Meiotic recombination in yeast: coronation of the double-strand-break repair model.
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Chromosomes, Fungal↗
Biomedical subjects
F Stahl
Publications and source records attributed to F Stahl.
Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme.
Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.
DNA↗
Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein.
Type II restriction endonucleases are dimers of two identical subunits that together form one binding site for the double-stranded DNA substrate. Cleavage within the palindromic recognition site occurs in the two strands of the duplex in a concerted manner, due to the action of two catalytic centers, one per subunit. To investigate how the two identical subunits of the restriction endonuclease EcoRV cooperate in binding and cleaving their substrate, heterodimeric versions of EcoRV with different amino acid substitutions in the two subunits were constructed. For this purpose, the ecorV gene was fused to the coding region for the glutathione-binding domain of the glutathione S-transferase and a His6-tag, respectively. Upon cotransformation of Escherichia coli cells with both gene fusions stable homo- and heterodimers of the EcoRV variants are produced, which can be separated and purified to homogeneity by affinity chromatography over Ni-nitrilotriacetic acid and glutathione columns. A steady-state kinetic analysis shows that the activity of a heterodimeric variant with one inactive catalytic center is decreased by 2-fold, demonstrating that the two catalytic centers operate independently from each other. In contrast, heterodimeric variants with a defect in one DNA-binding site have a 30- to 50-fold lower activity, indicating that the two subunits of EcoRV cooperate in the recognition of the palindromic DNA sequence. By combining a subunit with an inactive catalytic center with a subunit with a defect in the DNA-binding site, EcoRV heterodimers were produced that only nick DNA specifically within the EcoRV recognition sequence.
Base Sequence↗
The production and characterization of artificial heterodimers of the restriction endonuclease EcoRV.
A novel approach to studying the inter- and intrasubunit communication required for the activity of homodimeric proteins is described. It was developed for the restriction endonuclease EcoRV, but should also be useful for other homodimeric enzymes. Two ecorV genes encoding different EcoRV mutants are coexpressed in the same Escherichia coli cell leading to homo- and heterodimeric variants of the enzyme. The two ecorV genes carry either a 5' extension coding for the glutathione-S-transferase or a His6-tag. The EcoRV heterodimer produced in vivo is separated from the two EcoRV homodimers and purified to homogeneity by affinity chromatography. Purified EcoRV heterodimers are stable and are not subject to reassortment of the subunits. To investigate the interdependence of the two catalytic centers, EcoRV heterodimers consisting of one subunit with wild type sequence and one subunit with amino acid substitutions in the PD...(D/E)XK motif, characteristic for the active sites of many restriction endonucleases, were produced. While the homodimeric EcoRV active site mutants are catalytically inactive, the heterodimeric EcoRV variants with one active and one inactive catalytic center display a twofold reduced activity toward oligodeoxynucleotide substrates compared to the wild type, and preferentially nick supercoiled plasmid DNA. From these results we conclude that in the wild type enzyme both catalytic centers function independently of each other.
Biopolymers↗
The rat: an experimental animal in search of a genetic map.
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Animals↗
Haemangiomatosis in children: value of MRI during therapy.
MRI findings of 18 examinations of six children with haemangiomatosis and one with extended arteriovenous malformation are reported. Structures involved were the liver, liver and lung, periorbital area and the thigh. Response to interferon therapy, in particular, was assessed. With MRI the disease can be characterized and the extent of the lesions and size of the haemangiomas measured. Coronal views provide excellent demonstration of the involved structures in liver and lung haemangiomatosis. Two children showed response to interferon therapy with a reduction in lesion size and subsequently in number. Signal intensity decreased slightly on T2-weighted images. During treatment, however, no definitive fibrotic zones were seen. Following complete regression, signal intensity of the liver parenchyma was homogeneous in both weightings, that is, no fibrotic areas were visible 18 months after the beginning of treatment. Two children showed no response and one child died from congestive cardiac failure. The periorbital haemangioma was reduced in size and the lesion in the thigh might be classified as an arteriovenous malformation. In children MRI can replace CT as it is a reliable imaging modality for diagnosing haemangiomatosis and monitoring therapy.
Child↗
[Alcoholism and dentistry].
In this article the physical and psychological consequences of alcohol abuse are considered. In addition the interaction between alcohol abuse and oral health and dentistry is discussed.
Alcohol Drinking↗
Short- and long-term effects of perinatal interleukin-1 beta-application in rats.
Nervous, endocrine and immune systems are regarded as a complex functional unit, interacting by their specific chemical messengers-neurotransmitters, systemic hormones and hormone-like mediators of immune cells (cytokines). Cytokines are known to affect several endocrine axes. Interleukin-1 beta (IL) was administered in rats intraperitoneally twice daily from day 17 to 21 of pregnancy. Some of the IL-treated mothers were rapidly decapitated 1.5 h after the last injection. The fetuses were delivered by cesarean section. Maternal plasma CRF, ACTH and corticosterone were found to be significantly elevated. Fetal adrenal and thymus weights were lower, and plasma corticosterone did not differ from controls. Fetal plasma testosterone was decreased in males, androstenedione was increased in females. Open-field testing revealed a higher total locomotor activity of IL offspring than of controls. IL offspring showed worse results in Skinner box learning than controls. Sexual behavior was only affected in males, showing a higher percentage of female-type lordosis behavior after castration and estrogen treatment compared to controls. At the age of 6 months responsiveness to 'novel-environment stress' of IL groups was significantly lower than that of controls in terms of plasma corticosterone. These results indicate that prenatal treatment with IL-1 beta results in long-lasting alterations in psychomotor development, behavior as well as in the neuroendocrine system.
Adrenal Glands↗
Na(+)-dependent HCO3- transport and Na+/H+ exchange regulate pHi in human ciliary muscle cells.
We investigated intracellular pH (pHi) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4',5'-dimethylfluorescein (CDMF). The steady-state pHi was 7.09 +/- 0.04 (n = 12) in CO2/HCO3(-)-buffered and 6.86 +/- 0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11 +/- 0.06 pH units (n = 12) in the presence of CO2/HCO3- and by 0.91 +/- 0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pHi recovery, which was almost completely amiloride-sensitive in the absence of CO2/HCO3- but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25 +/- 0.02 (n = 4) in 6 min, while amiloride had no effect. The pHi recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pHi recovery was almost completely mediated by Na+/H+ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amiloride-insensitive pHi recovery was observed in CO2/HCO3(-)-buffered solution which was mediated by chloride-independent and chloride-dependent Na+ HCO3- cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter), was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pHi recovery after NH4Cl prepulse revealed Vmax = 0.57 pH units/min, Km = 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and Vmax = 0.19 pH units/min, Km = 14.3 mmol/liter extracellular sodium for the amiloride-insensitive component. We conclude that Na+/H+ exchange and chloride-independent and chloride-dependent Na(+)-HCO3- cotransport are involved in the pHi regulation of cultured human ciliary muscle cells.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Characterization of acetylcholine- and endothelin-induced calcium entry in cultured human ciliary muscle cells.
We characterized the effects of acetylcholine and endothelin on cultured human ciliary muscle cells, using the calcium-sensitive dye fura-2 to measure intracellular calcium and intracellular microelectrodes to measure the membrane potential. Both agonists, endothelin and acetylcholine, had a typical biphasic effect on the intracellular calcium concentration. Calcium peaked initially, because of its release from intracellular stores, and then reached a plateau, owing to entry of extracellular calcium. Endothelin-induced calcium entry was almost completely blocked by addition of extracellular La3+ (50 mumol/l) and Ni2+ (1 mmol/l). Acetylcholine-induced calcium entry was likewise almost completely abolished by La3+ and Ni2+. Both endothelin and acetylcholine led to an initial transient hyperpolarization with a subsequent depolarization. The hyperpolarization of the membrane potential had a time course similar to the initial calcium peak, while the depolarization occurred parallel to the calcium plateau. The depolarization induced by both agonists was reduced in the presence of La3+ and Ni2+. Verapamil (10 mumol/l) had no effect on either the calcium entry or the depolarization. Acetylcholine did not induce a [Ca2+]i peak when it was applied during the endothelin-induced [Ca2+]i plateau and vice versa. The [Ca2+]i plateau was not higher with concomitant than with single application of acetylcholine or endothelin. Thus, calcium entry and membrane depolarization induced by acetylcholine and endothelin seem to be mediated by a common La(3+)- and Ni(2+)-sensitive but verapamil-insensitive mechanism.
Acetylcholine↗
Synthesis of a cortisol-biotin conjugate and evaluation as a tracer in an immunoassay for salivary cortisol measurement.
Cortisol 3-(o-carboxymethyl)oxime (C3-CMO) and a commercially available biotin-hydrazide derivative were used to synthesize a C3-CMO-biotin conjugate. C3-CMO was converted into a N-hydroxysuccinimide ester derivative which in a second reaction step was allowed to interact with the hydrazide derivative of biotin. This simple-to-perform synthesis yielded a conjugate suitable for use as a tracer in immunoassays for cortisol measurement. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for variable end point determination by means of commercially available labeled avidin or streptavidin derivatives. Streptavidin-Europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA) throughout the study. In addition, colorimetric end point determination (ELISA) using streptavidin-alkaline phosphatase as a secondary probe was established and evaluated. Both forms of this non-isotopic assay showed excellent correlation with a commercially available radioimmunoassay adapted for salivary cortisol measurement. The lower detection limit was 0.43 nM for a 50 microliters salivary sample. The intra-assay coefficient of variation was 6.7, 4.7 and 4.0% at cortisol concentrations of 2.2, 5.5 and 13.2 nM, respectively (n = 37), and the corresponding inter-assay coefficients of variation were 9.0, 8.6 and 7.1% (n = 50). The competitive immunoassay requires 1.5 h incubation time and shows robust and reproducible performance. The C3-CMO-biotin conjugate allows for sensitive and flexible end point determination of salivary cortisol levels in immunoassays.
Biotin↗
Dehydroepiandrosterone (DHEA) levels in patients with prostatic cancer, heart diseases and under surgery stress.
DHEA levels in patients with prostatic cancer were significantly lower, but total and free testosterone (T) significantly higher as those in an age-matched control group. Therefore, the calculated quotients DHEA/free T and DHEA/T were especially different between both groups. DHEA and DHEAS levels in patients with heart diseases were also significantly lower but cortisol (F) levels were significantly higher as those in a control group. The quotients DHEA/F and DHEAS/F were also of greater significance between both groups than the hormone values alone. The response of DHEA and F levels in patients undergoing surgery showed an increase of both steroids under surgery. On the second postoperative day, however, F levels were still significantly higher but DHEA levels were significantly lower as the initial values. The differences between the initial values and those on the second postoperative day of F and DHEA showed a significant correlation, i.e. the higher the elevation of F levels above the initial values the greater was the diminution of DHEA levels below the initial values.
Adult↗
Elevation of cytosolic free calcium in cultured ciliary epithelial cells by histamine: effects of verapamil and staurosporine.
Treatment with histamine (10(-4) M) of cultured non-pigmented human ciliary epithelial cells led to a biphasic elevation of free intracellular calcium mediated by H1-receptors. The initial transient increase was due to Ca(2+)-release from intracellular calcium stores and could be blocked with a high concentration of verapamil (10(-4) M). The subsequent sustained elevation of cytoplasmic calcium caused by an influx of extracellular calcium was reduced by staurosporine (10(-7) M). We conclude that the sustained increase of cytoplasmic calcium by histamine may be partially mediated by activation of protein kinase C. Since depolarization of the cells had no effect on intracellular calcium, we conclude that typical voltage-operated calcium channels do not significantly influence intracellular calcium.
Alkaloids↗
Electrogenic sodium-bicarbonate cotransport in human ciliary muscle cells.
We investigated membrane voltage and intracellular pH (pHi) in cultured human ciliary muscle cells using a cell line (H7CM) and primary-cultured human ciliary muscle cells. 1) Resting potential was 58.9 +/- 1.0 mV in H7CM cells and 61.9 +/- 1.4 mV in primary cultures. The following data are from H7CM cells, but results from primary cultures were basically similar. 2) In HCO3(-)-CO2-buffered solution, removal of extracellular sodium resulted in a depolarization [change in membrane resistance (delta V) = 31.3 +/- 2.8 mV] that was less marked in the absence of HCO3(-)-CO2 (delta V = 0.5 +/- 2.6 mV) and reduced by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (delta V = 19.3 +/- 1.9 mV). 3) Removal of extracellular HCO3(-)-CO2 led to a depolarization (delta V = 13.2 +/- 0.8 mV) that was abolished in the absence of extracellular sodium and inhibited by DIDS. 4) Intracellular alkalinization led to a depolarization (delta V = 24.7 +/- 2.3 mV), and intracellular acidification resulted in a hyperpolarization (delta V = 9.4 +/- 1.1 mV) that was inhibited by DIDS and dependent on extracellular HCO3(-)-CO2 and sodium. 5) pHi backregulation after an acid load occurred in both the presence and absence of extracellular bicarbonate but not in the absence of extracellular sodium. Our data are consistent with an electrogenic Na(+)-HCO3- cotransport in human ciliary muscle cells, which is activated by intracellular acidification.
Bicarbonates↗
Endothelin-like immunoreactivity in the aqueous humour and in conditioned medium from cultured ciliary epithelial cells.
Endothelin-like immunoreactivity was detected in human (15.6 +/- 2.7 pg/ml) and bovine (11.1 +/- 0.98 pg/ml) aqueous humour of the eye. These concentrations are 2-3 times higher than the corresponding plasma levels. Cultured human nonpigmented ciliary epithelial cells released endothelin-like immunoreactivity with a maximum of 2.1 +/- 0.32 pg/(cm2* 48 h). The release was stimulated by fetal calf serum, thrombin, carbachol and phorbol ester and blocked by cycloheximide. Immunocytochemistry showed cytoplasmic staining of cultured human nonpigmented ciliary epithelial cells for endothelin-1. Endothelin-1 was shown to induce contractions in isolated human ciliary muscle by isometric force measurements. Endothelin in the aqueous humour may play a role in the regulation of intraocular pressure.
Animals↗
Membrane potentials in retinal capillary pericytes: excitability and effect of vasoactive substances.
Retinal capillary pericytes are believed to have a contractile function and to regulate retinal blood flow at the microvascular level. Membrane potential is an important control element for contractility in smooth muscle cells. In the present study, bovine retinal capillary pericytes have been grown in tissue culture and membrane potentials have been measured using glass microelectrodes. Resting potentials averaged -31 +/- 7 mV (n = 203). Relative K+ conductance was low, with a transference number for K+ of 0.16. Readdition of K+ to K(+)-depleted cells transiently hyperpolarized the membrane potential, probably by stimulating the electrogenic Na+/K+ transport. Repetitive spike-like depolarizations (action potentials) were induced by stimulating the Na+/K(+)-ATPase, by applying norepinephrine (10(-5) mol/l), and by adding 10 mmol/l Ba2+. These action potentials depended on the presence of extracellular Ca2+ and were inhibited by the Ca2+ antagonist nifedipine (10(-6) mol/l). Norepinephrine (10(-5) mol/l) depolarized the membrane by 7.4 +/- 3.5 mV (mean +/- SD, n = 49). This response was blocked by the alpha 1-antagonist prazosin (10(-5) mol/l). Histamine also led to a membrane depolarization of 8.6 +/- 2.8 mV (n = 49), which could be inhibited by the H1-antagonist diphenhydramine. Endothelin (10(-7) mol/l), vasopressin (10(-6) mol/l), and acetylcholine (10(-4) mol/l) had no major effects on membrane potential. The conclusion is that retinal capillary pericytes are excitable cells and react to several vasoactive substances.
Acetylcholine↗
Effect of histamine on the membrane potential of cultured human nonpigmented ciliary epithelial cells.
The effect of histamine on the membrane potential of cultured human nonpigmented ciliary epithelial cells was examined using intracellular microelectrodes. The resting voltage was Vm = 42 +/- 0.7 mV (n = 41). Histamine (10(-6) mol/l) induced transient hyperpolarization (V = 19.4 +/- 1.2 mV, n = 35) and subsequent depolarization when applied for more than 1 min. The histamine effect was completely inhibited by the H1 antagonist diphenhydramine (10(-6) mol/l), but not influenced by the H2 antagonist cimetidine (up to 10(-3) mol/l). When histamine was applied repeatedly in the absence of external calcium, the amplitude of the hyperpolarization decreased until finally no response was observed. Ba2+ (2 mmol/l) and quinidine (1 mmol/l), both inhibitors of potassium conductance, significantly reduced histamine-induced hyperpolarization. It was also reduced when cells were depolarized by 40 mmol/l extracellular potassium and almost completely abolished in the presence of 80 mmol/l external potassium. We conclude that H1 receptors mediate hyperpolarization by increasing potassium conductance, probably via transient release of intracellularly stored calcium.
Calcium↗
Effects of endothelin and calcium channel blockers on membrane voltage and intracellular calcium in cultured bovine trabecular meshwork cells.
The membrane voltage was measured in cultured bovine trabecular meshwork cells. External barium (Ba2+; 10 mM) evoked repetitive overshooting action potentials which were reversibly inhibited by 10(-6) M nifedipine but insensitive to tetrodotoxin (10(-5) M). In contrast, no action potentials could be induced in bovine corneal endothelial cells. The vasoactive peptide endothelin (10(-9)-10(-6) M) induced dose-dependent depolarizations. The intracellular calcium concentration measured by the fura-2 method increased after endothelin application. An initial peak was followed by a sustained plateau. The effect of endothelin on the membrane potential and intracellular calcium value is typical for smooth muscle cells showing a contractile response to the peptide. Recent studies have shown the contractility of bovine trabecular meshwork. We conclude that calcium-channel antagonists and endothelin may alter the aqueous humour outflow through the trabecular meshwork.
Animals↗