Genetic dissection of root formation in maize (Zea mays) reveals root-type specific developmental programmes

BACKGROUND: Maize (Zea mays) forms a complex root system comprising embryonic and post-embryonic roots. The embryonically formed root system is made up of the primary root and a variable number of seminal roots. Later in development the post-embryonic shoot-borne root system becomes dominant and is responsible together with its lateral roots for the major portion of water and nutrient uptake. Although the anatomical structure of the different root-types is very similar they are initiated from different tissues during embryonic and post-embryonic development. Recently, a number of mutants specifically affected in maize root development have been identified. These mutants indicate that various root-type specific developmental programmes are involved in the establishment of the maize root stock. SCOPE: This review summarizes these genetic data in the context of the maize root morphology and anatomy and gives an outlook on possible perspectives of the molecular analysis of maize root formation.     

Paternal origin of FGFR3 mutations in Muenke-type craniosynostosis

Muenke syndrome, also known as FGFR3-associated coronal synostosis, is defined molecularly by the presence of a heterozygous nucleotide transversion, c.749C>G, encoding the amino acid substitution Pro250Arg, in the fibroblast growth factor receptor type 3 gene (FGFR3). This frequently occurs as a new mutation, manifesting one of the highest documented rates for any transversion in the human genome. To understand the biology of this mutation, we have investigated its parental origin, and the ages of the parents, in 19 families with de novo c.749C>G mutations. All ten informative cases originated from the paternal allele (95% confidence interval 74–100% paternal); the average paternal age at birth overall was 34.7 years. An exclusive paternal origin of mutations, and increased paternal age, were previously described for a different mutation (c.1138G>A) of the FGFR3 gene causing achondroplasia, as well as for mutations of the related FGFR2 gene causing Apert, Crouzon and Pfeiffer syndromes. We conclude that similar biological processes are likely to shape the occurrence of this c.749C>G mutation as for other mutations of FGFR3 as well as FGFR2.S.V. Rannan-Eliya and I.B. Taylor contributed equally to this work.     

CD4<Superscript>+</Superscript> T cells kill Id<Superscript>+</Superscript> B-lymphoma cells: FasLigand-Fas interaction is dominant in vitro but is redundant in vivo

B-lymphoma cells express a highly tumor-specific antigen, monoclonal Ig, which is a promising target for immunotherapy. Previous work has demonstrated that B-lymphoma cells spontaneously process their endogenous monoclonal Ig and present variable (V) region peptides (Id-peptides) on their MHC class II molecules to CD4⁺ T cells. Id-specific CD4⁺ T cells protect mice against B-lymphoma cells in the absence of anti-idiotypic antibodies. The molecular mechanism by which Id-specific CD4⁺ T cells kill B-lymphoma cells is hitherto unknown. We here demonstrate in an Id-specific T-cell receptor (TCR)–transgenic mouse model that Id-specific CD4⁺ T cells induce apoptosis of Fas⁺ B-lymphoma cells in vitro by FasLigand (FasL)–Fas interaction. Moreover, the rare B lymphomas that had escaped rejection in TCR-transgenic mice had down-regulated their sensitivity to Fas-mediated apoptosis. Although these results suggest that FasL-Fas interaction is important, Id-specific CD4⁺ T cells could eliminate Id⁺ B-lymphoma cells in vivo by other mechanisms, since three independent ways of blocking FasL-Fas–mediated killing failed to abrogate tumor protection in TCR-transgenic mice. These results suggest that there are several redundant pathways by which Id-specific CD4⁺ T cells eliminate Id⁺ B-lymphoma cells in vivo, of which FasL-Fas interaction is only one.Supported by grants from the Norwegian Cancer Society, the Research Council of Norway, and the Multiple Myeloma Research Foundation.     

Inhibition of basal and interleukin-1-induced VCAM-1 expression by phospholipid hydroperoxide glutathione peroxidase and 15-lipoxygenase in rabbit aortic smooth muscle cells

Cytokines or hydroperoxides upregulate cell adhesion molecules (CAM) in early stages of atherosclerosis. VCAM-1 expression was therefore investigated in rabbit aortic smooth muscle cells (SMC) stably transfected either with phospholipid hydroperoxide glutathione peroxidase (PHGPx; SMCPHGPx) as a hydroperoxide-reducing enzyme or with 15-lipoxygenase (15-LOX; SMCLOX) as a hydroperoxide-producing enzyme. Transfected cells showed up to 3-fold enhanced PHGPx and a marked LOX activity, respectively, that was absent in controls. Intracellular hydroperoxides were 6-fold higher in SMCLOX than in SMC or SMCPHGPx. Intracellular protein thiols were decreased by 50 and 90% in SMCPHGPx and SMCLOX, respectively. Glutathione mixed disulfides were tentatively increased from SMC via SMCPHGPx to SMCLOX, accordingly. Thiol reduction with tris(2-carboxyethyl)phosphine completely restored protein thiols in SMCPHGPx, whereas in SMCLOX only 60% of control values were recovered. Basal VCAM-1 mRNA levels were decreased by 50% in SMCPHGPx and 75% in SMCLOX. VCAM-1-inducibility was abrogated in SMCLOX but not in SMCPHGPx. Accordingly, NFkappaB-driven reporter gene activation by IL-1 was unaffected in SMCPHGPx but abolished in SMCLOX. The data confirm that PHGPx overexpression dampens CAM expression either by lowering stimulatory hydroperoxides or by using hydroperoxides for protein modification. But hydroperoxides, when constitutively overproduced as in SMCLOX, inhibit CAM expression and render cells refractory to IL-1 stimulation likely due to oxidation of protein thiols of the signaling system.     

A novel transmembrane MSP-containing protein that plays a role in right ventricle development

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.     

Interferon-alpha inhibits interleukin-3-induced proliferation of Ba/F3 cells in a protein kinase R-dependent manner

We have previously shown that interferon-alpha (IFN-alpha) inhibits proliferation of Ba/F3 cells by interfering with the action of the mitogen interleukin-3 (IL-3) [Cell Signal 11 (1999) 769]. Here, we have characterised the role of protein kinase R (PKR), an IFN-alpha-inducible enzyme, in the mediation of IL-3-antagonistic IFN-alpha effects. Downregulation of PKR expression by antisense oligonucleotide treatment blocked IFN-alpha-induced growth inhibition. Reduction of PKR levels and overexpression of a dominant-negative PKR mutant correlated with diminished inhibitory IFN-alpha effects on the IL-3-dependent expression of a luciferase reporter construct, GAS-luc. Furthermore, increased nuclear levels of STAT1 (bound in ISGF3 complexes) were observed in PKR-depleted cells cultured with or without IFN-alpha. Together, our data indicate an essential role of PKR in the mediation of IL-3-antagonistic IFN-alpha effects on Ba/F3 cells. They also suggests that activation of STAT1, an essential mediator of IFN effects, is insufficient for growth inhibition if PKR is not expressed.     

Tumor attenuation by 2(6-hydroxynaphthyl)-beta-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products

We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide-dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70-97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.     

Expression, Isolation, and Crystallization of the Catalytic Domain of CopB, a Putative Copper Transporting ATPase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.