The Smc5/6 complex: more than repair?

Through its functions in chromosome replication, segregation, and repair, the Smc5/6 complex has a central role in the maintenance of genome stability. The complex is part of the family of structural maintenance of chromosome protein complexes that also includes cohesin and condensin. Mutations in any of these complexes disrupt chromosome segregation and render cells hypersensitive to different types of DNA damage. The chromosome mis-segregation phenotypes in cohesin and condensin mutants can be attributed to their functions in sister chromatid cohesion and chromosome condensation, respectively. Cohesin-dependent chromatid cohesion is also needed for DNA double-strand break repair, whereas condensin is required for repair of single-strand breaks. How Smc5/6 promotes chromosome stability is largely unknown. Accumulating data suggest that it prevents accumulation of aberrant DNA links between sister chromatids created during repair by homologous recombination. A long-standing idea is that it also has a role in the maintenance of nondamaged chromosomes. Here, we present an overview of the current knowledge of Smc5/6 and discuss a possible nonrepair role of the complex.

Expression of estrogen receptor-alpha and -beta in anterior vaginal walls of genuine stress incontinent women.

Our objective was to study the expression of estrogen receptor (ER) isoforms, ER-alpha and ER-beta, in the anterior vaginal wall of menopausal and fertile women with genuine stress incontinence (SI) by immunohistochemistry and Western blot analysis. Eighteen menopausal women with SI who either were or were not taking estrogen/progestin replacement therapy and 14 fertile women with SI who either were or were not taking contraceptives were enrolled in the study. Biopsies from the suburethral anterior vaginal wall were obtained at tension-free vaginal tape (TVT) operation. Monoclonal antibody to ER-alpha and polyclonal antibody to ER-beta were used to stain frozen sections of vaginal tissue. The receptor expressions were scored based on percentage of positive cells. ER-alpha was detected in vaginal epithelial, stromal and smooth muscle cells. In menopausal SI women ER-alpha was detected significantly more frequently in the vaginal walls of estrogen/progestin-treated patients than in those who were untreated. Fertile SI women had significantly higher expression of ER-alpha than menopausal SI women. ER-alpha was not observed in vaginal blood vessels. ER-beta was detected in epithelial and vascular smooth muscle cells of the vagina. No significant difference in ER-beta expression was observed between different groups of patients. The expression of ER-alpha was not correlated with that of ER-beta. Both ER-alpha and -beta were detected, indicating a potential role for both types of estrogen receptor in the human vaginal wall. The expression of ER-alpha, but not of ER-beta, in menopausal SI women was regulated by estrogen/progestin replacement therapy. The presence of ER-beta in vaginal vascular smooth muscle cells raises the possibility of vascular effects of estrogen on the human vaginal wall.

Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.

The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process. Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair. This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation. The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair. We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast. Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair. Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.

The role of profilin in actin polymerization and nucleotide exchange.

Properties of human profilin I mutated in the major actin-binding site were studied and compared with wild-type profilin using beta/gamma-actin as interaction partner. The mutants ranged in affinity, from those that only weakly affected polymerization of actin to one that bound actin more strongly than wild-type profilin. With profilins, whose sequestering activity was low, the concentration of free actin monomers observed at steady-state of polymerization [Afree], was close to that seen with actin alone ([Acc], critical concentration of polymerization). Profilin mutants binding actin with an intermediate affinity like wild-type profilin caused a lowering of [Afree] as compared to [Acc], indicating that actin monomers and profilin:actin complexes participate in polymer formation. With a mutant profilin, which bound actin more strongly than the wild-type protein, an efficient sequestration of actin was observed, and in this case, the [Afree] at steady state was again close to [Acc], suggesting that the mutant profilin:actin had a greatly lowered ability to incorporate actin subunits at the (+)-end. The results from the kinetic and steady-state experiments presented are consonant with the idea that profilin:actin complexes are directly incorporated at the (+)-end of actively polymerizing actin filaments, while they do not support the view that profilin facilitates polymer formation.

Prejunctional facilitatory and inhibitory modulation of parasympathetic nerve transmission in the rabbit urinary bladder.

Release of [3H]choline and muscle contraction in response to electrical field stimulation were measured from rabbit detrusor muscle strips previously loaded with [3H]choline. The importance of different stimulation frequencies (1 and 10 Hz) for activating either facilitatory or inhibitory prejunctional effects was examined in the presence of muscarinic and adrenergic (alpha2) receptor selective substances. At 1 Hz, neither [3H]choline overflow nor contraction was affected by the M1-selective receptor antagonist pirenzepine (10(-7) M), whereas overflow and contraction decreased at 10 Hz. The M1-selective receptor agonist McN-A-343 (10(-6) M) caused no significant changes except for reducing contractions at 10 Hz. The M2-selective receptor antagonist methoctramine (10(-6) M), on the other hand, increased overflow as well as contraction at both frequencies, most conspicuously at 1 Hz. Atropine (10(-7) M) caused a significant increase with respect to overflow only at 1 Hz, while quite the opposite effect occurred with respect to contractions (reduced only at 10 Hz). Clonidine (10(-6) M) induced inhibition of [3H]choline overflow at 10 Hz only, but without significantly changing contractile responses. The results show that in the rabbit urinary bladder a muscarinic autoreceptor mediated inhibition (M2) of the transmitter release dominates during low frequency stimulation and that a facilitation (M1) may be present at stimulations with higher frequencies. However, this amplification may also be influenced by alpha2-adrenoceptor mediated inhibition.

Characterization of a mutant profilin with reduced actin-binding capacity: effects in vitro and in vivo.

We are investigating structure-function relationships in profilin and actin by site-specific mutagenesis using a yeast, Saccharomyces cerevisiae, expression system to produce wild-type and mutant proteins. This paper shows that deleting proline 96 and threonine 97, which are located close to the major actin binding site on profilin, did not significantly alter the interaction between profilin and phosphatidylinositol 4,5-bisphosphate, nor did it affect the profilin:poly(L-proline) interaction. The mutant protein, however, had a lower capacity to bind to actin in vitro than wild-type profilin, though it showed a slightly increased profilin-enhanced nucleotide exchange on the actin. When microinjected into Swiss 3T3 mouse fibroblasts or porcine aortic endothelial cells, the mutant profilin did not change the organization of the microfilament system like the wild-type profilin did. This provides further evidence that profilin controls microfilament organization in the cell by interacting directly with actin.

Physicochemical characterisation of mangafodipir trisodium.

PURPOSE: To determine the structure and various physicochemical properties of mangafodipir (MnDPDP) trisodium, the active ingredient of Teslascan, a new-organ-specific contrast medium for MR imaging. MATERIAL AND METHODS: The structure of MnDPDP trisodium crystals was determined by X-ray crystallography. The possible existence of polymorphism in MnDPDP trisodium was evaluated by powder X-ray diffraction, optical microscopy, thermal analysis and IR spectroscopy. In addition, various spectroscopic techniques and physicochemical measurements were used for characterisation of MnDPDP trisodium. RESULTS: The crystallographic data obtained for MnDPDP trisodium show that the general core structure of the MnDPDP anion is similar to that seen in related substances. The metal coordination geometry is a distorted octahedron defined by 2 phenolate oxygens, 2 carboxylate oxygens and 2 amine nitrogens. The unit cell contains 2 MnDPDP anions, 6 sodium ions and 50 water molecules. The various spectroscopic data are consistent with the structure determined by X-ray crystallography. The product (Teslascan) has low viscosity, is isotonic with blood and has a physiological pH. CONCLUSION: MnDPDP trisodium is a crystalline, hygroscopic solid which is readily soluble in water. No evidence of polymorphism was seen in the samples studied.

Crystal size and properties of superparamagnetic iron oxide (SPIO) particles.

The properties of a superparamagnetic iron oxide (SPIO) model contrast agent have been studied. The test material, HEP-SPIO, contained iron oxide multicrystal agglomerates coated with heparin, polyanionic, naturally occurring glycosaminoglycan. Fractionation of the HEP-SPIO suspension showed the existence of colloidally stable particles ranging from approx. 100 nm down to single crystal sizes. The small (< 20 nm) particles represented the major number fraction of particles present, but only approx. 2% of the total iron oxide mass. The volume weighted average diameter of the individual iron oxide crystals forming the multicrystal agglomerates was found to be 11-12 nm using transmission electron microscopy and vibrating sample magnetometry (VSM) techniques. Comparable results were obtained with X-ray diffraction and Mössbauer spectroscopy. A number of additional SPIO properties could also be determined on a routine VSM, such as the distribution standard deviation for the log-normal distribution of crystal sizes, the magnetic susceptibility, the magnetic remanence, and the intrinsic magnetization (magnetic moment) of the iron oxide. These parameters are useful tools for evaluation of the magnetic characteristics and contrast efficacy of SPIO contrast agents.

Isolation and characterization of two mutants of human profilin I that do not bind poly(L-proline).

A simple procedure for the isolation of profilin mutants having a reduced capacity to bind poly(L-proline) is used to isolate two mutants of human profilin I, W3N and H133S. Binding of the mutants to poly(L-proline), actin, and phosphatidylinositol (4,5)-bisphosphate (PIP2) was studied. Both mutations abolished the poly(L-proline)-binding activity of profilin. This suggests that the arrangement of the N- and C-terminal helices forming the poly(L-proline)-binding site depends on the stabilizing interaction between W3 and W31 in the underlying beta-strand, and that the H133S mutation in the C-terminal helix also must have distorted the arrangement of the terminal helices. Both mutations caused a reduced affinity for actin, with the W3N replacement having the most pronounced effect. This shows that structural changes in the poly(L-proline)-binding region of profilin can affect the distantly located actin-binding site. Thus, ligands influencing the structure of the poly(L-proline)-binding site may regulate actin polymerization through profilin. This is consonant with the finding that PIP2, which changes the tryptophan fluorescence in wild-type profilin and dissociates the profilin:actin complex in vitro, binds more strongly to the W3N mutant profilin. Thus, the poly(L-proline)-binding surface represents a crucial regulatory site of profilin function.

In vivo and in vitro effects of muscarinic receptor antagonists on contractions and release of [3H]acetylcholine in the rabbit urinary bladder.

The functional effects of muscarinic receptor antagonists were examined in vivo and in vitro on the rabbit urinary bladder. Inhibitory effects on carbachol-evoked contractions of detrusor strips were pronounced for 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; -logIC50: 8.64), p-fluoro-hexahydro-sila-diphenidol (pFHHSiD; 7.84) and atropine (8.27), while they were less pronounced for pirenzepine (6.62) and methoctramine (5.36). 4-DAMP and methoctramine increased 3H overflow from [3H]choline-labelled strips in response to electrical stimulation, contrary to pirenzepine, which decreased the overflow. Concomitant contractions were markedly reduced by 4-DAMP and by pirenzepine, but not by methoctramine. The -logIC50 estimations for atropine-sensitive electrically evoked contractions revealed methoctramine (4.85) to be less potent on nerve-evoked contractions than on carbachol-evoked contractions, in contrast to pirenzepine (7.15) and 4-DAMP (9.15). The effects of the antagonists in anaesthetized rabbits resembled those in vitro. Thus, muscarinic receptors in the rabbit urinary bladder are heterogeneous; prejunctional facilitatory (M1) and inhibitory (M2) for acetylcholine release, and postjunctional muscarinic M3 receptors mediating contractile responses.

An experimental model for long-term examinations of urethral and uterine pressures in conscious dogs: effect of a highly selective compound contracting urethral smooth muscles.

An experiment model was designed for studies of urethral and uterine pressures in a conscious animal, thereby avoiding the interference of anesthesia on these responses. Dogs were trained to stand in cradles for an experimental period of 3 hours, during which urethral and uterine pressures and ECG were recorded. Substances were administered either orally or intravenously. Intravenously given phenylpropanolamine (PPA; 0.3 mg./kg.) and a newly synthetized compound S113 (0.5 mg./kg. intravenously) increased the urethral pressure by about 30 and 100%, respectively. Oral administration of PPA (3.5 mg./kg.) and S113 (6 mg./kg.) increased it by about 60 and 85%, respectively. In contrast to PPA, S113 evoked no cardiac effects. Whereas PPA had pronounced effects on uterine contractility, S113 had only minor effects. Furthermore, the responses in the experiments were stable and reproducible. This dog model seems to be useful for long-term recordings of urethral and uterine pressures.

Synthesis and characterization of iodixanol.

Iodixanol (Visipaque) is a new nonionic roentgen contrast medium intended for general use. Visipaque is a pharmaceutical formulation of iodixanol which is isotonic and iso-osmotic with blood. Two synthetic routes from 5-nitro-isophthalic acid to iodixanol are described. The chemical structure is confirmed spectroscopical data ((1)H-NMR, (13)C-NMR, FAB-MS, UV, IR and Raman). Chromotographic characteristics are related to the isomerism of iodixanol.

Physicochemical properties of iodixanol.

Iodixanol, the radiopaque in Visipaque, is a new nonionic, dimeric roentgen contrast medium for intravascualr use. Compared to aqueous solutions of nonionic monomers, which have higher osmolality than blood, aqueous solutions of iodixanol have a lower osmolality due to dimeric structure of the molecule. As a consequence of this advantageous property, solutions of all clinical concentrations of iodixanol can be made isotonic by the addition of salts of the key electrolytes sodium and calcium to the formulation. The viscosity of all iodixanol (Visipaque) solutions is less than or equal to that of iohexol (Omnipaque) 350 mg I/ml. Iodixanol itself is an amorphorus and hygroscopic solid which is freely soluble in water. Partition coefficients show that iodixanol is even more hydrophilic than the nonionic monomers such as iohexol. The high hydrophilicity and the good aqueous solubility of iodixanol are due to the hydroxyl group in the dimer linkage and the hydrophilic amide side chains of the molecule.

Magnetic characterization of iron oxides for magnetic resonance imaging.

The OMP particle and four additional iron oxide samples were investigated in terms of zero field cooled magnetization (ZFCM) curves and hysteresis loops. The observation of superparamagnetic blocking and the absence of magnetic remanence demonstrated that the samples are superparamagnetic at room temperature. The magnitude of the ZFCM peak temperatures are in qualitative agreement with the iron oxide crystal size. One sample deviated from the remaining four in that it had a significantly lower magnetic moment and an irregular ZFCM curve showing the presence of various less magnetic phases. The similar results obtained with the OMP particles and the individual OMP crystallites without the polymer support show that the superparamagnetic properties of the individual OMP crystallites are retained on the OMP particle. Depending on the application, the ZFCM experiment may be viewed as an alternative or a supplement to Mössbauer spectroscopy in studies of the superparamagnetic blocking of iron oxides.

Nasal mucosal congestion after treatment with bromocriptine.

Nine women were given bromocriptine a few days after delivery in order to inhibit lactation. Nasal airway resistance to airflow (NAR) was recorded and blood samples were taken before treatment with bromocriptine, 2 to 3 hours after the first dose of this drug, and after 3 to 5 days on this treatment. All the women had increased nasal congestion after bromocriptine and NAR rose significantly. The prolactin, estradiol, and progesterone hormone levels decreased significantly, but no significant difference was found in the levels of thyroid-stimulating hormone (TSH) and vasoactive intestinal polypeptide (VIP). The bromocriptine effect may be caused by different mechanisms.

A pharmacological in vitro study of the mouse urinary bladder at the time of acute change in bladder reservoir function after irradiation.

Mouse urinary bladder strips were investigated as to whether the acute change in bladder reservoir function seen after irradiation might be due to major changes in basic nerve and smooth muscle functions. The release mechanism of acetylcholine, cholinergic and non-cholinergic nerve activation explored by indomethacin and potassium channel activation were investigated. It was concluded that the normal mouse bladder is partly cholinergically and partly non-cholinergically innervated. The role of acetylcholine is of the same importance as in other rodents. However, it was not possible to distinguish any difference between normal and irradiated mouse bladders in respect to nerve and smooth muscle function.

Effects of parasympathetic decentralization on some nerve-mediated functions in the feline urinary bladder.

Isolated detrusor muscle from control cats and cats parasympathetically decentralized for 3 and 10 weeks responded to electrical field stimulation by tetrodotoxin-sensitive, frequency-dependent contractions. There were no significant differences in the frequencies producing 50% response. However, the amplitude of the scopolamine-resistant contraction was distinctly lower in decentralized bladder preparations than in controls. Decentralized detrusor muscle (10 weeks) showed no increased response to alpha-adrenoceptor stimulation (phenylephrine, clonidine, noradrenaline), but the sensitivity to carbachol (at 3 and 10 weeks) was significantly decreased compared with controls. The release of [3H]noradrenaline was decreased by carbachol 10(-7) to 10(-5) M both in controls and in decentralized (10 weeks) detrusor muscle, the decrease being significantly more pronounced in the decentralized group at carbachol concentrations of 10(-7) and 10(-6) M. There were no differences between controls and decentralized bladders of muscarinic receptor concentration, but decentralization significantly reduced the affinity for the radioligand used to label the receptors. Thus, the main changes caused by parasympathetic decentralization of the feline bladder seem to be a reduced post-junctional, but an increased prejunctional response to muscarinic receptor stimulation.

Contribution to suicide risk assessment: II. On the practice of suicide risk assessment.

A simple method for predicting new crises after parasuicide is introduced. This method makes it possible, without any previous knowledge, to filter out 50% of the risk patients, who may then receive special preventive activities. An examination of the practice of evaluating the danger of suicide shows that such judgments are based on four criteria: (1) risk groups, (2) crises and crisis progression, (3) suicidal development, and (4) the presuicidal syndrome.