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Herman P Spaink

Publications and source records attributed to Herman P Spaink.

At least 19 recordsLinked to original sources
PubMed2005

Gene expression profiling of the long-term adaptive response to hypoxia in the gills of adult zebrafish.

Low oxygen levels (hypoxia) play a role in clinical conditions such as stroke, chronic ischemia, and cancer. To better understand these diseases, it is crucial to study the responses of vertebrates to hypoxia. Among vertebrates, some teleosts have developed the ability to adapt to extremely low oxygen levels. We have studied long-term adaptive responses to hypoxia in adult zebrafish. We used zebrafish that survived severe hypoxic conditions for 3 wk and showed adaptive behavioral and phenotypic changes. We used cDNA microarrays to investigate hypoxia-induced changes in expression of 15,532 genes in the respiratory organs (the gills). We have identified 367 differentially expressed genes of which 117 showed hypoxia-induced and 250 hypoxia-reduced expressions. Metabolic depression was indicated by repression of genes in the TCA cycle in the electron transport chain and of genes involved in protein biosynthesis. We observed enhanced expression of the monocarboxylate transporter and of the oxygen transporter myoglobin. The hypoxia-induced group further included the genes for Niemann-Pick C disease and for Wolman disease [lysosomal acid lipase (LAL)]. Both diseases lead to a similar intra- and extracellular accumulation of cholesterol and glycolipids. The Niemann-Pick C protein binds to cholesterol from internal lysosomal membranes and is involved in cholesterol trafficking. LAL is responsible for lysosomal cholesterol degradation. Our data suggest a novel adaptive mechanism to hypoxia, the induction of genes for lysosomal lipid trafficking and degradation. Studying physiological responses to hypoxia in species tolerant for extremely low oxygen levels can help identify novel regulatory genes, which may have important clinical implications.

Adaptation, Physiological↗
PubMed2005

Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation.

Recent studies show that the partitioning of the small GTPase H-Ras in different types of membrane microdomains is dependent on guanosine 5'-triphosphate (GTP)-loading of H-Ras. Detailed knowledge about the in vivo dynamics of this phenomenon is limited. In this report, the effect of the activation of H-Ras on its microdomain localization was studied by single-molecule fluorescence microscopy. Individual human H-Ras molecules fused to the enhanced yellow fluorescent protein (eYFP) were imaged in the dorsal plasma membrane of live mouse cells and their diffusion behavior was analyzed. The diffusion of a constitutively inactive (S17N) and constitutively active (G12V) mutant of H-Ras was compared. Detailed analysis revealed that for both mutants a major, fast-diffusing population and a minor, slow-diffusing population were present. The slow-diffusing fraction of the active mutant was confined to 200 nm domains, which were not observed for the inactive mutant. In line with these results we observed that the slow-diffusing fraction of wild-type H-Ras became confined to 200 nm domains upon insulin-induced activation of wild-type H-Ras. This activation-dependent localization of H-Ras to 200 nm domains, for the first time directly detected in live cells, supports the proposed relationship between H-Ras microdomain localization and activation.

Animals↗
PubMed2005

Genomic annotation and expression analysis of the zebrafish Rho small GTPase family during development and bacterial infection.

The zebrafish genomic sequence database was analyzed for the presence of genes encoding members of the Rho small GTPases. The analysis shows the presence of 32 zebrafish Rho genes representing one or more homologs of the human RHOA, RND3, RHOF, RHOG, RHOH, RHOJ, RHOU, RHOV, CDC42, RAC1, RAC2, RAC3, RND1, RHOBTB1, RHOBTB2, RHOBTB3, and RHOT1 genes. By expression analysis using reverse transcriptase-PCR we show that at least 20 of the predicted zebrafish small GTPase genes are expressed in the adult stage. Interestingly, only 5 of these were found to be expressed at early embryonic stages, including rhoab, rhoad, cdc42a, cdc42c, and rac1a. We observed a strong upregulation of zebrafish rhogb expression after Mycobacterium marinum infection of adult fish. This complete annotation study provides a firm basis for the use of zebrafish as a model for analysis of Rho GTPase function in vertebrate development and the innate immune system.

Amino Acid Sequence↗
PubMed2005

Programmed cell death during the transition from multicellular structures to globular embryos in barley androgenesis.

Androgenesis represents one of the most fascinating examples of cell differentiation in plants. In barley, the conversion of stressed uninucleate microspores into embryo-like structures is highly efficient. One of the bottlenecks in this process is the successful release of embryo-like structures out of the exine wall of microspores. In the present work, morphological and biochemical studies were performed during the transition from multicellular structures to globular embryos. Exine wall rupture and subsequent globular embryo formation were observed only in microspores that divided asymmetrically. Independent divisions of the generative and the vegetative nuclei gave rise to heterogeneous multicellular structures, which were composed of two different cellular domains: small cells with condensed chromatin structure and large cells with normal chromatin structure. During exine wall rupture, the small cells died and their death marked the site of exine wall rupture. Cell death in the small cell domain showed typical features of plant programmed cell death. Chromatin condensation and DNA degradation preceded cell detachment and cytoplasm dismantling, a process that was characterized by the formation of vesicles and vacuoles that contained cytoplasmic material. This morphotype of programmed cell death was accompanied by an increase in the activity of caspase-3-like proteases. The orchestration of such a death program culminated in the elimination of the small generative domain, and further embryogenesis was carried out by the large vegetative domain. To date, this is the first report to show evidence that programmed cell death takes part in the development of microspore-derived embryos.

Apoptosis↗
PubMed2005

Transcriptome profiling of adult zebrafish at the late stage of chronic tuberculosis due to Mycobacterium marinum infection.

The Mycobacterium marinum-zebrafish infection model was used in this study for analysis of a host transcriptome response to mycobacterium infection at the organismal level. RNA isolated from adult zebrafish that showed typical signs of fish tuberculosis due to a chronic progressive infection with M. marinum was compared with RNA from healthy fish in microarray analyses. Spotted oligonucleotide sets (designed by Sigma-Compugen and MWG) and Affymetrix GeneChips were used, in total comprising 45,465 zebrafish transcript annotations. Based on a detailed comparative analysis and quantitative reverse transcriptase-PCR analysis, we present a validated reference set of 159 genes whose regulation is strongly affected by mycobacterial infection in the three types of microarrays analyzed. Furthermore, we analyzed the separate datasets of the microarrays with special emphasis on the expression profiles of immune-related genes. Upregulated genes include many known components of the inflammatory response and several genes that have previously been implicated in the response to mycobacterial infections in cell cultures of other organisms. Different marker genes of the myeloid lineage that have been characterized in zebrafish also showed increased expression. Furthermore, the zebrafish homologs of many signal transduction genes with relationship to the immune response were induced by M. marinum infection. Future functional analysis of these genes may contribute to understanding the mechanisms of mycobacterial pathogenesis. Since a large group of genes linked to immune responses did not show altered expression in the infected animals, these results suggest specific responses in mycobacterium-induced disease.

Animals↗
PubMed2005

Analysis of promoter activity of the early nodulin Enod40 in Lotus japonicus.

Our comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a stringent regulation of the Enod40 promoter in the root cortex and root hairs in response to Nod factors. Interestingly, the L. japonicus Enod40-2 promoter fragment also shows symbiotic activity in the reverse orientation. Deletion analyses of the Glycine max (Gm) Enod40 promoter revealed the presence of a minimal region -185 bp upstream of the transcription start. Stable transgenic L. japonicus reporter lines were used in bioassays to test the effect of different compounds on early symbiotic signaling. The responses of prGmEnod40 reporter lines were compared with the responses of L. japonicus (Lj) reporter lines based on the LjNin promoter. Both reporter lines show very early activity postinoculation in root hairs of the responsive zone of the root and later in the dividing cells of nodule primordia. The LjNin promoter was found to be more responsive than the GmEnod40 promoter to Nod factors and related compounds. The use of prGmEnod40 reporter lines to analyze the effect of nodulin genes on the GmEnod40 promoter activity indicates that LJNIN has a positive effect on the regulation of the Enod40 promoter, whereas the latter is not influenced by ectopic overexpression of its own gene product. In addition to pointing to a difference in the regulation of the two nodulin genes Enod40 and Nin during early time points of symbiosis, the bioassays revealed a difference in the response to the synthetic cytokinin 6-benzylaminopurine (BAP) between alfalfa and clover and L. japonicus. In alfalfa and clover, Enod40 expression was induced upon BAP treatment, whereas this seems not to be the case in L. japonicus; these results correlate with effects at the cellular level because BAP can induce pseudonodules in alfalfa and clover but not in L. japonicus. In conclusion, we demonstrate the applicability of the described L. japonicus reporter lines in analyses of the specificity of compounds related to nodulation as well as for the dissection of the interplay between different nodulin genes.

Gene Expression Regulation, Plant↗
PubMed2004

Time-lapse tracking of barley androgenesis reveals position-determined cell death within pro-embryos.

Following abiotic stress to induce barley (Hordeum vulgare L.) androgenesis, the development of 794 enlarged microspores in culture was monitored by time-lapse tracking. In total, 11% of the microspores tracked developed into embryo-like structures (type-I pathway), 36% formed multicellular structures (type-II pathway) and 53% of the microspores followed gametophytic divisions, accumulated starch and died in the first days of tracking (type-III pathway). Despite the microspore fate, enlarged microspores showed similar morphologies directly after stress treatment. Ultrastructural analysis, however, revealed two morphologically distinct cell types. Cells with a thin intine layer and an undifferentiated cytoplasm after stress treatment were associated with type-I and type-II pathways, whereas the presence of differentiated amyloplasts and a thick intine layer were associated with the type-III pathway. Tracking revealed that the first morphological change associated with embryogenic potential was a star-like morphology, which was a transitory stage between uninucleate vacuolated microspores after stress and the initiation of cell division. The difference between type-I and type-II pathways was observed during the time they displayed the star-like morphology. During the transition phase, embryo-like structures in the type-I pathway were always released out of the exine wall at the opposite side of the pollen germ pore, whereas in the type-II pathway multicellular structures were unable to break the exine and to release embryo-like structures. Moreover, by combining viability studies with cell tracking, we show that release of embryo-like structures was preceded by a decrease in viability of the cells positioned at the site of exine wall rupture. These cells were also positively stained by Sytox orange, a cell death indicator. Thereby, we demonstrate, for the first time, that a position-determined cell death process marks the transition from a multicellular structure into an embryo-like structure during barley androgenesis.

Cell Death↗
PubMed2004

DNA computing using single-molecule hybridization detection.

DNA computing aims at using nucleic acids for computing. Since micromolar DNA solutions can act as billions of parallel nanoprocessors, DNA computers can in theory solve optimization problems that require vast search spaces. However, the actual parallelism currently being achieved is at least a hundred million-fold lower than the number of DNA molecules used. This is due to the quantity of DNA molecules of one species that is required to produce a detectable output to the computations. In order to miniaturize the computation and considerably reduce the amount of DNA needed, we have combined DNA computing with single-molecule detection. Reliable hybridization detection was achieved at the level of single DNA molecules with fluorescence cross-correlation spectroscopy. To illustrate the use of this approach, we implemented a DNA-based computation and solved a 4-variable 4-clause instance of the computationally hard Satisfiability (SAT) problem.

Algorithms↗
PubMed2004

Isoform-specific differences in rapid nucleocytoplasmic shuttling cause distinct subcellular distributions of 14-3-3 sigma and 14-3-3 zeta.

Nucleocytoplasmic transport of proteins plays an important role in the regulation of many cellular processes. Differences in nucleocytoplasmic shuttling can provide a basis for isoform-specific biological functions for members of multigene families, like the 14-3-3 protein family. Many organisms contain multiple 14-3-3 isoforms, which play a role in numerous processes, including signalling, cell cycle control and apoptosis. It is still unclear whether these isoforms have specialised biological functions and whether this specialisation is based on isoform-specific ligand binding, expression regulation or specific localisation. Therefore, we studied the subcellular distribution of 14-3-3 sigma and 14-3-3 zeta in vivo in various mammalian cell types using yellow fluorescent protein fusions and isoform-specific antibodies. 14-3-3 sigma was mainly localised in the cytoplasm and only low levels were present in the nucleus, whereas 14-3-3 zeta was found at relatively higher levels in the nucleus. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the 14-3-3 proteins rapidly shuttle in and out of the nucleus through active transport and that the distinct subcellular distributions of 14-3-3 sigma and 14-3-3 zeta are caused by differences in nuclear export. 14-3-3 sigma had a 1.7x higher nuclear export rate constant than 14-3-3 zeta, while import rate constants were equal. The 14-3-3 proteins are exported from the nucleus at least in part by a Crm1-dependent, leptomycin B-sensitive mechanism. The differences in subcellular distribution of 14-3-3 that we found in this study are likely to reflect a molecular basis for isoform-specific biological specialisation.

14-3-3 Proteins↗
PubMed2004

Different subcellular localization and trafficking properties of KNOX class 1 homeodomain proteins from rice.

Genes of the KN1-like homeobox (KNOX) class 1 encode transcription factors involved in shoot apical meristem development and maintenance. We studied the subcellular localization of Green Fluorescent Protein-tagged rice KNOX proteins (Oskn1-3) after particle bombardment of onion and rice cells and after transformation of Arabidopsis and rice with constitutive and inducible expression constructs. In all test systems, the three rice KNOX proteins showed nuclear and cytoplasmic localization patterns. However, Oskn1 additionally showed in some cells a distribution over punctae moving randomly in the cytosol. Use of an inducible expression system indicated a nuclear presence of Oskn1 in cells of the shoot apical meristem and post-transcriptional down-regulation in early leaf primordia. Arabidopsis and rice test systems were used to study effects of plant hormones and auxin transport inhibition on KNOX protein localization. Application of GA3 or 1-NAA shifted protein localization completely to the cytoplasm and resulted in loss of the punctae formed by Oskn1. Conversely, NPA application induced a complete nuclear localization of the KNOX proteins. To study intercellular movement of the KNOX proteins we set up a novel co-bombardment assay in which trafficking of untagged KNOX proteins was visualized through the co-trafficking of green fluorescent or blue fluorescent marker proteins. In multiple independent experiments Oskn1 trafficked more extensively to neighboring cells than Oskn2 and Oskn3. Differences in the localization and trafficking properties of Oskn1, Oskn2 and Oskn3 correlate with differences in mRNA localization patterns and functional differences between the rice KNOX genes and their putative orthologues from other species.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2004

Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish.

The zebrafish genomic sequence database was analysed for the presence of genes encoding members of the Toll-like receptors (TLR) and interleukin receptors (IL-R) and associated adaptor proteins containing a TIR domain. The resulting predictions show the presence of one or more counterparts for the human TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, IL-1R and IL-18R genes and one copy of the adaptor genes MyD88, MAL, TRIF and SARM. In contrast to data for the pufferfish Fugu rubripes, zebrafish has two genes that are highly similar to human TLR4. In addition, one fish-specific TLR group can be distinguished that is closely related to the Drosophila melanogaster Toll-9 gene. The sequence of cloned cDNAs for TLR4, TLR2 and MyD88 show the same intron-exon organisation as in the human counterparts. Expression analysis using reverse transcriptase-PCR (RT-PCR) shows that 17 of the predicted zebrafish TLR genes and all the genes encoding adaptor proteins are expressed in the adult stage. A subset of the TLR genes are expressed at higher levels in fish infected with the pathogen Mycobacterium marinum. The induced genes include the homologues of the human TLR1 and TLR2 genes, whose functions are associated with mycobacterial infections, underscoring the suitability of zebrafish as a model for analysis of the vertebrate innate immune system.

Animals↗
PubMed2004

Single-molecule imaging of the H-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane.

In the last decade evidence has accumulated that small domains of 50-700 nm in diameter are located in the exoplasmic leaflet of the plasma membrane. Most of these domains supposedly consist of specific sets of lipids and proteins, and are believed to coordinate signal transduction cascades. Whether similar domains are also present in the cytoplasmic leaflet of the plasma membrane is unclear so far. To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein. Using single-molecule fluorescence microscopy, trajectories of individual molecules diffusing in the cytoplasmic leaflet of the plasma membrane were recorded. From these trajectories, the diffusion of individual membrane-anchored enhanced yellow fluorescent protein molecules was studied in live cells on timescales from 5 to 200 ms. The results show that the diffusion of 30-40% of the molecules is constrained in domains with a typical size of 200 nm. Neither breakdown of actin nor cholesterol extraction changed the domain characteristics significantly, indicating that the observed domains may not be related to the membrane domains identified so far.

3T3 Cells↗
PubMed2004

Has2 is required upstream of Rac1 to govern dorsal migration of lateral cells during zebrafish gastrulation.

The large extracellular polysaccharide Hyaluronan (HA) and its synthesizing enzymes (Has) have been implicated in regulating the migratory potential of metastatic cancer cells. Here, we analyze the roles of zebrafish Has2 in normal development. Antisense morpholino oligonucleotide (MO)-mediated knockdown of zebrafish Has2 leads to the loss of HA, and severe migratory defects during gastrulation, somite morphogenesis and primordial germ cell migration. During gastrulation, ventrolateral cells of has2 morphant embryos fail to develop lamellipodia and to migrate dorsally, resulting in a blockage of dorsal convergence, whereas extension of the dorsal axis is normal. The effect is cell autonomous, suggesting that HA acts as an autocrine signal to stimulate the migration of HA-generating cells. Upon ectopic expression in axial cells, has2 causes the formation of supernumerary lamellipodia and a blockage of axis extension. Epistasis analyses with constitutively active and dominant-negative versions of the small GTPase Rac1 suggest that HA acts by Rac1 activation, rather than as an essential structural component of the extracellular matrix. Together, our data provide evidence that convergence and extension are separate morphogenetic movements of gastrulation. In addition, they suggest that the same HA pathways are active to auto-stimulate cell migration during tumor invasion and vertebrate embryogenesis.

Animals↗
PubMed2003

Structural motifs in the RNA encoded by the early nodulation gene enod40 of soybean.

The plant gene enod40 is highly conserved among legumes and also present in various non-legume species. It is presumed to play a central regulatory role in the Rhizobium-legume interaction, being expressed well before the initiation of cortical cell divisions resulting in nodule formation. Two small peptides encoded by enod40 mRNA as well as its secondary structure have been shown to be key elements in the signalling processes underlying nodule organogenesis. Here results concerning the secondary structure of mRNA of enod40 in soybean are presented. This study combined a theoretical approach, involving structure prediction and comparison, as well as structure probing. Our study indicates five conserved domains in enod40 mRNA among numerous leguminous species. Structure comparison suggests that some domains are also conserved in non-leguminous species and that an additional domain exists that was found only in leguminous species developing indeterminate nodules. Enzymatic and chemical probing data support the structure for three of the domains, and partially for the remaining two. The rest of the molecule appears to be less structured. Some of the domains include motifs, such as U-containing internal loops and bulges, which seem to be conserved. Therefore, they might be involved in the regulatory role of enod40 RNA.

Base Sequence↗
PubMed2003

Specific activation of ERK pathways by chitin oligosaccharides in embryonic zebrafish cell lines.

Chitin oligosaccharides (COs) play a role in plant development and are presumed to affect body plan formation during vertebrate embryogenesis. The mechanisms of COs recognition and cellular processes underlying embryonic development are still not understood. We analyze the possible link with the mitogen-activated protein kinase pathway that is conserved in evolution through the plant and animal kingdom and has been implicated in diverse cellular processes, including cell growth, proliferation, differentiation, survival, and vertebrate development. We show that in vivo stimulation of embryonic zebrafish cells ZF13 and ZF29 with chitin tetrasaccharides at 10-9 M concentration transiently induced activation/phosphorylation of extracellular regulated kinases (ERKs), with a maximum after 15 min. Furthermore the biological specificity of chitin tetrasaccharides and various derivatives was examined. The replacement of one or two GlcNAc residues of the chitin backbone by glucose and fucosylation of chitin tetrasaccharides at the reducing terminus caused a complete loss of their activity. We also tested a chitin tetrasaccharide analogue in which the oxygen atoms in glycosidic linkages were replaced by sulfur atoms. This analog, which could not be enzymatically hydrolyzed, was as potent an inducer as chitin tetrasaccharide. These results suggest that the observed activation of ERKs is chitin tetrasaccharide-specific and does not require further enzymatic processing. We examined possible signaling pathways leading to ERK activation by COs by use of phosphospecific antibodies and inhibitors. We conclude that a high-affinity CO receptor system exists that links to the Raf, MEK, and ERK pathway in zebrafish cells.

Animals↗
PubMed2003

Auxin distribution in Lotus japonicus during root nodule development.

For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.

Alphaproteobacteria↗
PubMed2003

14-3-3 isoforms and pattern formation during barley microspore embryogenesis.

The members of the 14-3-3 isoform family have been shown to be developmentally regulated during animal embryogenesis, where they take part in cell differentiation processes. 14-3-3 isoform-specific expression patterns were studied in plant embryogenic processes, using barley (Hordeum vulgare L.) microspore embryogenesis as a model system. After embryogenesis induction by stress, microspores with enlarged morphology showed higher viability than non-enlarged ones. Following microspore culture, cell division was only observed among the enlarged microspores. Western blot and immunolocalization of three barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C were carried out using isoform-specific antibodies. The level of 14-3-3C protein was higher in enlarged microspores than in non-enlarged ones. A processed form of 14-3-3A was associated with the death pathway of the non-enlarged microspores. In the early embryogenesis stage, 14-3-3 subcellular localization differed among dividing and non-dividing microspores and the microspore-derived multicellular structures showed a polarized expression pattern of 14-3-3C and a higher 14-3-3A signal in epidermis primordia. In the late embryogenesis stage, 14-3-3C was specifically expressed underneath the L(1) layer of the shoot apical meristem and in the scutellum of embryo-like structures (ELSs). 14-3-3C was also expressed in the scutellum and underneath the L(1) layer of the shoot apical meristem of 21 d after pollination (DAP) zygotic embryos. These results reveal that 14-3-3A processing and 14-3-3C isoform tissue-specific expression are closely related to cell fate and initiation of specific cell type differentiation, providing a new insight into the study of 14-3-3 proteins in plant embryogenesis.

14-3-3 Proteins↗