Ca2+ influx via the L-type Ca2+ channel during tail current and above current reversal potential in ferret ventricular myocytes.
(73/4084)
1. Current through L-type Ca2+ channels (ICa) was measured electrophysiologically at the same time as Ca2+ influx was measured by trapping entering Ca2+ with a high concentration of indo-1 (> 1 mM) in ferret ventricular myocytes. 2. Na+-free conditions prevented Na+-Ca2+ exchange and K+ currents were blocked by Cs+ and TEA. Thapsigargin (5 microM) prevented Ca2+ uptake and release by the sarcoplasmic reticulum. ICa was pre-activated by brief pulses to +120 mV (the equilibrium potential for Ca2+, ECa), followed by steps to different membrane potentials (Em, -80 to +100 mV), in some cases in the presence of the Ca2+ channel agonist FPL-64176. 3. Integrated ICa ( 82 ICa) was linearly related to the change in the concentration of Ca2+ bound to indo-1, which was assessed by the fluorescence difference signal DeltaFd (Fd = F500 - F400). This created an internal calibration of DeltaFd as a measure of Ca2+ influx. 4. The DeltaFd/ 82 ICadt relationship was virtually unchanged at all measurable inward ICa (at Em from -80 to +50 mV). This indicates that the fractional current carried by Ca2+ and channel selectivity are unchanged over this Em range, and also that the selectivity for Ca2+ is very high. 5. Ca2+ influx was readily detected by DeltaFd beyond the ICa reversal potential (+65 to +100 mV) and was not abolished until Em was +120 mV (i.e. ECa). This is explained by the fact that inward Ca2+ flux at the ICa reversal potential is exactly balanced by outward Cs+ current through the Ca2+ channels and can be described by classic Goldman flux analysis with a Ca2+/Cs+ selectivity of the order of 5000. 6. This result also emphasizes that net Ca2+ influx via Ca2+ channels occurs over a voltage range where the net channel current is outward. (+info)
TspO of rhodobacter sphaeroides. A structural and functional model for the mammalian peripheral benzodiazepine receptor.
(74/4084)
The function and specific structural aspects of the tryptophan-rich sensory protein (TspO) of Rhodobacter sphaeroides 2.4.1 were studied using site-directed mutagenesis involving some 17 different amino acids. The choice of these amino acids changes was dictated from an analysis of the TspO family of proteins derived from the data bases. These studies demonstrated the importance of several highly conserved tryptophan residues in the sensory transduction pathway involving TspO through the proposed binding of an intermediate(s) in the tetrapyrrole biosynthesis pathway. These studies also revealed that the substitution of one or several of the amino acid residues dramatically affected, either directly or indirectly, the levels of TspO in the membranes of R. sphaeroides. Mounting evidence is presented suggesting that TspO normally forms a dimer within the bacterial outer membrane, and the dimer form of TspO may be the active form for TspO function. Because our earlier studies provided us with a functional framework within which to view these amino acid substitutions, we are able to suggest a preliminary model for TspO structure-function. Not only do these studies tell us more about TspO, but they also shed light on the TspO homologue, the drug-binding component of the mitochondrial peripheral benzodiazepine receptor. Mounting evidence draws numerous parallelism between these proteins and supports the significance of using TspO as a model for the structure and function of the mitochondrial protein. (+info)
Regression of U-87 MG human glioblastomas in nude mice after treatment with a cytotoxic somatostatin analog AN-238.
(75/4084)
Receptors for somatostatin (SST) found on brain tumors could be used for targeting of chemotherapeutic agents. This study was conducted to investigate the effects of targeted cytotoxic SST analogue AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201), a potent derivative of doxorubicin (DOX) linked to somatostatin analogue RC-121, on the growth of SST receptor-positive U-87 MG human glioblastomas. Nude mice bearing U-87 MG xenografts received i.v. saline or equimolar doses of AN-238 or AN-201 (150 nmol/kg). Experiments also included groups that were administered RC-121 prior to the injection of AN-238, and groups injected with AN-162, a cytotoxic SST analogue similar to AN-238 but containing DOX instead of AN-201. Tumor volume, weight, and burden were determined. The effect of AN-238 and AN-201 on the survival time of nude mice bearing orthotopically implanted U-87 MG tumors was also evaluated. The binding of AN-238 to U-87 MG tumors was determined by radioreceptor assay and SST receptor (SSTR) subtype by reverse transcription-PCR. Nineteen days after a single administration of AN-238 the growth of U-87 MG tumors in nude mice was significantly inhibited (P = 0.00168), whereas two injections of AN-238 produced the regression of tumors (P = 0.0046). AN-201 was toxic and ineffective at the same dose. The antitumor effect on AN-238 could be blocked by pretreatment of the tumor-bearing mice with RC-121. The mean survival time of nude mice inoculated orthotopically with U-87 MG cells into the brain was significantly prolonged by treatment with AN-238 (P = 0.0099). AN-162 failed to inhibit significantly the growth of U-87 MG xenografts. High affinity binding sites for SST and mRNA for SST-2 receptor subtype were detected in U-87 MG tumors. Cytotoxic SST analogue AN-238 can be targeted to SST receptors on U-87 MG human glioblastomas to produce powerful inhibition of growth. (+info)
The role of the CRH type 1 receptor in autonomic responses to corticotropin- releasing hormone in the rat.
(76/4084)
The involvement of the corticotropin-releasing hormone (CRH) type 1 receptor in CRH-induced cardiac responses was studied in freely moving rats. Intracerebroventricular (icv) infusion of 2 microg CRH under resting conditions resulted in a significant increase in heart rate (HR), but did not significantly affect the PQ interval of the electrocardiogram. This effect involves sympathetic nervous system (SNS) activation, since CRH-treatment resulted in a marked increase in plasma norepinephrine (NE) and epinephrine (E), and sympathetic blockade by subcutaneously injected atenolol (1 mg/kg), a beta1-selective adrenergic antagonist, completely prevented the CRH-induced tachycardia. CRH infusion after sympathetic blockade resulted in an elongation of the PQ interval, indicating CRH-induced vagal activation. Gross locomotor activity (GA) was determined to study its possible indirect effects on cardiac activity. Although CRH induced a marked increase in GA, this effect followed the tachycardiac response, indicating that the HR response was not a consequence of increased locomotor activity, but was a direct effect of icv CRH. Treatment with CP-154,526 (icv, 10 or 25 microg), a selective CRH type 1 receptor antagonist, did not affect baseline HR, plasma NE and E, whereas it partially blocked the CRH-induced increase in HR, plasma NE and E levels. CP-154,526 treatment had no significant effects on baseline or CRH-induced changes in GA. These results indicate that CRH activates the sympathetic nervous system at least in part via the CRH type 1 receptor. (+info)
Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon.
(77/4084)
The structure of phycoviolobilin, the photoactive chromophore of alpha-phycoerythrocyanin, is incompatible with a chromophore ligation to the apoprotein via SH-addition (cysteine) to a Delta3, 3(1)-double bond of the phycobilin. The two putative phycoerythrocyanin lyase genes of Mastigocladus laminosus, pecE and pecF, were overexpressed in Escherichia coli. Their action has been studied on the addition reaction of phycocyanobilin to apo-alpha-phycoerythrocyanin (PecA). In the absence of the components of alpha-PEC-phycoviolobilin lyase PecE and PecF, or in the presence of only one of them, phycocyanobilin binds covalently to PecA forming a fluorescent chromoprotein with a red-shifted absorption (lambda(max)=641 nm) and low photoactivity (<10%). In the presence of both PecE and PecF, a chromoprotein forms which by its absorption (lambda(max)=565 nm) and high photoreversible photochromism (100% type I) has been identified as integral alpha-phycoerythrocyanin. We conclude that PecE and PecF jointly catalyze not only the addition of phycocyanobilin to PecA, but also its isomerization to the native phycoviolobilin chromophore. (+info)
Not acute but chronic hypertriglyceridemia is associated with impaired endothelium-dependent vasodilation: reversal after lipid-lowering therapy by atorvastatin.
(78/4084)
There is controversy regarding the relation between hypertriglyceridemia (HTG) and endothelial function. This study was designed to investigate endothelial function in a patient group with chronic HTG, before and during lipid-lowering therapy by atorvastatin. In addition, the effects of acute HTG on endothelial function were studied in normolipidemic individuals. Eight male patients with chronic HTG were studied before and after 6 weeks of lipid-lowering treatment with 80 mg atorvastatin once daily. Ten age-matched control subjects were studied at baseline and immediately after a high-dose infusion of artificial triglycerides. The endothelium-dependent response to serotonin was attenuated in the HTG group, whereas the response to acetylcholine was comparable to the response in the control group. The response to the endothelium-independent vasodilator nitroprusside was comparable in both groups. In response to atorvastatin therapy, serum triglyceride and cholesterol levels decreased significantly by 43% (paired t test, P=0.017) and 38% (paired t test, P=0.012), respectively. After 6 weeks of treatment, the forearm blood flow response to serotonin improved from 63% to 106% (ANOVA, P<0.001). Induction of acute HTG in the control subjects did not affect the forearm blood flow responses to serotonin and nitroprusside; however, the response to acetylcholine was paradoxically increased. In conclusion, patients with chronic HTG have an impaired endothelium-dependent vasodilation to serotonin that is normalized after 6 weeks of lipid-lowering therapy by atorvastatin. (+info)
Sequence specific alkylation of DNA by hairpin pyrrole-imidazole polyamide conjugates.
(79/4084)
BACKGROUND: Pyrrole-imidazole polyamides are synthetic ligands that recognize predetermined sequences in the minor groove of DNA with affinities and specificities comparable to those of DNA-binding proteins. As a result of their DNA-binding properties, polyamides could deliver reactive moieties for covalent reaction at specific DNA sequences and thereby inhibit DNA-protein interactions. Site-specific alkylation of DNA could be a useful tool for regulating gene expression. As a minimal first step, we set out to design and synthesize a class of hairpin polyamides equipped with DNA alkylating agents and characterize the specificity and yield of covalent modification. RESULTS: Bis(dichloroethylamino)benzene derivatives of the well-characterized chlorambucil (CHL) were attached to the gamma turn of an eight-ring hairpin polyamide targeted to the HIV-1 promoter. We found that a hairpin polyamide-CHL conjugate binds and selectively alkylates predetermined sites in the HIV promoter at subnanomolar concentrations. Cleavage sites were determined on both strands of a restriction fragment containing the HIV-1 promoter, revealing good specificity and a high yield of alkylation. CONCLUSIONS: The ability of polyamide-CHL conjugates to sequence specifically alkylate double-stranded DNA in high yield and at low concentrations sets the stage for testing their use as regulators of gene expression in cell culture and ultimately in complex organisms. (+info)
Novel streptopyrroles from Streptomyces rimosus with bacterial protein histidine kinase inhibitory and antimicrobial activities.
(80/4084)
A series of halogenated pyrrolo [2,1-b] [1,3] benzoxazines (1 approximately 9) was isolated from fermentations of an actinomycete strain X10/78/978 (NCIMB40808), identified as Streptomyces rimosus, during a microbial extract screening programme to identify inhibitors of bacterial histidine kinase. The structures of these compounds were elucidated by spectroscopic methods including the HMQC, HMBC and INADEQUATE NMR experiments. The structure of 1 was confirmed by X-ray crystallographic studies. Compounds 5 and 6 were produced in fermentations in the presence of NaBr and NaI respectively. The most abundant member of the series, streptopyrrole, 1, inhibited the nitrogen regulator II (NRII) histidine kinase from Escherichia coli with an IC50 of 20 microM and exhibited antimicrobial activity against a range of bacteria and fungi. (+info)