Elucidation of the mechanism of the regulatory function of the Ig1 module of the fibroblast growth factor receptor 1

The extracellular part of the fibroblast growth factor (FGF) receptor (FGFR) consists of up to three Ig modules (Ig1-Ig3), in which the Ig2 and Ig3 modules determine affinity and specificity for FGF and heparin. The FGFR isoforms lacking the Ig1 module have higher affinity for FGF and heparin than the triple Ig-module isoforms, suggesting that the Ig1 module is involved in the regulation of the FGFR-ligand interaction. We show here by surface plasmon resonance and NMR analyses that the Ig1 module binds to the Ig2 module, and identify by NMR the binding sites involved in the Ig1-Ig2 interaction. The identified binding site in the Ig2 module was found to be in the area of the FGF-Ig2 and Ig2-heparin contact sites, thus providing direct structural evidence that the Ig1 module functions as a competitive autoinhibitor of the FGFR-ligand interaction. Furthermore, the Ig1 binding site of the Ig2 module overlaps the Ig2-Ig2 contact site. This suggests that the function of the Ig1 module is not only regulation of the FGFR-ligand binding affinity but also prevention of spontaneous FGFR dimerization (through a direct Ig2-Ig2 interaction) in the absence of FGF.     

Investigation of the mechanism of the methylmalonyl-CoA mutase reaction with the substrate analogue: ethylmalonyl-CoA.

1. Ethylmalonyl-CoA was found to be a substrate for methylmalonyl-CoA mutase from Propionibacterium shermanii, the product being mainly (2R)-methylsuccinyl-CoA along with some (2S)-diastereoisomer. 2. The relevant 1H-nuclear magnetic resonance signals of methylsuccinic acid and of its dimethyl ester were assigned to the diastereotopic methylene hydrogens using sterospecifically dideuterated specimens of known configuration. 3. [2(-2)H1]Ethylmalonyl-CoA was converted by methylmalonyl-CoA mutase in 2H2O mainly to (2R, 3S)-[3(-2)H1]methylsuccinyl-CoA. No dideuterated product was observed. 4. Starting from (1R)-[1(-2)H1]-ethathanol, (1S)-[1(-2)H1]ethanol and [2H6] ethanol the following deuterated specimens of ethylmalonic acid were synthesised and characterised: (3S)-[3(-2)H1], (3R)-[3(-2)H1] and [3(-2)H2, 4(-2)H3], respectively. 5. Conversion of (3S)-[3(-2)H1]-ethylmalonyl-CoA (70% 2H1 and 2% 2H2 species) on the mutase in water afforded mainly (2R)-[2(-2)H1]methylsuccinyl-CoA along with some (2S)-diastereoisomer. No deuterium loss was observed. 6. Methylmalonyl-CoA mutase converted (3R)-[3(-2)H1]ethylmalonyl-CoA (81% 2H1 and 2% 2H2 species) to the following methylsuccinyl-CoA species: 33% [3(-2)H1], the deuterium being in the threo position with respect to the methyl group; 21% [2(-2)H1]; 46% unlabelled. The ratio of the species with (2R) and (2S) configuration was about 60:40. 7. Reaction of [3(-2)H2, 4(-2)H3]ethylmalonyl-CoA (94.5% [2H5] species) with the mutase gave the following labelled methylsuccinyl-CoA species:53.4% [methyl-2H3, 2(-2)H1, 3(-2)H1], the 3-deuterium being in the threo position with respect to the methyl group; 37.6% [methyl-2H3, 2(-2)H1]; 5% [methyl(-2)H3, 2(-2)H1, 2(-2)H1, 3(-2)H1] the 3-deuterium being in erythro position with respect to the methyl group; 4% [methyl(-2)H3, 3(-2)H1]. The ratio of the species with (2R) and (2S) configuration was about 70:30. 8. Implications of these findings for the mechanism of the rearrangements catalysed by coenzyme B12 are discussed.     

The nature of the lengthening of the G2 phase in the mitotic cycle of parenchymal cells of the mouse liver

The phenomenon of G2 phase prolongation was found in the population of mouse hepatocytes. In normal postnatal liver growth, G2 phase prolongation in not pronounced and occurs in a small fraction of proliferating hepatocytes. In case of liver regeneration after removal of 2/3 of the organ, G2 phase prolongation is observed in a population of hepatocytes, which response to the proliferative stimulus first. Estimation of individual variation in expression of prolonged G2 phase along with the detailed analysis of the structure of the process of proliferation in the main population of hepatocytes (cells with normal G2 phase) allows to define the biological meaning of the "G2-population" observed. The prolongation of G2 phase may result from non-specific cell damage in mitotic cycles, caused by destruction of trophic relations in liver during its growth and regeneration.     

The nature of the acid-volatile selenium in the liver of the male rat

1. The properties of rat liver acid-volatile selenium have been compared with those of H(2)Se and (CH(3))(2)Se. 2. In model experiments oxidation-sensitive H(2) (75)Se was trapped quantitatively under anaerobic conditions in 0.1m-AgNO(3), and (CH(3))(2) (75)Se was trapped quantitatively in 8m-HNO(3). The acid-labile selenium of a liver homogenate, and of a microsomal fraction, was found to behave quite unlike (CH(3))(2) (75)Se and in a manner indistinguishable from H(2) (75)Se. 3. It was concluded that the acid-volatile material is certainly not (CH(3))(2)Se and that it is probably H(2)Se. 4. The significance of these findings is discussed in relation to current knowledge about the metabolism and detoxication of selenium, and a scheme is proposed which incorporates this knowledge with recent observations on the interactions between trace amounts of selenium and tocopherol, and the production of acute selenium deficiency by Ag(+) in vitamin E-deficient rats.     

Purification and properties of the synthetase catalyzing the biotination of the aposubunit of transcarboxylase from Propionibacterium shermanii

The synthetase that attaches biotin to the aposubunit of transcarboxylase (biotin-[methylmalonyl-CoA-carboxyltransferase]ligase) (EC 6.3.4.9) was purified to homogeneity by ion-exchange chromatography on cellulose DE-52 and CM-cellulose. The synthetase is a monomer of molecular weight 30,000. The pH and temperature optima for the synthetase are 6.0 and 37 degrees C, respectively. The apparent Km for the substrates ATP, biotin, and apo 1.3 S subunit of apotranscarboxylase are 38, 2.0, and 0.9 microM, respectively. Ni2+, Co2+, Zn2+, or Mn2+ could replace Mg2+ in the reaction. The affinity of synthetase toward metals is as follows: Zn2+ greater than Ni2+ greater than Mn2+ greater than Co2+ greater than Mg2+, and the activity with Zn2+ was much greater than that with the other divalent metals. EDTA completely inactivates the enzyme. The metals are necessary not only for the catalytic activity but also for the storage stability of the enzyme. The synthetase shows absolute specificity toward ATP.     

The effect of the size of the irradiation field on the severity and outcome of the radiation injury of swine skin

Swine skin areas of 12.56 cm2, 50 cm2 and 100 cm2 were exposed to beta-particles from 90Sr + 90Y (100 Gy). The increase in size of the exposed site caused a considerable increase in the degree of the affection and a change in the regeneration rate. Epithelialization of 12.56 cm2 skin field was completed by the 14th-16th week, and it was absent after 4.5 years in the fields of 50 and 100 cm2.     

Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors play crucial roles in excitatory synaptic transmission as well as in excitotoxicity. A growing body of evidence suggests that the regulation of both subunit composition and the number of NMDA receptors reaching the surface membrane are tightly regulated. Recently, we have shown that the third membrane domains (M3) of both NR1 and NR2B subunits contain endoplasmic reticulum (ER) retention signals that prevent the unassembled subunits from leaving the ER. Furthermore, these membrane domains together with NR1 M4 are necessary for negating the ER retention signals found in M3 of NR1 and NR2B. In this addendum, we present new electrophysiological data showing that mutation of the HLFY motif, located immediately after M4 of the NR2B subunit, abolishes the surface trafficking of full-length NR1/NR2B complexes (supporting previous immunofluorescent experiments from our lab); however, the deletion of the NR2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the NR2B subunit, NR2B truncated before M4 and NR2B M4, were co-expressed together with the NR1 subunit. These observations will help to uncover the processes involved in the assembly of NR1 and NR2 subunits into functional NMDA receptors.     

Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors play crucial roles in excitatory synaptic transmission as well as in excitotoxicity. A growing body of evidence suggests that the regulation of both subunit composition and the number of NMDA receptors reaching the surface membrane are tightly regulated. Recently, we have shown that the third membrane domains (M3) of both NR1 and NR2B subunits contain endoplasmic reticulum (ER) retention signals that prevent the unassembled subunits from leaving the ER. Furthermore, these membrane domains together with NR1 M4 are necessary for negating the ER retention signals found in M3 of NR1 and NR2B. In this addendum, we present new electrophysiological data showing that mutation of the HLFY motif, located immediately after M4 of the NR2B subunit, abolishes the surface trafficking of full-length NR1/NR2B complexes (supporting previous immunofluorescent experiments from our lab); however, the deletion of the NR2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the NR2B subunit, NR2B truncated before M4 and NR2B M4, were co-expressed together with the NR1 subunit. These observations will help to uncover the processes involved in the assembly of NR1 and NR2 subunits into functional NMDA receptors.     

Use of aequorin for the appraisal of the hypothesis of the release of calcium from the sarcoplasmic reticulum induced by a change of pH in skinned cardiac cells

A change of pH did not modify the sensitivity of aequorin to Ca2+, but an increase of pH enhanced the Ca2+ sensitivity of the myofilaments of a skinned canine cardiac Purkinje cell. The tension-pCa curve did not present any hysteresis when a given [free Ca2+] was reached from a higher versus from a lower [free Ca2+] in the presence of pH 6.60, 7.10 or 7.40. A rapid variation of pH in either direction failed to induce Ca2+ release from the sarcoplasmic reticulum (SR). The proton ionophores CCCP and gramicidin also failed to induce Ca2+ release from the SR. Increase of pH from 7.10 to 7.40 enhanced Ca2+ accumulation into the SR and, thereby, augmented the Ca2+ content of the SR. Consequently, the amplitude of a subsequent Ca2+ release triggered by a rapid increase of [free Ca2+] at the outer surface of the SR was increased. Conversely, a decrease of pH from 7.10 to 6.60 diminished the Ca2+ accumulation into the SR, the Ca2+ content of the SR and the amplitude of a subsequent Ca2+-induced release of Ca2+ from the SR. In addition, the optimum [free Ca2+] for triggering Ca2+-induced release of Ca2+ was shifted to higher [free Ca2+] values by a decrease of pH from 7.40 to 7.10 or 7.10 to 6.60. This may help to explain the enhancement of the aequorin light transient during acidosis in the intact cardiac muscle inasmuch as acidosis may increase the [free Ca2+] trigger at the outer surface of the SR by inhibiting Na+-Ca2+ exchange across the sarcolemma.     

Calcium paradox of aldosteronism and the role of the parathyroid glands

The hypercalciuria and hypermagnesuria that accompany aldosteronism contribute to a fall in plasma ionized extracellular Ca2+ and Mg2+ concentrations ([Ca2+]o and [Mg2+]o). Despite these losses and the decline in extracellular levels of these cations, total intracellular and cytosolic free Ca2+ concentration ([Ca2+]i) is increased and oxidative stress is induced. This involves diverse tissues, including peripheral blood mononuclear cells (PBMC) and plasma. The accompanying elevation in plasma parathyroid hormone (PTH) and reduction in bone mineral density caused by aldosterone (Aldo)-1% NaCl treatment (AldoST) led us to hypothesize that Ca2+ loading and altered redox state are due to secondary hyperparathyroidism (SHPT). Therefore, we studied the effects of total parathyroidectomy (PTx). In rats receiving AldoST, without or with a Ca2+-supplemented diet and/or PTx, we monitored urinary Ca2+ and Mg2+ excretion; plasma [Ca2+]o, [Mg2+]o, and PTH; PBMC [Ca2+]i and H2O2 production; plasma alpha1-antiproteinase activity; total Ca2+ and Mg2+ in bone, myocardium, and rectus femoris; and gp91(phox) labeling in the heart. We found that 1) the hypercalciuria and hypermagnesuria and decline (P < 0.05) in plasma [Ca2+]o and [Mg2+]o that occur with AldoST were not altered by the Ca2+-supplemented diet alone or with PTx; 2) the rise (P < 0.05) in plasma PTH with AldoST, with or without the Ca2+-supplemented diet, was prevented by PTx; 3) increased (P < 0.05) PBMC [Ca2+]i and H2O2 production, increased total Ca2+ in heart and skeletal muscle, and fall in bone Ca2+ and Mg2+ and plasma alpha1-antiproteinase activity with AldoST were abrogated (P < 0.05) by PTx; and 4) gp91(phox) activation in right and left ventricles at 4 wk of AldoST was attenuated by PTx. AldoST is accompanied by SHPT, with parathyroid gland-derived calcitropic hormones being responsible for Ca2+ overload in diverse tissues and induction of oxidative stress. SHPT plays a permissive role in the proinflammatory vascular phenotype.     

Connections of the representational area of the distal section of the forelimb in the rat sensorimotor cortex

Using horseradish peroxidase, studies have been made on the distribution of retrogradely labeled nervous cells in the sensorimotor cortex of rats. The enzyme was injected into electrophysiologically identified zone of representation of the distal part of the forelimb in areas S2 and S1. It was found that this zone in S2 contains afferent connections mainly from representation of the same extremity in S1 and only a few afferents from other areas of S1, S2 and M1 of the same hemisphere. Single labeled neurones were found in areas S2, S1 and M1 of the contralateral hemisphere. Representation of the forelimb in S1 receives mainly cortical afferents from the same region of S1 and from single cells of homologous zones S2 of the same and S1 of the contralateral hemisphere. Connections from S1 to S2 are more numerous than the opposite ones. In contrast to cats and monkeys, in rats afferent cortical fibers to zone S2 pass not only from the third layer, but also from the fifth and sixth layers of the cortex. It is suggested that during progressive development of the neocortex in mammals, the increase in the degree of separation of neurones (which give origin to corticofugal and cortical connections) among different layers of the cortex takes place.     

Small-angle X-ray scattering study of the ATP modulation of the structural features of the nucleotide binding domains of the CFTR in solution

Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding site for several candidate drugs in the disease treatment. We studied the structural properties of recombinant NBD1, NBD2, and an equimolar NBD1/NBD2 mixture in solution by small-angle X-ray scattering. We demonstrated that NBD1 or NBD2 alone have an overall structure similar to that observed for crystals. Application of 2 mM ATP induces a dimerization of NBD1 but does not modify the NBD2 monomeric conformation. An equimolar mixture of NBD1/NBD2 in solution shows a dimeric conformation, and the application of ATP to the solution causes a conformational change in the NBD1/NBD2 complex into a tight heterodimer. We hypothesize that a similar conformation change occurs in situ and that transition is part of the gating mechanism. To our knowledge, this is the first direct observation of a conformational change of the NBD1/NBD2 interaction by ATP. This information may be useful to understand the physiopathology of cystic fibrosis.     

A postulated role of the near amino-terminal domain of the ryanodine receptor in the regulation of the sarcoplasmic reticulum Ca(2+) channel

To test the hypothesis that interactions among several putative domains of the ryanodine receptor (RyR) are involved in the regulation of its Ca(2+) release channel, we synthesized several peptides corresponding to selected NH(2)-terminal regions of the RyR. We then examined their effects on ryanodine binding and Ca(2+) release activities of the sarcoplasmic reticulum isolated from skeletal and cardiac muscle. Peptides 1-2s, 1-2c, and 1 enhanced ryanodine binding to cardiac RyR and induced a rapid Ca(2+) release from cardiac SR in a dose-dependent manner. The order of the potency for the activation of the Ca(2+) release channel was 1-2c > 1 > 1-2s. Interestingly, these peptides produced significant activation of the cardiac RyR at near zero or subactivating [Ca(2+)], indicating that the peptides enhanced the Ca(2+) sensitivity of the channel. Peptides 1-2c, 1-2s, and 1 had virtually no effect on skeletal RyR, although occasional and variable extents of activation were observed in ryanodine binding assays performed at 36 degrees C. Peptide 3 affected neither cardiac nor skeletal RyR. We propose that domains 1 and 1-2 of the RyR, to which these activating peptides correspond, would interact with one or more other domains within the RyR (including presumably the Ca(2+)-binding domain) to regulate the Ca(2+) channel.     

Two pathways for the degradation of the H2 subunit of the asialoglycoprotein receptor in the endoplasmic reticulum

An intermediate of 35 kD accumulates transiently during ER degradation of the H2 subunit of the asialoglycoprotein receptor; it is derived by an endoproteolytic cleavage in the exoplasmic domain near the transmembrane region. In the presence of cycloheximide all of the precursor H2 is converted to this intermediate, which is degraded only after cycloheximide is removed (Wikstrom, L., and H. F. Lodish. 1991. J. Cell Biol. 113:997-1007). Here we have generated mutants of H2 that do not form the 35-kD fragment, either in transfected cells or during in vitro translation reactions in the presence of pancreatic microsomes. In transfected cells the kinetics of ER degradation of these mutant proteins are indistinguishable from that of wild-type H2, indicating the existence of a second pathway of ER degradation which does not involve formation of the 35-kD fragment. Degradation of H2 in the ER by this alternative pathway is inhibited by TLCK or TPCK, but neither formation nor degradation of the 35-kD fragment is blocked by these reagents. As determined by NH2-terminal sequencing of the 35-kD fragment, formed either in transfected cells or during in vitro translation reactions in the presence of pancreatic microsomes, the putative cleavage sites are between small polar, uncharged amino acid residues. Substitution of the residues NH2- or COOH-terminal to the cleavage site by large hydrophobic or charged ones decreased the amount of 35-kD fragment formed and in some cases changed the putative cleavage site. Cleavage can also be affected by amino acid substitutions (e.g., to proline or glycine) which change protein conformation. Therefore, the endoprotease that generates the 35-kD fragment has specificity similar to that of signal peptidase. H2a and H2b are isoforms that differ only by a pentapeptide insertion in the exoplasmic juxtamembrane region of H2a. 100% of H2a is degraded in the ER, but up to 30% of H2b folds properly and matures to the cell surface. The sites of cleavage to form the 35-kD fragment are slightly different in H2a and H2b. Two mutant H2b proteins, with either a glycine or proline substitution at the position of insertion of the pentapeptide in H2a, have metabolic fates similar to that of H2a. These mutations are likely to change the protein conformation in this region. Thus the conformation of the juxtamembrane domain of the H2 protein is important in determining its metabolic fate within the ER.     

Self-organization in the determination of the size of the axial structures in the embryogenesis of the clawed toad

Experiments were performed using X. laevis embryos during gastrulation and neurulation (stages 10, 11 1/2, 12 1/2, 13 1/2, 15 and 18). Part of presumptive epidermis and lateral plate mesoderm was removed, and embryos raised until stage 25. The size of axial structures (notochord, somite mesoderm, central nervous system) was determined using serial histological sections and compared with that of control embryos. In experimental embryos, the size of axial structures was decreased. Until a specific stage of development, close correlation was found between the volume of embryonic compartment corresponding to a particular, structure and the volume of presumptive epidermis and lateral plate mesoderm. This stage is individual for each axial organ: middle gastrula (stage 11 1/2) for notochord, late gastrula (stage 12 1/2) for somite mesoderm, and late neurula (stage 18) for central nervous system. This data suggest that differentiation pattern of ecto-mesodermal rudiment is subject to regulation during gastrulation-neurulation, and subdivision of ectoderm and mesoderm into axial and non-axial tissues is a self-organizing process.     

Effect of the lethal Bacillus anthracis toxin on phagocytosis and the dynamics of the change in the enzyme activity of the antioxidative system of peritoneal mononuclear phagocytes in mice with differing hereditary immunity to anthrax

It was demonstrated, that the lethal in vitro suppressed the phagocytic activity of peritoneal mononuclear phagocyte (Mph) and enhanced the level activity of glutathione peroxidase to H2O2 (GP-H2O2) in Mph of resistant to anthrax BALB mice. In Mph BALB the authors observed dependent-dose enhancement of GP-H2O2 activity and reduction of the ratio of level glutathione reductase (GR) to GP-H2O2 (GR/GP-H2O2). The enhancement of activity GR-H2O2 in Mph CBA was not dependent on the doses of toxin. The coefficient GR/GP-H2O2 was similar to the control. The mechanisms of hereditary resistance to anthrax were discussed.     

Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin

Human gene-2 (H2) relaxin is a member of the insulin-relaxin peptide superfamily. Because of the potential clinical applications of H2 relaxin, there is a need for novel analogs that have improved biological activity and receptor specificity. In this respect, we have chemically assembled chimeric peptides consisting of the B-chain of H2 relaxin in combination with A-chains from other insulin/relaxin family members. The peptides were prepared using solid phase peptide synthesis together with regioselective disulfide bond formation and characterized by RP-HPLC, MALDI-TOF MS and amino acid analysis. Their in vitro activity was assessed in RXFP1 or RXFP2 expressing cells. Replacement of the H2 relaxin A-chain resulted in parallel losses of binding affinity and activity on RXFP1. Not surprisingly H1A-H2B demonstrated the highest activity as the H1 A-chain shares high homology with H2 relaxin whereas INSLA-H2B, which shows low homology, had very poor activity. Importantly A-chain replacements had a dramatic effect on RXFP2 activity similar to previous results demonstrating different modes of activation of A-chain variants on RXFP1 and RXFP2. H3A-H2B is particularly interesting as it displays moderate activity at RXFP1 but poor activity at RXFP2 indicating that it may be a template for specific RXFP1 agonist development. Our study confirms that the activity of H2 relaxin at both RXFP1 and RXFP2 relies on interactions with both the B- and A-chains, and also provide new biochemical insights into the mechanism of relaxin action that the A-chain needs to be in native or near-native form for strong RXFP1 or RXFP2 agonist activity.     

Use of selectively trypsinized nucleosome core particles to analyze the role of the histone "tails" in the stabilization of the nucleosome

Using immobilized trypsin and an appropriate fractionation procedure, we have been able to prepare, for the first time, nucleosome core particles containing selectively trypsinized histone domains. The particles thus obtained: [(H3T-H4T)2-2(H2AT-H2BT)].DNA; [(H3-H4)2-2(H2AT-H2BT)].DNA; [H3T-H4T)2-2(H2A-H2B)].DNA (where T means trypsinized), together with the non-trypsinized controls have been characterized using the following techniques: analytical ultracentrifugation, circular dichroism, thermal denaturation and DNAse I digestion. The major aim of this study was to analyze the role of the amino-terminal regions (the histone "tails") on the stability of the nucleosome in solution. The data obtained from this analysis clearly show that stability of the nucleosome core particle to dissociation (below a salt concentration of 0.7 M-NaCl) is not affected by the presence or the absence of any of the N-terminal regions of the histones. Furthermore, these histone regions make very little contribution, if any, to the conformational transition that nucleosomes undergo in this range of salt concentrations. They play, however, a very important role in determining the thermal stability of the particle, as reflected in the dramatic alterations exhibited by the melting profiles upon selective removal of these tails by trypsinization. The melting data can be explained by a simple hypothesis that ascribes interaction of H2A/H2B and H3/H4 tails to particular regions of the nucleosomal DNA.