Search PubMed⌕ Search

Biomedical subjects

B Robert

Publications and source records attributed to B Robert.

At least 109 records · Page 6Linked to original sources
PubMed1996

Evidence that embryonic kidney cells expressing flk-1 are intrinsic, vasculogenic angioblasts.

Renal glomerular capillary tufts have been believed to arise from angiogenic ingrowth of extrinsic vessels. We found, however, that when embryonic day 12 (E12) mouse kidneys were maintained in culture for 6 days and then grafted into anterior eye chambers of adult transgenic ROSA26 host mice (which carry the beta-galactosidase transgene), glomerular endothelial cells within the grafts were predominantly of intrinsic, kidney origin. To identify potential endothelial precursors, we immunolabled kidneys with antibodies against the vascular endothelial growth factor receptor, flk-1. Numerous discrete cells expressing flk-1 were scattered throughout the nephrogenic mesenchyme of both E12 and newborn kidneys, and with development these cells became concentrated in microvessels, glomerular vascular clefts, and glomerular tufts. In adults, flk-1 was weakly expressed in glomeruli but absent elsewhere. To examine abilities of flk-1-positive cells to establish glomeruli, E12 kidneys were grafted into kidney cortices of adult and newborn ROSA26 hosts. Grafts into adults resulted in few glomeruli containing host-derived endothelium, whereas a majority of glomeruli grafted into newborns contained host cells. Cells of graft origin were found in vessels forming in renal cortices of newborn hosts, but not in adults. Our findings indicate that embryonic kidney cells expressing flk-1 are angioblasts that create microvessels and glomeruli by vasculogenesis.

Animals↗
PubMed1996

Expression of endothelin precursor genes in human trophoblast in culture.

We have shown previously the presence of immunoreactive endothelin in cultured trophoblastic cells from human term placenta as well as in the trophoblast-conditioned medium. To confirm whether or not the differentiated syncytiotrophoblast is a site for endothelin synthesis, we investigated, by reverse transcription and polymerase chain reaction, the expression of the three preproendothelin genes in 3-day cultured trophoblast. While no endothelin-2 precursor mRNA was detected, preproendothelin-1 mRNA was found to be expressed by the trophoblast. The endothelin-3 precursor gene was also expressed, but at low level and it was detected only after Southern blotting and oligonucleotide hybridization. The ability of trophoblast in culture to express the endothelin precursor genes supports the idea that, in human term placenta, villous syncytiotrophoblast that lines the intervillous space containing maternal blood acts as an endothelial layer.

Base Sequence↗
PubMed1996

ELK and LERK-2 in developing kidney and microvascular endothelial assembly.

Eph family receptor tyrosine kinases direct neuronal cell targeting, bundling and intercellular aggregation activity, yet their role in mammalian kidney development has been unexplored to date. We recently identified expression of ELK (Eph-like kinase) receptors in cultured human renal microvascular endothelial cells (HRMEC), and showed that ELK mediates their in vitro assembly into capillary-like structures in response to the exogenous ligand, LERK-2. Here we identify expression of the ELK ligand, LERK-2, in HRMEC and in primitive vascular structures of developing murine kidney. ELK and LERK-2 are expressed on endothelial progenitor cells of primitive microvasculature in a pattern similar to that of the VEGF receptor, flk-1. ELK LERK-2 and flk-1 antigens are also displayed on the branching ureteric bud epithelium; ELK and LERK-2 expression persists in mature collecting ducts, glomeruli and arterioles. To explore whether renal-derived endothelial cells may distinguish LERK-2 from the angiogenic Eck ligand, LERK-1 (B61), and whether endothelial cells from different sources may distinguish among Eph receptor ligands, we compared HRMEC and human umbilical vein endothelial cell (HUVEC) responses in an in vitro capillary-like assembly assay. HRMEC endothelial cells assembled capillary-like structures in response to LERK-2, but not LERK-1, under conditions that promoted HUVEC to assemble in response to LERK-1, but not LERK-2. Therefore, responses mediated through specific Eph family receptors (ELK and Eck) are discriminated by endothelial cells from different vascular bed sources. ELK and its ligand, LERK-2, are spatially and temporally coordinated in expression and may function in morphogenesis of the renal microvasculature.

Animals↗
PubMed1995

Time-resolved and steady-state spectroscopic analysis of membrane-bound reaction centers from Rhodobacter sphaeroides: comparisons with detergent-solubilized complexes.

The spectroscopic analysis of the antenna-deficient Rhodobacter sphaeroides strain RCO1 has been extended to an investigation of the kinetics and spectroscopy of primary charge separation. Global analysis of time-resolved difference spectra demonstrated that the rate of charge separation in membrane-bound reaction centers is slightly slower than in detergent-solubilized reaction centers from the same strain. A kinetic analysis of the decay of the primary donor excited state at single wavelengths was carried out using a high repetition rate laser system, with the reaction centers being maintained in the open state using a combination of phenazine methosulfate and horse heart cytochrome c. The kinetics of primary charge separation in both membrane-bound and solubilized reaction centers were found to be non-monoexponential, with two exponential decay components required for a satisfactory description of the decay of the primary donor excited state. The overall rate of charge separation in membrane-bound reaction centers was slowed if the primary acceptor quinone was reduced using sodium ascorbate. This slowing was caused, in part, by an increase in the relative amplitude of the slower of the two exponential components. The acceleration in the rate of charge separation observed on removal of the reaction center from the membrane did not appear to be caused by a significant change in the electrochemical properties of the primary donor. The influence of the environment of the reaction center on primary charge separation is discussed together with the origins of the non-monoexponential decay of the primary donor excited state.

Ascorbic Acid↗
PubMed1995

Symmetric structural features and binding site of the primary electron donor in the reaction center of Chlorobium.

The protein binding interactions of the constituent bacteriochlorophyll a molecules of the primary electron donor, P840, in isolated reaction centers from Chlorobium limicola f thiosulphatophilum and the electronic symmetry of the radical cation P840+. were determined using near-infrared Fourier transform (FT) Raman spectroscopy excited at 1064 nm. The FT Raman vibrational spectrum of P840 indicates that it is constituted of a single population of BChl a molecules which are spectrally indistinguishable. The BChl a molecules of P840 are pentacoordinated with only one axial ligand on the central Mg atom, and the pi-conjugated C2 acetyl and C9 keto carbonyls are free of hydrogen-bonding interactions. The FT Raman spectrum of P840+. exhibits a 1707 cm-1 band attributable to a BChl a C9 keto carbonyl group vibrational frequency that has upshifted 16 cm-1 upon oxidation of P840; this upshift is exactly one-half of that expected for the one-electron oxidation of monomeric BChl a in vitro. The 16 cm-1 upshift, thus, indicates that the resulting +1 charge is equally shared between two BChl a molecules. This situation is markedly different from that of the oxidized primary donor of the purple bacterial reaction center of Rhodobacter sphaeroides, (i) which exhibits a 1717 cm-1 band that has upshifted 26 cm-1, indicating an asymmetric distribution of the resulting +1 charge over the two constituent BChl a molecules, and (ii) whose H-bonding pattern with respect to the pi-conjugated carbonyl groups is asymmetric.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1995

Biochemical and spectroscopic characterization of the B800-850 light-harvesting complex from Rhodobacter sulphidophilus and its B800-830 spectral form.

We demonstrate that the B800-830 spectral form of the B800-850 peripheral light-harvesting complex of Rhodobacter sulphidophilus, which is formed at low ionic strengths in the presence of the zwitterionic detergent LDAO, results from a local modification of the bacteriochlorophyll binding site and not the dissociation of the complex. This perturbation does not result in significant changes to the interactions between the pigments as studied by circular dichroism or fluorescence spectroscopy; however, modifications in the pigment binding sites are inferred from changes in the preresonance Raman spectrum. Specifically, an alteration of the hydrogen bonding of the 2-acetyl group of at least one of the bacteriochlorophyll groups that make up the 850 nm absorbing pair is observed. This implies an alteration in the conformation of the C-terminal domain of the alpha-polypeptide, in which are located the two tyrosyl residues that are believed to act as H-bond donors to these groups, induced by the protein-bound detergent in the absence of bound cations. We suggest that the ability of this complex to form an 800-830 complex is linked to the presence of an aspartyl residue immediately upstream of the tyrosyl residues. This study therefore provides a further illustration of the importance of hydrogen bonds to the 2-acetyl group of the bacteriochlorophyll in the determination of its spectral properties; furthermore, we provide a description of a conformational change that is able to modulate chromophore binding in these complexes.

Bacteriochlorophylls↗
PubMed1995

Resonance Raman spectroscopy of the photosystem II light-harvesting complex of green plants: a comparison of trimeric and aggregated states.

Resonance Raman spectroscopy was performed on photosystem II light-harvesting complex (LHCII) in trimeric and oligomeric states with various excitation conditions. From these studies, it can be concluded that the structure of LHCII is altered during the trimer/oligomer transition. These structural changes affect the conformation of a population of carotenoid within the complex, which becomes twisted in the oligomeric form. Moreover, the interactions assumed by a chlorophyll a and a chlorophyll b are modified during the oligomerization process. This induces the formation of an H-bond to a formyl group of a chlorophyll b molecule and to a keto group of a chlorophyll a molecule. The extent to which these H-bonds to chlorophyll relate to the formation of the quencher cannot yet be precisely established. However, the structural changes they evidence may play a role in the control of the energy flux by LHCII complexes.

Carotenoids↗
PubMed1995

Structure and properties of the bacteriochlorophyll binding site in peripheral light-harvesting complexes of purple bacteria.

In this paper, we have examined, using FT resonance Raman spectroscopy, the bacteriochlorophyll (BChl) binding sites in the peripheral light-harvesting complexes extracted from a number of purple bacterial strains. A comparison of interactions of the BChl molecules with their binding sites in these LH2 complexes, together with the primary sequences of the alpha and beta polypeptides, allows three amino acids to be proposed to be involved in the hydrogen bonding of the 9-keto carbonyl of one of the 850-nm-absorbing pair of BChl molecules. Specifically, we show that one keto carbonyl group, which is strongly hydrogen bonded in Rhodobacter sphaeroides LH2, is involved in much weaker interactions in the LH2 complexes from all the other species studied (i.e., Rhodobacter capsulatus, Rubrivivax gelatinosus, Rhodopseudomonas palustris, Rhodopseudomonas acidophila, and Rhodopseudomonas cryptolactis). This is correlated with the presence of three polar amino acids in the primary sequence of the alpha polypeptide of Rb. sphaeroides which are absent in the sequences from all the other bacteria and probably close to a chromophore. These three residues are a serine at position -4, a threonine at position +6 and another serine at position +17 (numbering relative to the conserved histidine, considered as position 0), in the alpha polypeptide of Rb. sphaeroides. Furthermore, the study of the interactions in natural B800-820 complexes shows that the two 2-acetyl groups of the 820-nm-absorbing BChl molecules are free from hydrogen-bonding interactions. In the light of previous site-selected mutagenesis studies, the lack of such hydrogen bonds seems to be a general phenomenon, associated with the 820-nm absorption of LH2 complexes, and suggests that hydrogen-bonding interactions have a precise molecular role in finely tuning the functional properties of these complexes.

Amino Acid Sequence↗
PubMed1995

Recombinant adenovirus-mediated gene transfer to genitourinary epithelium in vitro and in vivo.

Transitional cell carcinoma (TCC) of the bladder is associated with characterized lesions in dominant and recessive oncogenes. The understanding of the molecular basis of tumorigenesis in these instances makes possible the application of gene therapy strategies for TCC. In this regard, the ability to directly access the epithelium of the genitourinary (GU) tract via the urethra provides a practical means to implement these various gene therapy approaches. We thus explored vector strategies to accomplish direct in vivo transduction of GU epithelium. Initially, three human (HT 1197, HT 1376, T24) and one mouse (MBT-2) TCC cell lines were transduced using a recombinant adenoviral vector expressing the firefly luciferase reporter gene, rAd-CMV-Luc. In these studies, reporter gene expression was found to be significantly elevated above background for all four cell lines. Of note, the TCC cell lines HT 1197 and HT 1376 showed expression levels comparable with the cervical carcinoma cell line HeLa, a cell line previously shown to be highly susceptible to recombinant adenovirus-mediated gene transduction. An in vitro time course for T24 and MBT-2 using rAd-CMV-Luc showed peak expression 1 day after transduction for the T24 line and 3 days after transduction for the MBT-2 line, with detectable levels of expression persisting for at least 7 days. As a next step, human and mouse primary tissue deriving from the GU epithelium were transduced using rAd-CMV-Luc. In this assay, luciferase expression levels significantly above background were observed in both instances.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1994

Modification of a hydrogen bond to a bacteriochlorophyll a molecule in the light-harvesting 1 antenna of Rhodobacter sphaeroides.

Site-directed mutagenesis has been used to examine the function of a highly conserved aromatic residue, alpha Trp43, in the light-harvesting 1 antenna of the photosynthetic bacterium Rhodobacter sphaeroides. In this antenna alpha Trp43 is thought to be located near the putative binding site for bacteriochlorophyll; in this work it was changed to both Tyr and Phe, and in each case the main near-infrared absorbance peak was shifted to the blue, from 876 nm to 865 nm and then to 853 nm, respectively. Resonance Raman spectroscopy of the resulting complexes shows a shift of one component of the 1640-cm-1 peak to 1632 cm-1 for the Tyr mutant and to 1660 cm-1 for the Phe mutant. This demonstrates a strengthening of an existing H bond for the Tyr change and a breakage of this bond for the change to Phe. The 1640-cm-1 peak has been previously assigned to H-bonded C2 acetyl carbonyl groups of both bacteriochlorophylls in the light-harvesting 1 antenna dimer [Robert, B. & Lutz, M. (1985) Biochim. Biophys. Acta 807, 10-21]. These results indicate that one of these H bonds is to alpha Trp43, placing this residue in close proximity to the bacteriochlorophyll a macrocycle with which it interacts. The existence of this bond places constraints on the conformation of the alpha polypeptide, and a model of an alpha beta heterodimer is presented incorporating these data.

Bacteriochlorophylls↗
PubMed1994

Structure and binding site of the primary electron acceptor in the reaction center of Chlorobium.

In isolated, chlorosome-free reaction centers from Chlorobium limicola f thiosulphatophilum, a chlorin pigment exhibits a Qy absorption band at 672 nm (Feiler, U., Nitschke, W., & Michel, H. (1992) Biochemistry 31, 2608-2614). To characterize the chemical nature of this chlorin pigment and its interactions within the reaction-center protein, selective enhancement of its Raman scattering was achieved by resonant excitation within its Soret band. This is the first time that structural studies of this pigment were performed on the native reaction-center protein. The obtained resonance Raman spectra were consistent with a single population of a chlorophyll a(-like) pigment, possessing a vinyl group on ring I, but not with bacteriochlorophyll c or bacteriophaeophytin c. The stretching frequencies of the C9-keto carbonyl of this pigment indicates that it is H-bonded to the reaction-center protein. The strength of this H-bond is very close to those of the keto carbonyls of the primary electron acceptors in purple bacterial reaction centers and D1/D2 particles. Since in membranes of Chlorobiaceae a transient bleaching at 670 nm is due to the primary acceptor in the reaction center (Nuijs, A. M., Vasmel, H., Joppe, H. L. P., Duysens, L. N. M., & Amesz, J. (1985a) Biochim. Biophys. Acta 907, 24-34), we thus conclude that the primary acceptor in Chlorobium reaction centers is the characterized chlorophyll a(-like) pigment.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1994

Thermodynamics of membrane polypeptide oligomerization in light-harvesting complexes and associated structural changes.

Investigation of the equilibrium between the dissociated B777 form of the light-harvesting complex of Rhodospirillum rubrum and the oligomeric B820 form demonstrates that the B777 form consists of bacteriochlorophyll a (BChl) bound to the alpha or beta polypeptide chains; this binding appears to be reasonably stable at room temperature with little dissociation to free BChl and polypeptides. Analysis of the reaction order for the B777 association reaction to form B820 shows that this reaction requires four components, presumably two alpha-B777 units and two beta-B777 units, implying that the B820 subunit contains four BChl molecules. Estimations of the enthalpy and entropy changes associated with the tetramerization give values of, respectively, -175 kJ mol-1 and -0.46 kJ and mol-1 K-1. Soret resonance Raman and Fourier transform preresonance Raman spectra of BChl in detergent together with those of the B777, B820 and native B873 forms of the light harvesting complex illustrate significant changes occurring to the environments of the C-2 acetyl groups (Fischer numbering system) during dissociation to form B820 and a loss of order in the C-9 keto environments on formation of B777. Attenuated total reflectance Fourier transform infrared absorption spectra of the three antennae forms demonstrate little perturbation of the approximately 50% alpha-helical secondary structure during dissociation. These observations are discussed in terms of the energetics of membrane protein folding and the structure of the light harvesting complex.

Bacterial Proteins↗
PubMed1994

Blue shifts in bacteriochlorophyll absorbance correlate with changed hydrogen bonding patterns in light-harvesting 2 mutants of Rhodobacter sphaeroides with alterations at alpha-Tyr-44 and alpha-Tyr-45.

A combination of Fourier-Transform (FT) resonance Raman spectroscopy and site-directed mutagenesis has been used to examine the function of two highly conserved aromatic residues, alpha-Tyr-44 and alpha-Tyr-45, in the light-harvesting 2 (LH2) complex of the photosynthetic bacterium Rhodobacter sphaeroides. In LH2 complexes, aromatic residues located at positions alpha-44 and alpha-45 are thought to be located near the putative binding site for bacteriochlorophyll, and alterations at these positions are known to produce blue shifts in bacteriochlorophyll absorbance. In the present work, mutant LH2 complexes carrying the alterations alpha-Tyr-44-->Phe, alpha-Tyr-45-->Phe and alpha-Tyr-44,-45-->Phe,Leu were examined. FT resonance Raman spectroscopy of the resulting complexes shows the breakage of a hydrogen bond to the 2-acetyl carbonyl group of one of the B850 bacteriochlorophylls in the LH2 complex; in the double mutant, breakage of a second bond is probable. These results suggest that one of these hydrogen bonds is to alpha-Tyr-44, placing this residue in close proximity to ring I of one of the B850 bacteriochlorophyll a pigments. The breakage of one, then two, 2-acetyl carbonyl hydrogen bonds correlates well with the shift in the absorbance of the B850 pigments of 11 nm then 26 nm at 77 K. Thus a consistency between literature theoretical calculations and the observations from both absorption and FT resonance Raman spectroscopy is demonstrated.

Bacteriochlorophylls↗
PubMed1994

Changes in primary donor hydrogen-bonding interactions in mutant reaction centers from Rhodobacter sphaeroides: identification of the vibrational frequencies of all the conjugated carbonyl groups.

Specific changes in the hydrogen-bonding states of the primary donor, P, in reaction centers from Rhodobacter sphaeroides bearing mutations near P were determined using near-infrared excited Fourier transform (FT) Raman spectroscopy. This technique, using 1064-nm excitation, provides the preresonantly enhanced vibrational spectrum of P in its reduced state selectively over the contributions of the other reaction center chromophores and protein and yields structural information concerning P and its hydrogen-bonding interactions. The mutations studied were as follows: Leu M160-->His, Leu L131-->His, the D9 double mutant (Leu M160-->His + Leu L131-->His), Phe M197-->His, and His L168-->Phe. These mutations were designed to introduce new, or to break existing, hydrogen bonds to the C9 and C2 carbonyl groups of P. On the basis of previous assignments [Mattioli, T. A., Hoffmann, A., Robert, B., Schrader, B., & Lutz, M. (1991) Biochemistry 30, 4648-4654], the FT Raman spectra of these mutants show the predicted changes in hydrogen bond interactions of P carbonyl groups with the protein. The results of this study have permitted us to unambiguously identify the C2 and C9 carbonyl vibrators of P in Rb. sphaeroides. The genetically introduced hydrogen bond interactions are discussed in terms of other physicochemical properties of P including the redox potential and electronic asymmetry in the P+ state. It is discussed that changes in protein hydrogen bonding to the conjugated carbonyl groups of P alone are not the sole factor that contributes to the sizeable modifications of the P/P+ redox midpoint potentials, and that the chemical nature of the hydrogen bond donor plays a significant role in this modification.

Carbon↗
PubMed1994

Site-specific mutagenesis of the reaction centre from Rhodobacter sphaeroides studied by Fourier transform Raman spectroscopy: mutations at tyrosine M210 do not affect the electronic structure of the primary donor.

The effects of mutation of residue tyrosine M210 on the primary donor bacteriochlorophylls have been investigated by near infrared FT-Raman spectroscopy in reaction centres purified from an antenna-deficient strain of Rhodobacter sphaeroides. We find that mutation at the M210 position does not significantly perturb the distribution of the unpaired electron over the pair of bacteriochlorophyll molecules which constitute the primary donor radical cation. We conclude, therefore, that the effects of mutation of tyrosine M210 on the rate and asymmetry of primary electron transfer in reaction centres cannot be ascribed to a change in the electronic structure of the primary donor.

Bacteriochlorophylls↗
PubMed1994

Developmental control of IFN-alpha expression in murine embryos.

The expression of IFN-alpha transcripts was investigated in murine embryos, fetuses, and fetal annexes in mid and late pregnancy. We have shown by Northern blot analysis, reverse transcription-polymerase chain reaction, and in situ hybridization the presence of IFN-alpha transcripts in mouse placenta, fetus, and newborn. From the 14th day of gestation until birth, a typical IFN-alpha transcript (1.2 kb) is found in the fetus. A transcript of larger size (2.2 kb) appears near birth and is present in the newborn mouse. Fetal annexes between the 10th and 21st days of gestation also express IFN-alpha. From the 10th day until birth, the 1.2-kb IFN-alpha mRNA species is present, as well as unusually large transcripts: 4 and 7 kb. To localize IFN-alpha transcripts, in situ hybridization was performed, using 35S-IFN-alpha antisense RNA probe in comparison with the sense RNA probe. The tissue pattern of IFN-alpha transcription in fetuses shows a clear labeling of many epithelia, such as skin, ependyme, and intestine glandular epithelium. A possible relation with cellular differentiation is discussed.

Animals↗
PubMed1994

Msx1 is close but not allelic to either Hm or Hx on mouse chromosome 5.

The Msx1 homeobox locus has been mapped in relation to the mutations hammer-toe (Hm) and hemimelic extra toes (Hx). Msx1 is expressed in the developing limb, while limb development is affected by the Hm and Hx mutations. Hm and Hx are very tightly linked loci. In interspecific crosses, the segregation of either mutation was followed in relation to polymorphic alleles of Msx1, Il6, and En2, to give a fine map around the mutant loci. Our results show that Msx1 is not allelic to either of the mutations, but is located about 3 cM from them. Il6 did not recombine with either Hm or Hx and, therefore, provides a point of access for the analysis of these mutations at the molecular level.

Alleles↗