral protuberance might actually be valid at the specific level only. Only the discovery of a second species in this genus will decide on this issue.

Type material
Holotype: male (OC.2913): soft parts dissected in glycerine in a sealed slide, valves stored dry in a micropalaeontological slide. Allotype: female (OC.2914), dissected and stored as the holotype. Paratypes: two males and one female dissected and stored as the holotype (OC.2915-2917), two males (OC.2920, 2921) and three females (OC.2918, 2919, 2922) stored in toto dry in a micropalaeontological slide after use for SEM; ca 50 males and females stored in toto in 80% alcohol (OC.2923) in one tube, ca 20 males and females stored in toto in 80% alcohol (SAM.A45530).

Repository
Holotype, allotype, dissected and dried paratypes, and one tube with wet paratypes are in the Ostracod Collection of the Royal Belgian Institute of Natural Sciences (Brussels, Belgium). One tube with wet paratypes is in the South African Museum (Cape Town, South Africa).   Sars, 1924.

Derivation of name
The species is named after my friend and colleague, Luc ''brenne'' Brendonck (University of Leuven, Belgium), in recognition of his major contribution to the knowledge of Anostracan biology, especially in southern Africa, and in fond memory of years of friendship and rewarding collaboration.

Diagnosis
Large species, with globular carapace, very hirsute and pitted. Anterior margin of both valves produced towards the ventral side. Anterior calcified inner lamellae wide. Anterior part in dorsal view pointed like a rostrum, greatest width (. half the length) in the middle. Prehensile palps (male) asymmetrical, with terminal segment in right palp larger than in the left palp. T1-palp in female short and wide. Hemipenis with dorsal lobe of the lateral shield large, ventral lobe of the lateral shield long and rectangular, distally produced towards the ventral side. T2 stoutly built. Caudal ramus slender.

Additional description of male
Carapace ( Figure 2N) in dorsal view rounded, with greatest width situated in the middle and being more than half of the length; posterior part rounded, anterior part pointed, in both dorsal and ventral view like a rostrum ( Figure 2K), RV slightly overlapping LV. Ventrally ( Figure 2M), RV overlapping LV in the middle. Valves in external view ( Figure 2E, F) highly arched, greatest height situated slightly behind the middle; anterior margin ventrally produced, posterior margin nearly straight; ventral margin strongly sinuous; valve surface very hirsute, except in the centre, and pitted to varying degrees.
RV ( Figure 2D) with anterior margin ventrally produced, posterior margin broadly rounded, nearly straight; dorsal margin highly arched; ventral margin strongly sinuous in the middle. Calcified part of the inner lamella very wide anteriorly, narrower along the posterior side. Anterior valve margin with clear, but submarginal, selvage ( Figure 2I), antero-ventrally with some traces of inner lists; posterior margin without selvage, but with striking inner list, not running parallel to the valve margin.
LV ( Figure 2C) with outline similar to that of RV. Anterior calcified inner lamella wide, with remnants of inner lists, but no selvage; posterior calcified inner lamella narrower, also with large inner list not running parallel to valve margin.
Central muscle scars with mandibular scars large and with scar pattern of adductor muscles conforming to that of the subfamily. A1 ( Figure 3C) seven-segmented and conform to the subfamily. Terminal segment about as long as wide, all natatory setae long. Aesthetasc Ya less than twice as long as the short seta on this segment. Rome organ minute. A2 ( Figure 3B) with natatory setae reaching well beyond tip of terminal claws; aesthetasc Y short. Apical chaetotaxy ( Figure 3A) with typical sexual dimorphic characters, but with claw G 1 short; seta of y 3 about twice as long as the aesthetasc and seta z3 as long as claw z1 (seta is longer in Cypris).
Md with coxa elongated ( Figure 3E). Md-palp ( Figure 4A) four-segmented. First segment with large respiratory plate and a group of four apical setae: one long and smooth, one short, narrow and smooth (alpha-seta), the latter flanked by one large ''s''seta, set with a double row of setulae, and by one short ''s''-seta. Second segment with two groups of apical setae: an internal group, consisting of a short and hirsute beta-seta, three long and smooth and one long and barbed setae, external group consisting of two long and one shorter setae. Penultimate segment with four dorsal, subapical setae; apically with one narrow and hirsute gamma-seta of intermediate length, two centrally situated smooth setae and three subequal and subapical setae on the internal side. Terminal segment (not shown) with four claw-like and with two or three short and slender setae, all apically inserted.
Mx1 with three endites, a two-segmented palp ( Figure 3D) and a large respiratory plate. Second palp segment about three times as long as basal width. Third endite with two smooth apical claws and with a long and stout lateral seta, apart from the normal apical setae.
Right prehensile palp ( Figure 4D) with elongated basal segment, carrying one apical sensory seta; terminal segment three-dimensionally curved; when flattened in a slide broad, with a narrow apex and with distal margin showing a blunt angle, apically with one broad sensory organ.
Left prehensile palp ( Figure 4E) with basal segment slightly shorter than that of the right palp; terminal segment with broad base, rapidly narrowing towards the tip, the latter bearing a single sensory organ. T2 ( Figure 5B) stout, with seta d 1 more than three times as long as d 2 ; all segments short, wide and hairy. Second segment with one short apical seta. Penultimate segment divided; segment 3A with one subapical seta; segment 3B with one large and one minute apical setae. Fourth segment with one lateral seta and, apart from the apical claw, with one other apical clawlike seta; apical claw short and stout and only in its distal half set with a double row of spines. T3 ( Figure 5A) a cleaning limb with an apical pincer and without further special features. Caudal ramus ( Figure 5D) with ramus narrow and curved, proximally swollen, distally carrying two claws and two setae and with ventral margin serrated with minute setulae. Attachment of the caudal ramus ( Figure 5C) distally split into a short ventral and a longer dorsal branch.  Hemipenis ( Figure 3F, G) with a broadly rounded medial shield, asymmetrically expanded towards the ventral side and with a ventral protuberance; dorsal lobe of the lateral shield symmetrically rounded, ventral lobe of the lateral shield long and distally expanding (but see Figure 3G with pointed vls). Internal anatomy with the normal labyrinth, consisting of the elongated parts ''a'' and ''c'' and the rounded hinge-joint ''b'', followed by the three to five ''8''-shaped coils of the inner spermiductus, situated distally from the labyrinth, the sclerotized semi-circular loop and the various hollow trabecules, leading to the bursa copulatrix.

Additional description of female
Valves (Figure 2A, B, G, H, J) basically as in the male, but in general somewhat larger and even higher. Carapace in dorsal and ventral view ( Figure 2K, M) with greatest width situated in the middle; RV frontally and ventrally overlapping LV. A2 ( Figure 3A) basically as in the male, apart from the normal sexual dimorphism in the apical chaetotaxy. T1 ( Figure 4C) with palp undivided, carrying the normal unequal three apical setae and no lateral setae.
T2 somewhat plumper and heavier than in the male. Genital lobe undivided and without specific characteristics. Ovaria on both sides curved upwards.
For measurements see Table I.

Phylogenetic analysis
When all characters are given equal weight (51), the Maximum Parsimony (MP) method can only partly resolve the trees. In addition, in both MP and distance methods, bootstrap values supporting the Cypris/Globocypris and the Pseudocypris/Mnementh clusters are very low ( Figure 6A: 53 and 63, respectively). Allocating higher weight to the valve characters offers strong bootstrap support for the Cypris/Globocypris clade (100), but the Pseudocypris/Mnementh cluster remains only weakly supported ( Figure 6B: 60). Allocating higher weight to the soft part features dramatically changes the tree topology, with Cypris now clustering with Pseudocypris (bootstrap 580) instead of with Globocypris, which now becomes a basal taxon. In this ''weighted soft part'' tree, nodes have high bootstrap support in both MP and distance methods  ( Figure 6C). CI, HI and RI are lowest in the analyses with equal weight for all characters, they score better in the ''weighted valve'' trees and are best in the ''weighted soft part'' tree (Table II).

Taxonomic history of Cypridinae
The taxonomic history of the Cypridinae is confused. Hartmann and Puri (1974) Wouters (1983) created Tanganyikacypridinae for the genus Tanganyikacypris. This subfamily was later lowered to the rank of tribe and lodged in the Megalocypridinae by Wouters et al. (1989). Martens (1990Martens ( , 1992 grouped four genera in the nominal tribe Cypridini: Cypris, Pseudocypris, Globocypris Klie, and a new genus Ramotha Martens. The latter genus comprised a number of large African species, previously referred to either Cypris, Eucypris, or Strandesia, i.e. to no less than three different subfamilies within the Cyprididae. This illustrates the taxonomic confusion in cypridid ostracods, even amongst relatively large species. Matzke-Karasz and Martens (2007) meanwhile discovered additional appendages in females of Afrocypris barnardi, much like in Liocypris grandis and therefore transferred Afrocypris to the Liocyprinidinae. The positions of the other genera presently lodged within the Cypridinae (Riocypris, Afrocypris, Chlamydotheca, and Bennelongia) are still largely uncertain.
Position of Mnementh n. gen.
In all of the phylogenetic analyses performed here, Mnementh n. gen. clusters closest to Pseudocypris and, to a lesser extent, to Ramotha. It seems fairly clear that Mnementh and Pseudocypris are sister-taxa, but their mutual relationship to Ramotha remains less clear. The weighted soft part tree indicates that Mnementh n. gen. is in fact derived from a lineage intermediate to those of Ramotha and Pseudocypris. But in the weighted valve tree, the three genera form a separate clade (albeit without support), while in the general tree Ramotha is situated basally to the four other genera in the subfamily. Apart from the seemingly strong association between Pseudocypris and Mnementh n. gen., not much can be deduced at present regarding the phylogenetic position of the genera in the Cypridinae.

Morphological mosaic evolution in Cypridinae?
Of the two ''weighted'' analyses, the valve tree is closest in topology to the general tree, and it thus seems that the phylogenetic reconstruction is most influenced by valve characters, such as presence or absence of selvages and inner lists. The topology of the resulting tree in the weighted soft part analysis is different, with Cypris being closest to Pseudocypris and Globocypris being allocated a basal position within the clade. Characters used here are mostly associated with the stout walking leg (T2), where the penultimate segment can be fused or divided and the setae d 1 and d 2 can have different relative lengths. Such characters are also used in other subfamilies of the Cyprididae (e.g. the Eucypridinae) and have demonstrated their merit to characterize genera. Apparently, character evolution has not been congruent, and this high level of uncertainty in the dataset is expressed by the weak support of the tree resulting from the general analyses. It basically means that if the evolution of the valves is assumed to have dominated cladogenesis in this group, then several reversals in soft part characters must have occurred.
On the other hand, if it is assumed that soft part characters are more conservative, then it follows that many reversals in valve characters (such as loss and re-appearance of selvages and inner lists) must have occurred during the history of this clade. It has been suggested that soft parts are more conservative than valves in most Cyprididae (Martens 1998), whereas the inverse is true in, for example, Candonidae (Pinto et al. 2005). In this view of character evolution within the Cyprididae, the weighted soft part tree in Figure 6C would best reflect the reality of the evolutionary history of these genera. This is also the most robust tree with the highest CI and RI and the lowest HI. However, apart from this rule of thumb on conservative evolution of soft parts (which remains to be quantitatively demonstrated), there is no criterion that can be used to discriminate between the different topologies in Figure 6.
The difference in topologies between the two ''weighted'' trees, however, unequivocally demonstrates that the valve and soft part ''modules'' (Danielopol et al. 1990) can evolve independently from each other. This in itself does not have to be a surprising result, as previous studies have already shown that different structures in ostracods can have independent developmental programmes. For example, Gonzalez Mozo et al. (1996) showed that the five natatory setae on the A2 in Herpetocypris can evolve independently from each other, while Yin et al. (1996) found that different setae in clones of Limnocythere inopinata react differently to similar environmental changes.

Genus concepts
In this particular case of the Cypridinae, however, the incongruence in evolution between the two sets of characters could have an effect on the question of what constitutes a genus in Ostracoda in general.
Ever since the first attempt to integrate palaeontological and neontological ostracod classifications by Hartmann and Puri (1974), there is a strongly held belief by ostracod taxonomists that new taxa (species, genera, or above) need to have uniting features in both valves and soft parts. Incongruence in the evolution of both modules could force changes in practical ostracod taxonomy. There would be problems in the application of the classic genus concepts.
Two of the classical genus concepts are the empirical concept (practicality) and the phenetic concept (similarity), both widely applied in the first half of the 20th century. The phylogenetic concept (viewed more narrowly as ''cladistic concept'' by Dubois (1988)) adds biological relevance to genera, in that it requires a group of species to be more than morphologically similar and practically applicable; a genus in this view constitutes a monophyletic clade of species. Dubois' synthetic concept (1988) requires a genus to be a genetic, phylogenetic, and ecological unit, which can be identified by a single criterion, that of hybridizability (species belong to the same genus if they can produce viable hybrids).
To test validity of genera, then, one of the following criteria can be used: (1) to rely on morphological similarity of species; (2) to attempt phylogenetic analyses; or (3) to carry out hybridization experiments.
Morphological similarity is applied in the first two concepts and generally lacks biological relevance, as both concepts are prone to produce polyphyletic genera. Dubois' hybridizability concept, intellectually appealing as it may be, is seldom applicable as hybridization experiments on invertebrates are difficult: most species are indeed known from preserved museum specimens only. The phylogenetic concept is best embedded in present-day systematic research, as it requires application of the same phylogenetic techniques used at all taxonomic levels, and using all types of data (morphological, molecular,…). A major problem in this concept is the definition of a general cut-off point that defines genera. In the case of distance-criteria in tree building, for example, does one define genera at 5, 10 or 15% of similarity? This subjective decision caused some taxonomists to abandon all Linnean hierarchical levels altogether and to only work with non-hierarchical clades (see http:// www.ohio.edu/phylocode/ but also Sluys et al. (2004)). However, this phylocode approach also relies on robust phylogenies. If morphological phylogenies are not robust, because of incongruent evolution between different sets of characters, than validity of genera as well as of phylocode clades in such groups can be seriously weakened.
Taking the Cypridini as an example, the possible solutions are: 1. Collapse all genera within this tribe into one, large genus. This is not advisable, as a lot of information would be lost. In the present model group, Cypris, Ramotha, and Pseudocypris are monophyletic clades comprising several species each and this hierarchical clustering would be lost if they are no longer considered separate genera. 2. Decide on priority amongst modules of characters. If soft parts in Cyprididae are indeed (generally) more conservative than valves, then it would be possible to increase robustness of phylogenies by adding more weight to such characters. It is possible to test this assumption by quantitatively comparing the degree of morphological evolution of both character modules at a higher taxonomic level. 3. Improve phylogenies, either by incorporating new morphological characters, or by combining morphology with molecular markers. This seems rather straightforward, but again the problem is that most taxa are represented by preserved museum specimens from which DNA is (thus far) difficult to extract.

Conclusions
The present phylogenetic analysis of a small group of cypridinid ostracods (five genera, ca 30 species) has shown that character evolution might have occurred in a mosaic way, with incongruence between soft part and valve modules. This poses problems for phylogenetic reconstructions of the evolutionary history of the group, but also for modern classification, as at least phylogenetic genus-concepts depend on robust phylogenies.