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B Ni

Publications and source records attributed to B Ni.

At least 55 records · Page 3Linked to original sources
PubMed1994

Cloning and expression of a cDNA encoding a brain-specific Na(+)-dependent inorganic phosphate cotransporter.

We have isolated a brain-specific cDNA that encodes a Na(+)-dependent inorganic phosphate (Pi) cotransporter (BNPI). The nucleotide sequence of BNPI predicts a protein of 560 amino acids with 6-8 putative transmembrane-spanning segments that is approximately 32% identical to the rabbit kidney Na(+)-dependent Pi cotransporter. Expression of BNPI mRNA in Xenopus oocytes results in Na(+)-dependent Pi transport similar to that reported for the recombinantly expressed or native kidney Na(+)-dependent cotransporter. RNA blot analysis reveals that BNPI mRNA is expressed predominantly (if not exclusively) in brain, and in situ hybridization histochemistry reveals BNPI transcripts in neurons of the cerebral cortex, hippocampus, and cerebellum. Furthermore, we have confirmed the presence of saturable Na(+)-dependent Pi cotransport in cultured cerebellar granule cells. Together, these data demonstrate the presence of a specific neuronal Na(+)-dependent transport system for Pi in brain.

Amino Acid Sequence↗
PubMed1993

Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca(2+)-binding sites. In our efforts to find the location of these Ca2+ sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca2+ ions in Ca(2+)-regenerated bR were examined. The important results are as follows: (i) The removal of Ca2+ from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Tyr-185, was replaced by a non-H-bonding amino acid (Phe). (ii) Similar to the observation in wild-type bR, the binding of Ca2+ to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (iii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca2+. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the extent of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Tyr-185 was replaced with Phe(Y185F). The above results suggest that the two Ca2+ ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. The results further suggest that, while the interaction between Tyr-185 and the high-affinity Ca2+ ions is relatively short range and specific (with more coupling to the Ca2+ ion in the second affinity site), between the charged residues and Ca2+ ions it seems to be of the electrostatic (e.g., ion-ion) long range, nonspecific type. Although neither Asp-85, Asp-212, nor Arg-82 is individually directly involved in the binding of Ca2+ in these two sites, they might all participate in it. Together with the protonated Schiff base, the charged residues along with Tyr-185 and one or two Ca2+ ions (and probably a few water molecules) seem to form an electrostatically coupled system that is part of a cavity that controls the color and function of bR.

Amino Acid Sequence↗
PubMed1993

Modulation of a neuronal calmodulin mRNA species in the rat brain stem by reserpine.

Reserpine evokes transsynaptic impulse activity by depleting catecholaminergic neurotransmitters in the rat brain. Previous studies suggest a relationship between catecholaminergic activity and calmodulin concentration. In this report we employ Northern blot analysis to examine the effect of a single subcutaneous injection of reserpine on levels of calmodulin mRNA species which are preferentially expressed in neurons of the rat brain. Regional differences in mRNA levels were also investigated by in situ hybridization and drug-induced changes were noted particularly in specific regions of the rat brain stem. The riboprobe used in the in situ hybridization study recognized a 4.0 kilobase neuronal calmodulin mRNA species (NGB1), which was derived from the rat CaM1 gene. A calmodulin radio-immunoassay was utilized to demonstrate a drug-induced increased in calmodulin protein levels in a region which included the brain stem.

Animals↗
PubMed1992

Pathways of proton release in the bacteriorhodopsin photocycle.

The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.

Bacteriorhodopsins↗
PubMed1992

Structures of aspartic acid-96 in the L and N intermediates of bacteriorhodopsin: analysis by Fourier transform infrared spectroscopy.

The light-induced difference Fourier transform infrared spectrum between the L or N intermediate minus light-adapted bacteriorhodopsin (BR) was measured in order to examine the protonated states and the changes in the interactions of carboxylic acids of Asp-96 and Asp-115 in these intermediates. Vibrational bands due to the protonated and unprotonated carboxylic acid were identified by isotope shift and band depletion upon substitution of Asp-96 or -115 by asparagine. While the signal due to the deprotonation of Asp-96 was clearly observed in the N intermediate, this residue remained protonated in L. Asp-115 was partially deprotonated in L. The C = O stretching vibration of protonated Asp-96 of L showed almost no shift upon 2H2O substitution, in contrast to the corresponding band of Asp-96 or Asp-115 of BR, which shifted by 9-12 cm-1 under the same conditions. In the model system of acetic acid in organic solvents, such an absence of the shift of the C = O stretching vibration of the protonated carboxylic acid upon 2H2O substitution was seen only when the O-H of acetic acid is hydrogen-bonded. The non-hydrogen-bonded monomer showed the 2H2O-dependent shift. Thus, the O-H bond of Asp-96 enters into hydrogen bonding upon conversion of BR to L. Its increased hydrogen bonding in L is consistent with the observed downshift of the O-H stretching vibration of the carboxylic acid of Asp-96.

Acetates↗
PubMed1992

Developmental expression of neuronal calmodulin mRNA species in the rat brain analyzed by in situ hybridization.

The temporal and spatial distribution of calmodulin mRNAs which are preferentially expressed in neurons was determined during postnatal development of rat central nervous system. Expression of these mRNAs was strongly detected in the developing neocortex, hippocampus, and cerebellum. Differences in the pattern of expression of a 1.8 and 4.0 kb neuronal calmodulin mRNA species were identified in the developing cerebellum. Expression of the smaller mRNA appeared to correlate with proliferating and developing cerebellar granule neurons while the larger mRNA was present in the mature granule neuron population. A transient elevation in the neuronal calmodulin mRNA species was observed in the superior and inferior colliculus and in the thalamus at postnatal days 5 and 10.

Aging↗
PubMed1992

Molecular cloning of calmodulin mRNA species which are preferentially expressed in neurons in the rat brain.

A cDNA clone designated NGB, which was isolated from a rat brain expression library, detected two mRNA species of 1.8 and 4.0 kb which are highly enriched in brain tissue. cDNAs NGB1 and NGB2 corresponding to these two mRNAs have been isolated and characterized. Sequence data showed that both mRNA species contain the same open reading frames but differ in their 3' untranslated regions. The open reading frame encodes a calmodulin protein of 148 amino acids. Both mRNA species are derived from the rat CaMI gene by utilization of different polyadenylation addition sites. Analysis of the 3' untranslated sequence which is unique to the larger mRNA species revealed a putative AU-rich 'destabilizer' sequence which is thought to be involved in mechanisms of selective mRNA breakdown. In situ hybridization studies revealed that the two calmodulin mRNAs are expressed strongly in neuronal cells in the adult rat brain. Levels of the two mRNA species increased during early postnatal development.

Aging↗
PubMed1992

A residue substitution near the beta-ionone ring of the retinal affects the M substates of bacteriorhodopsin.

The switch in the bacteriorhodopsin photocycle, which reorients access of the retinal Schiff base from the extracellular to the cytoplasmic side, was suggested to be an M1----M2 reaction (Váró and Lanyi. 1991. Biochemistry. 30:5008-5015, 5016-5022). Thus, in this light-driven proton pump it is the interconversion of proposed M substates that gives direction to the transport. We find that in monomeric, although not purple membrane-lattice immobilized, D115N bacteriorhodopsin, the absorption maximum of M changes during the photocycle: in the time domain between its rise and decay it shifts 15 nm to the blue relative to the spectrum at earlier times. This large shift strongly supports the existence of two M substates. Since D115 is located near the beta-ionone ring of the retinal, the result raises questions about the possible involvement of the retinal chain or protein residues as far away as 10 A from the Schiff base in the mechanism of the switching reaction.

Bacteriorhodopsins↗
PubMed1992

The two consecutive M substates in the photocycle of bacteriorhodopsin are affected specifically by the D85N and D96N residue replacements.

The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Váró and Lanyi, Biochemistry 30, 5016-5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild-type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M2 but not M1 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to 400 nm, suggesting that this species in turn is affected by D85. These results are consistent with the suggestion that M1 and M2 are pre-switch and post-switch states, respectively.

Bacteriorhodopsins↗
PubMed1992

[Sampling of expiratory benzene and gas chromatography].

The method of sampling alveolar air and gas chromatography of trace benzene is reported. The lowest detectable concentration of benzene is 7.9 x 10(-4) mg/m3 in 100 ml volume. Method parameters are r = 0.999.9, mean recovery 95.14% and mean relative deviation (CV) 4.94%. 42 non-occupational and 48 occupational workers who exposed to benzene were studied by this method. The concentrations of exhaled benzene are respectively 0.016 +/- 0.011, 0.138 +/- 0.093 (before workshift) and 10.17 +/- 4.11 mg/m3 (after the end of the workshift). The results showed that this method is a simplified method for sampling alveolar air and analysis. Besides for expiratory benzene, this method can be also applied to monitoring of atmospheric trace benzene.

Benzene↗
PubMed1991

Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.

Asparagine↗