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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasmids pCB20 and pCB22 were used for cloning and expression of the Bac brevis 7882 neutral protease gene in Bac. subtilis cells. The protease-containing fragments of 13 and 14 kb were cloned in pCB20 plasmid based on replication region of Streptococci plasmid pSM19035. Expression of the gene was shown to take place in Bac. subtilis. Application of vegetative promoters of the previously identified expression unit EU19035 greatly increases the expression of the protease in Bac. subtilis. Bac. subtilis cells, expressing the gene of Bac. brevis neutral protease, do not sporulate, are considerably larger than the cells which do not contain the gene and form multicellular structures.
Mol Biol (Mosk)
PMID:[Cloning and expression in Bacillus subtilis of the gene for neutral protease of Bacillus brevis]. 212 22

Several sequences, resembling vegetative promoters and ribosome-binding sites of Bacilli were found in the primary structure of the replication region of Streptococci plasmid pSM19035. Promoterless alpha-amylase gene of Bac. amyloliquefaciens and lambda cI857 gene, supplied with BamHI site upstream of the initiator ATG-codon, were used for functional characterization of the structures. As a result, Bac. subtilis synthesized alpha-amylase up to 0.5 g/l, and lambda-repressor up to 3% of the intracellular water-soluble protein. The repressor, synthesized in Bac. subtilis, regulates lambda PR promoter in the cells. Plasmid pCB22 is constructed for the convenience of usage of the found expression unit, called EU19035. The plasmid has BamHI and BgIII sites on different distances from the ribosome-binding site.
Mol Biol (Mosk)
PMID:[Expression unit in the region of replication initiation in the streptococcal plasmid pSM19035]. 212 23

Amino acid sequence of neutral metalloprotease from Bac. brevis has been compared with that of Bac. amyloloquefaciens, Bac. cereus, Bac. subtilis, Bac. stearothermophilis, Bac. thermoproteolyticus (thermolysine). A sequence region from N-40 to N-1 with a significant degree of homology allowed to predict the processing site of the propart of Bac. brevis enzyme. The sequence comparison allows to put Bac. brevis enzyme within the evolutionary branch of enzymes, which includes thermolysin and proteases of Bac. cereus and Bac. stearothermophilus. Using automated Edman degradation the N-terminal sequence of Bac. brevis protease has been determined. It does not differ from the sequence predicted from the nucleotide sequence of the gene. It was shown that, when Bac. brevis gene coding for thermostable protease is expressed on a plasmid vector in Bac. subtilis cells at 37 degrees C, enzyme forms possessing low activity are secreted. The enzyme may be significantly activated without an additional cleavage or processing and the activation includes numerous conformation transition states of the protein molecule.
Mol Biol (Mosk)
PMID:[Analysis of the structure of Bacillus brevis neutral proteinase and its biosynthesis in Bacillus subtilis cells]. 212 74

We investigated the effects of the physiological neurotransmitter norepinephrine on the contractile properties and Ca2+ dynamics of isolated cardiac myocytes, with particular emphasis on possible interactions between alpha- and beta-adrenergic effects. Individual rat ventricular myocytes were electrically stimulated at a frequency of 1 Hz. Norepinephrine (10(-9) to 10(-5) M) increased extent and velocity of shortening and decreased the contraction duration. beta-Adrenergic activation gave a greater enhancement of extent and velocity of shortening than did norepinephrine alone (i.e. alpha plus beta). Neither alpha 1 nor alpha 2 adrenergic activation individually produced a significant impact upon contraction. Using suspensions of myocytes loaded with Quin-2, we also studied resting levels of cytosolic Ca2+ ([ Ca2+]c), the increase of [Ca2+]c due to caffeine-addition (as an index of sarcoplasmic reticulum Ca2+ content) and the subsequent increase in [Ca2+]c due to depolarization with 30 mM K+ (as an index of sarcolemmal voltage-dependent Ca2+ channel activity). Norepinephrine decreased resting [Ca2+]c, increased sarcoplasmic reticulum Ca2+ content and increased Ca2+ channel activity. beta-Adrenergic activation produced the same effect on resting [Ca2+]c and sarcoplasmic reticulum content, but gave significantly greater activation of sarcolemmal Ca2+ channel activity, than did norepinephrine (alpha plus beta). By contrast, alpha-adrenergic stimulation had no effect on resting [Ca2+]c or sarcoplasmic reticulum Ca2+ content. We conclude that beta-mediated effects predominate in the action of the physiological agonist norepinephrine on cardiac myocytes. However, alpha (specifically alpha 1)-adrenergic effects are significant in diminishing the potentiation of the extent and velocity of shortening, and of depolarization-induced entry of Ca2+ into the cell, which is seen on beta-stimulation alone. Thus, there may be an intrinsic feedback effect in the actions of norepinephrine on the cardiac myocyte.
J Mol Cell Cardiol 1990 Jan
PMID:Interactive alpha- and beta-adrenergic actions of norepinephrine in rat cardiac myocytes. 215 52

The ability of alpha 1a- and alpha 1b-adrenergic receptor subtypes to stimulate [3H]inositol phosphate [( 3H]InsP) formation was examined in collagenase-dispersed hepatocytes and renal cells. alpha 1-Adrenergic receptor binding sites were labeled with 125I-BE 2254, and the proportion of alpha 1a and alpha 1b subtypes was determined with chloroethylclonidine (CEC) and WB 4101. Hepatocytes contained only alpha 1b-adrenergic receptors, whereas renal cells had approximately equal proportions of both subtypes. Pretreatment of renal cells with CEC selectively inactivated the alpha 1b subtype, leaving a homogeneous population of alpha 1a receptors. Norepinephrine stimulated [3H]InsP accumulation to a similar extent in both hepatocytes and renal cells. Pretreatment with CEC inactivated this response completely in hepatocytes but only partially in renal cells. WB 4101 was 1000-fold more potent in inhibiting the [3H]InsP response in renal cells than hepatocytes; however, some of this difference was due to rapid metabolism of WB 4101 by hepatocytes. After correction for metabolism, WB 4101 was still 11-fold more potent in inhibiting norepinephrine-stimulated [3H]InsP formation in hepatocytes (alpha 1b) than in CEC-pretreated renal cells (alpha 1a). These results demonstrate that both alpha 1a- and alpha 1b-adrenergic receptor subtypes activate formation of [3H]InsP, although the molecular mechanisms by which these responses occur remain to be determined.
Mol Pharmacol 1990 Jun
PMID:Alpha 1-adrenergic receptor subtypes and formation of inositol phosphates in dispersed hepatocytes and renal cells. 216 16

Norepinephrine (NE) increased formation of [3H]inositol phosphates ( [3H]InsPs) in primary cultures of neuronal and glial cells from 1-day-old rat brain. This response appeared to be mediated by alpha 1-adrenergic receptors, because prazosin was 40-fold more potent than yohimbine in blocking it. Pretreatment with pertussis toxin (PTX) dose-dependently decreased this response by 70-80%. The IC50 for PTX (7 ng/ml) was similar to that for blocking of alpha 2-adrenergic receptor-mediated decreases in cyclic AMP accumulation in the same cells. PTX pretreatment caused only a small, not statistically significant, inhibition of the [3H]InsP response to the muscarinic cholinergic receptor agonist carbachol in these cells. Radioligand binding studies showed that both neuronal and glial cultures contained mixed populations of alpha 1a- and alpha 1b-adrenergic receptor subtypes. Selective inactivation of the alpha 1b population by chloroethylclonidine reduced NE-stimulated [3H]InsP formation by 25 +/- 6%. Pretreatment with both PTX and chloroethylclonidine caused additive decreases (90 +/- 3%) in the NE response. NE-stimulated [3H]InsP formation was partially dependent on extracellular calcium, because it was decreased 64 +/- 6% by removal of calcium and 56 +/- 13% by addition of 1 mM CdCl2, although it was not affected by 1 microM nifedipine. These results suggest that NE stimulates [3H]InsP formation in neuronal and glial cultures through a pertussis toxin-sensitive guanine nucleotide-binding protein. This response appears to be mediated primarily by the alpha 1a subtype and may be subsequent to calcium influx.
Mol Pharmacol 1990 Aug
PMID:Pertussis toxin inhibits norepinephrine-stimulated inositol phosphate formation in primary brain cell cultures. 216 6

Glucocorticoids are known to have marked effects on blood pressure regulation, predominantly through altering cardiovascular sensitivity to noradrenaline. However, the molecular mechanisms underlying this action remain unclear. As part of our studies into these we have measured alpha 1-adrenergic receptor binding using the ligand [3H]prazosin in plasma membrane fractions of aortas prepared from control, adrenalectomized and dexamethasone-treated adrenalectomized rats. In controls there were 50 +/- 8 (S.E.M.; n = 6) fmol alpha 1-adrenergic receptors/mg membrane protein (Bmax) with a dissociation constant (Kd) of 0.52 +/- 0.10 nM (n = 6). Adrenalectomy 8 days before tissue preparation caused a 40% decrease in Bmax and a 60% decrease in Kd. Dexamethasone replacement after adrenalectomy returned these values close to those of controls. Noradrenaline competed for the [3H]prazosin-binding sites. Computer analysis by a non-linear curve-fitting program (LIGAND) showed that noradrenaline binding was to a heterogeneous population of high- and low-affinity receptors with Kd values of 1.87 +/- 0.73 microM and 0.48 +/- 0.12 mM (n = 5) respectively. Guanosine thiotriphosphate (GTP[S]) caused the conversion of high-affinity to low-affinity binding, consistent with the model of the high-affinity sites being coupled to a G protein. After adrenalectomy, noradrenaline binding was to a homogeneous population of low-affinity receptors; hence, the effect of GTP[S] was no longer apparent, suggesting that under these conditions the alpha 1-adrenergic receptors were unable to couple to a G protein. The two-site model of binding and GTP[S] effect was returned by dexamethasone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Endocrinol 1990 Aug
PMID:Effect of glucocorticoids on alpha 1-adrenergic receptor binding in rat vascular smooth muscle. 216 9

Primary structure of DNA fragment of 2355 b.p., encoding metalloprotease gene of Bac. brevis, had been determined. Open reading frame for a protein with size of 528 amino acid residues was found in this sequence. The encoding protein is homologous to metalloproteases of Bac. stearothermophilus, Bac. cereus, Bac. subtilis and Bac. amyloliquefaciens. The structure of Bac. brevis metalloprotease gene reveals that this enzyme is synthesised as pre-pro-protease with signal peptide and pro-region, which are cut during its synthesis. The proposed size of mature protease is 304 amino acid residues. The residues, essential for catalysis, binding of Zn ion and Ca ions were found on the basis of Bacilli metalloproteases structures comparison.
Mol Biol (Mosk)
PMID:[Structure of the Bacillus brevis metalloprotease gene]. 229 Apr 27

The effects of prolonged exposure to combined mild hypoxia (Po2 230 +/- 20 mmHg) and acidosis (pH: 6.8 +/- 0.05) were studied in guinea-pig left ventricular myocardium superfused in vitro. Only Purkinje fibers were impaled by microelectrodes. Triggered activity developed in depolarized Purkinje fibers after 48 +/- 9 min of exposure to hypoxic and acid conditions and was initiated either by short periods of rapid electrical driving or by the background slow Purkinje automaticity. Triggered activity occurred when a delayed afterdepolarization attained its threshold potential and terminated after a subthreshold afterdepolarization. Interaction between triggered activity and slow background automaticity was observed until 90 to 180 min of exposure to hypoxic and acid conditions. These effects were reversed by replacement in standard conditions (Po2 510 +/- 20 mmHg; pH 7.35 +/- 0.05). Norepinephrine (1 X 10(-6)M) significantly accelerated the rate of discharge of triggered foci and led to a stable sustained triggered activity. Increasing extracellular Ca2+ concentration aggravated the effects of combined mild hypoxia and acidosis and led to the occurrence of early afterdepolarizations initiating triggered activity. In addition abnormal automaticity developed in quiescent fibers without any triggering action potential. Lidocaine and verapamil suppressed the triggered activity following a subthreshold afterdepolarization. Their effects were reversed on wash-out. It is concluded that prolonged exposure to combined mild hypoxia and acidosis induces triggered activity by a basic mechanism common to other situations leading to a calcium overload and showing such behaviour.
J Mol Cell Cardiol 1986 Dec
PMID:Triggered activity induced by combined mild hypoxia and acidosis in guinea-pig Purkinje fibers. 243 59

Techniques routinely utilized in this laboratory for recording currents through single ionic channels of isolated atrial and ventricular rat cardiomyocytes are described. Emphasis is placed in two main areas: first, on methods for obtaining a sufficient yield of Ca++-tolerant myocytes suitable for patch clamp experiments, and secondly, on methods for analyzing the temporal characteristics of 'patched' ionic channels. These methods were used on acetylcholine activated K+ channels in isolated atrial myocytes and on an inwardly-rectifying K+ channel in ventricular myocytes. The latter is an example of a hormonally modulated K+ channel, since its activity could be substantially increased by norepinephrine. Analysis of the closed and open time distributions suggested that one of the closed states of this channel is markedly abbreviated by norepinephrine, whereas the open state is nearly unaffected. Norepinephrine was effective when channel activity was recorded from on-cell patches and the hormone was added to the solution bathing the cell membrane outside of the patched area. This indicates that a second messenger substance is probably mediating the action of norepinephrine.
Mol Cell Biochem
PMID:Patch clamp analysis of chemically activated and modulated ionic channels in isolated mammalian cardiomyocytes. 245


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