Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice

Inheritance of an inactivated form of the VHL tumor suppressor gene predisposes patients to develop von Hippel–Lindau disease, and somatic VHL inactivation is an early genetic event leading to the development of sporadic renal cell carcinoma. The VHL gene was disrupted by targeted homologous recombination in murine embryonic stem cells, and a mouse line containing an inactivated VHL allele was generated. While heterozygous VHL (+/−) mice appeared phenotypically normal, VHL −/− mice died in utero at 10.5 to 12.5 days of gestation (E10.5 to E12.5). Homozygous VHL −/− embryos appeared to develop normally until E9.5 to E10.5, when placental dysgenesis developed. Embryonic vasculogenesis of the placenta failed to occur in VHL −/− mice, and hemorrhagic lesions developed in the placenta. Subsequent hemorrhage in VHL −/− embryos caused necrosis and death. These results indicate that VHL expression is critical for normal extraembryonic vascular development.

Vitamin A in serum is a survival factor for fibroblasts

Murine 3T3 cells arrest in a quiescent, nondividing state when transferred into medium containing little or no serum. Within the first day after transfer, fibroblasts can be activated to proliferate by platelet-derived growth factor (PDGF) alone; cells starved longer than 1 day, however, are activated only by serum. We demonstrate that endogenous vitamin A (retinol) or retinol supplied by serum prevents cell death and that retinol, in combination with PDGF, can fully replace serum in activating cells starved longer than 1 day. The physiological retinol derivative 14-hydroxy-4,14-retro-retinol, but not retinoic acid, can replace retinol in rescuing or activating 3T3 cells. Anhydroretinol, another physiological retinol metabolite that acts as a competitive antagonist of retinol, blocks cell activation by serum, indicating that retinol is a necessary component of serum. It previously has been proposed that activation of 3T3 cells requires two factors in serum, an activation factor shown to be PDGF and an unidentified survival factor. We report that retinol is the survival factor in serum.

Probing the assembly of transcription initiation complexes through changes in σN protease sensitivity

The alternative bacterial σN RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins. Little is known about how sigma factors respond to their ligands or how the responses lead to transcription. To examine the liganded state of σN, the assembly of end-labeled Klebsiella pneumoniae σN into holoenzyme, closed promoter complexes, and initiated transcription complexes was analyzed by enzymatic protein footprinting. V8 protease-sensitive sites in free σN were identified in the acidic region II and bordering or within the minimal DNA binding domain. Interaction with core RNA polymerase prevented cleavage at noncontiguous sites in region II and at some DNA binding domain sites, probably resulting from conformational changes. Formation of closed complexes resulted in further protections within the DNA binding domain, suggesting close contact to promoter DNA. Interestingly, residue E36 becomes sensitive to proteolysis in initiated transcription complexes, indicating a conformational change in holoenzyme during initiation. Residue E36 is located adjacent to an element involved in nucleating strand separation and in inhibiting polymerase activity in the absence of activation. The sensitivity of E36 may reflect one or both of these functions. Changing patterns of protease sensitivity strongly indicate that σN can adjust conformation upon interaction with ligands, a property likely important in the dynamics of the protein during transcription initiation.

Two domains within σN (σ54) cooperate for DNA binding

The σ-N (σN) subunit of the bacterial RNA polymerase is a sequence specific DNA-binding protein. The RNA polymerase holoenzyme formed with σN binds to promoters in an inactive form and only initiates transcription when activated by enhancer-binding positive control proteins. We now provide evidence to show that the DNA-binding activity of σN involves two distinct domains: a C-terminal DNA-binding domain that directly contacts DNA and an adjacent domain that enhances DNA-binding activity. The sequences required for the enhancement of DNA binding can be separated from the sequences required for core RNA polymerase binding. These results provide strong evidence for communication between domains within a transcription factor, likely to be important for the function of σN in enhancer-dependent transcription.

Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy

To formally test the hypothesis that the granulocyte/macrophage colony-forming unit (GM-CFU) cells can contribute to early hematopoietic reconstitution immediately after transplant, the frequency of genetically modified GM-CFU after retroviral vector transduction was measured by a quantitative in situ polymerase chain reaction (PCR), which is specific for the multidrug resistance-1 (MDR-1) vector, and by a quantitative GM-CFU methylcellulose plating assay. The results of this analysis showed no difference between the transduction frequency in the products of two different transduction protocols: “suspension transduction” and “stromal growth factor transduction.” However, when an analysis of the frequency of cells positive for the retroviral MDR-1 vector posttransplantation was carried out, 0 of 10 patients transplanted with cells transduced by the suspension method were positive for the vector MDR-1 posttransplant, whereas 5 of 8 patients transplanted with the cells transduced by the stromal growth factor method were positive for the MDR-1 vector transcription unit by in situ or in solution PCR assay (a difference that is significant at the P = 0.0065 level by the Fisher exact test). These data suggest that only very small subsets of the GM-CFU fraction of myeloid cells, if any, contribute to the repopulation of the hematopoietic tissues that occurs following intensive systemic therapy and transplantation of autologous hematopoietic cells.

Molecular mechanisms underlying differential odor responses of a mouse olfactory receptor

The prevailing paradigm for G protein-coupled receptors is that each receptor is narrowly tuned to its ligand and closely related agonists. An outstanding problem is whether this paradigm applies to olfactory receptor (ORs), which is the largest gene family in the genome, in which each of 1,000 different G protein-coupled receptors is believed to interact with a range of different odor molecules from the many thousands that comprise “odor space.” Insights into how these interactions occur are essential for understanding the sense of smell. Key questions are: (i) Is there a binding pocket? (ii) Which amino acid residues in the binding pocket contribute to peak affinities? (iii) How do affinities change with changes in agonist structure? To approach these questions, we have combined single-cell PCR results [Malnic, B., Hirono, J., Sato, T. & Buck, L. B. (1999) Cell 96, 713–723] and well-established molecular dynamics methods to model the structure of a specific OR (OR S25) and its interactions with 24 odor compounds. This receptor structure not only points to a likely odor-binding site but also independently predicts the two compounds that experimentally best activate OR S25. The results provide a mechanistic model for olfactory transduction at the molecular level and show how the basic G protein-coupled receptor template is adapted for encoding the enormous odor space. This combined approach can significantly enhance the identification of ligands for the many members of the OR family and also may shed light on other protein families that exhibit broad specificities, such as chemokine receptors and P450 oxidases.

Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula

The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium–host pairs and are sufficient to cause a range of early responses involved in the host developmental program. Early events in the signal transduction of NFs are not well defined. We have previously reported that Medicago sativa root hairs exposed to NF display sharp oscillations of cytoplasmic calcium ion concentration (calcium spiking). To assess the possible role of calcium spiking in the nodulation response, we analyzed M. truncatula mutants in five complementation groups. Each of the plant mutants is completely Nod− and is blocked at early stages of the symbiosis. We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. Another mutant, altered in the DMI3 gene, has a similar mutant phenotype to dmi1 and dmi2 mutants but displays normal calcium spiking. The calcium behavior thus implies that the DMI3 gene acts either downstream of calcium spiking or downstream of a common branch point for the calcium response and the later nodulation responses. Two additional mutants, altered in the NSP and HCL genes, which show root hair branching in response to NF, are normal for calcium spiking. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.

Confidential inquiry into quality of care before admission to intensive care

Objective: To examine the prevalence, nature, causes, and consequences of suboptimal care before admission to intensive care units, and to suggest possible solutions.

In vitro roles of invariant helix–turn–helix motif residue R383 in σ54 (σN)

In vitro DNA-binding and transcription properties of σ54 proteins with the invariant Arg383 in the putative helix–turn–helix motif of the DNA-binding domain substituted by lysine or alanine are described. We show that R383 contributes to maintaining stable holoenzyme–promoter complexes in which limited DNA opening downstream of the –12 GC element has occurred. Unlike wild-type σ54, holoenzymes assembled with the R383A or R383K mutants could not form activator-independent, heparin-stable complexes on heteroduplex Sinorhizobium meliloti nifH DNA mismatched next to the GC. Using longer sequences of heteroduplex DNA, heparin-stable complexes formed with the R383K and, to a lesser extent, R383A mutant holoenzymes, but only when the activator and a hydrolysable nucleotide was added and the DNA was opened to include the –1 site. Although R383 appears inessential for polymerase isomerisation, it makes a significant contribution to maintaining the holoenzyme in a stable complex when melting is initiating next to the GC element. Strikingly, Cys383-tethered FeBABE footprinting of promoter DNA strongly suggests that R383 is not proximal to promoter DNA in the closed complex. This indicates that R383 is not part of the regulatory centre in the σ54 holoenzyme, which includes the –12 promoter region elements. R383 contributes to several properties, including core RNA polymerase binding and to the in vivo stability of σ54.

Triplex forming oligonucleotide targeted to 3′UTR downregulates the expression of the bcl-2 proto-oncogene in HeLa cells

The bcl-2 proto-oncogene is overexpressed in a variety of human cancers and plays an important role in programmed cell death. Recent reports implied that the 3′-untranslated region (3′UTR) functions effectively in the regulation of gene expression. Here, we attempt to assay the ability of triplex forming oligonucleotides (TFOs) to inhibit expression of a target gene in vivo and to examine the potential of the 3′UTR of the bcl-2 proto-oncogene in the regulation of bcl-2 gene expression. To do this, we have developed a novel cellular system that involves transfection of a Doxycyclin inducible expression plasmid containing the bcl-2 ORF and the 3′UTR together with a TFO targeted to the 3′UTR of the bcl-2 proto-oncogene. Phosphorothioate-modified TFO targeted to the 3′UTR of the bcl-2 gene significantly downregulated the expression of the bcl-2 gene in HeLa cells as demonstrated by western blotting. Our results indicate that blocking the functions of the 3′UTR using the TFO can downregulate the expression of the targeted gene, and suggest that triplex strategy is a promising approach for oligonucleotide-based gene therapy. In addition, triplex-based sequence targeting may provide a useful tool for studying the regulation of gene expression.

Peroxide Sensors for the Fission Yeast Stress-activated Mitogen-activated Protein Kinase Pathway

The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast.

Vasopressin mRNA localization in nerve cells: Characterization of cis-acting elements and trans-acting factors

mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein/RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules. In vitro interaction studies with cytosolic extracts demonstrated specific binding of a protein, enriched in nerve cell tissues, to the radiolabeled dendritic localizer sequence probe. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions are far from clear but may include functions such as translational silencing. Presumably, the translational state of mRNAs subject to dendritic sorting is influenced by external stimuli. PABP thus could be a component required to regulate local synthesis of the VP precursor and possibly of other proteins.

Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack.

We develop a unifying theory of hypoxia tolerance based on information from two cell level models (brain cortical cells and isolated hepatocytes) from the highly anoxia tolerant aquatic turtle and from other more hypoxia sensitive systems. We propose that the response of hypoxia tolerant systems to oxygen lack occurs in two phases (defense and rescue). The first lines of defense against hypoxia include a balanced suppression of ATP-demand and ATP-supply pathways; this regulation stabilizes (adenylates) at new steady-state levels even while ATP turnover rates greatly decline. The ATP demands of ion pumping are down-regulated by generalized "channel" arrest in hepatocytes and by "spike" arrest in neurons. Hypoxic ATP demands of protein synthesis are down-regulated probably by translational arrest. In hypoxia sensitive cells this translational arrest seems irreversible, but hypoxia-tolerant systems activate "rescue" mechanisms if the period of oxygen lack is extended by preferentially regulating the expression of several proteins. In these cells, a cascade of processes underpinning hypoxia rescue and defense begins with an oxygen sensor (a heme protein) and a signal-transduction pathway, which leads to significant gene-based metabolic reprogramming-the rescue process-with maintained down-regulation of energy-demand and energy-supply pathways in metabolism throughout the hypoxic period. This recent work begins to clarify how normoxic maintenance ATP turnover rates can be drastically (10-fold) down-regulated to a new hypometabolic steady state, which is prerequisite for surviving prolonged hypoxia or anoxia. The implications of these developments are extensive in biology and medicine.

Novel retinoic acid receptor ligands in Xenopus embryos.

Retinoids are a large family of natural and synthetic compounds related to vitamin A that have pleiotropic effects on body physiology, reproduction, immunity, and embryonic development. The diverse activities of retinoids are primarily mediated by two families of nuclear retinoic acid receptors, the RARs and RXRs. Retinoic acids are thought to be the only natural ligands for these receptors and are widely assumed to be the active principle of vitamin A. However, during an unbiased, bioactivity-guided fractionation of Xenopus embryos, we were unable to detect significant levels of all-trans or 9-cis retinoic acids. Instead, we found that the major bioactive retinoid in the Xenopus egg and early embryo is 4-oxoretinaldehyde, which is capable of binding to and transactivating RARs. In addition to its inherent activity, 4-oxoretinaldehyde appears to be a metabolic precursor of two other RAR ligands, 4-oxoretinoic acid and 4-oxoretinol. The remarkable increase in activity of retinaldehyde and retinol as a consequence of 4-oxo derivatization suggests that this metabolic step could serve a critical regulatory function during embryogenesis.