Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors.

Pharmacological interventions that increase myofiber size counter the functional decline of dystrophic muscles. We show that deacetylase inhibitors increase the size of myofibers in dystrophin-deficient (MDX) and alpha-sarcoglycan (alpha-SG)-deficient mice by inducing the expression of the myostatin antagonist follistatin in satellite cells. Deacetylase inhibitor treatment conferred on dystrophic muscles resistance to contraction-coupled degeneration and alleviated both morphological and functional consequences of the primary genetic defect. These results provide a rationale for using deacetylase inhibitors in the pharmacological therapy of muscular dystrophies.

"Leaching or not leaching": an alternative approach to antimicrobial materials via copolymers containing crown ethers as active groups.

In this work, new copolymers containing either MMA and 18C6 crown-ether pendants, or PEG, MMA and 18C6 crown-ether pendants were synthesized to test the idea that sequestering structural alkali-earth ions from the bacterial outer membrane (OM) may lead to bacterial death. The copolymers were obtained either via uncontrolled radical polymerization or ATRP; the latter approached allowed us to produce not only linear copolymers but also branched Y-like structures. After checking for the capability of complexing magnesium and calcium ions, the antimicrobial activity of all copolymers was tested placing their casted plaques in contact with pure water E. coli suspensions. All plaques adsorbed alkali-earth ions and killed bacteria, albeit with different antimicrobial efficiencies. Differences in the latter characteristic were attributed to different plaque roughness. The role of the 18C6 crown-ether pendants was elucidated by pre-saturating plaques with Mg/Ca ions, the marked reduction in antimicrobial efficiency indicating that losing the latter from OM due to surface complexation does play an important role in killing bacteria at short (<5 h) contact times. At longer times, the mode of action is instead related to the poly-cationic nature acquired by the plaques due to ion sequestering.

Protein cross talking through osmotic work: the free energy of formation of the MgADP-myosin complexes at the muscle protein osmotic pressure.

A method is presented to determine the energy of formation of the myosin-ADP complexes at the muscle protein osmotic pressure. It is found that, at 18 kP, the putative protein osmotic pressure in skeletal muscle, the increase of MgADP from 0.05 to 2 mmolal, increases the free energy of myosin-ADP and of myosin-(ADP)2 by 0. 756 and by 9.85 kJ/mol, respectively, and decreases the free energy of myosin by 8.34 kJ erg/mol. It is pointed out that the local changes of water chemical potential, induced by the binding of MgADP to myosin, can be sensed by other structures of the contractile machinery, which per se may even be insensitive to MgADP. Cross talking between macromolecules can thus be achieved by changes of the water chemical potential.

Is nebulin truly a component of the thin filament?

Thin filaments were prepared from rabbit and beef skeletal muscle with three different procedures, both at high and low ionic strength. Nebulin was always found to be associated with the myosin fraction and was always absent from the thin filament fraction.

On the elastic properties of tetramethylrhodamine F-actin.

(Iodoacetamido)tetramethylrhodamine disrupts F-actin. At the 1:1 fluorophore to actin (as monomer) ratio approximately 80% of the protein becomes non-sedimentable. The fluorescent, non-sedimentable actin copolymerizes with G-actin to yield fluorescent filaments. The tensile strength of these filaments changes with the ratio of the fluorescent non-sedimentable actin to the G-actin, being 1.6 pN, 2.9 pN and 3.6 pN at the 1/4, 2/3 and 1/1 ratios, respectively. These tensile strengths are approximately two orders of magnitude lower than those obtained by decoration of F-actin with phalloidin.

Dissecting the free energy of formation of the 1:1 actomyosin complex.

The behaviour of solutions of pure myosin, of pure F-actin and of the equimolar mixture of myosin and of F-actin is studied. It is found that the chemical potential of the two proteins, in separate solutions, increases monotonically with the increase of protein osmotic pressure. A method is presented to determine the chemical potential of the 1:1 actin-myosin complex formed from equimolar solutions of myosin and of F-actin (as monomer). This is the first evaluation of the chemical potential of actomyosin under conditions similar to those of skeletal muscle. It is found that the filament suspensions of myosin and of the 1:1 actin-myosin complex display a high non-ideal behavior as well as distinctly different energy profiles as a function of protein osmotic pressure. This supports the hypothesis that, in muscle: (a) detached cross-bridge change significantly their free energy when sarcomere is shifting from the relaxed to the active or to the rigor state; and (b) the cross-bridge attachment-detachment process is accompanied by changes of muscle protein osmotic pressure.

Metabolic stability of glutaraldehyde cross-linked peptide DNA condensates.

The stability of peptide DNA condensates was examined after introducing glutaraldehyde to cross-link surface amine groups. A 20 amino acid peptide (CWK(18)) was used to condense DNA into small (70 nm) condensates. The reaction between glutaraldehyde and peptide DNA condensates was indirectly monitored using a fluorescence-based assay to establish reaction completion in 4-5 h when using glutaraldehyde-to-peptide ratios of 1 to 4 mol equiv. Higher levels of glutaraldehyde cross-linking led to significant increases in particle size. The improved stability imparted by glutaraldehyde cross-linking was demonstrated by the increased resistance of DNA condensates to shear stress induced fragmentation. The cross-linked condensates were also significantly more resistant to in vitro metabolism by serum endonucleases. A decrease in the magnitude of transient gene expression was determined for cross-linked DNA condensates which also resulted in a 10-day steady-state expression when cross-linking with 4 mol equiv of glutaraldehyde. The results suggest that cross-linking DNA condensates may provide a means to alter the time course of transient gene expression by inhibiting DNA metabolism.

Stability of peptide-condensed plasmid DNA formulations.

Low molecular weight homogeneous peptides were used to form peptide/DNA condensates. A peptide possessing 18 lysines was found to protect plasmid DNA from serum endonuclease and sonicative-induced degradation whereas a shorter peptide possessing 8 lysines dissociated in 0.1 M sodium chloride and failed to protect DNA from enzymatic degradation. Peptide-condensed DNA showed no change in the ratio of supercoiled to circular DNA following 100 W sonication for up to 60 s and was able to transfect HepG2 cells with equivalent efficiency as untreated condensed plasmid DNA. Alternatively, uncondensed plasmid DNA was rapidly fragmented by sonication and serum endonucleases and resulted in negligible gene expression following condensation with peptide. Cationic lipid/DNA complexes were only partially effective at stabilizing DNA in serum compared to the complete stabilization afforded by peptide/DNA condensation. These results indicate that the stabilization afforded by condensation with a peptide protects DNA during formulation and preserves its structure in serum. These functions are important to achieve optimal gene expression from a nonviral gene delivery system.

Strategies for maintaining the particle size of peptide DNA condensates following freeze-drying.

The particle size of peptide DNA condensates were studied after freeze-drying and rehydration as a function of sugar excipient, concentration, pH, DNA concentration, and peptide condensing agent. In the absence of an excipient, freeze-dried 50 microg/ml AlkCWK(18) (iodoacetic acid alkylated Cys-Typ-Lys(18)) DNA condensates formed large fibrous flocculates on rehydration. Of the sugars tested as lyoprotectants, sucrose proved most effective at preserving particle size during rehydration. The addition of 5 wt/vol% sucrose preserved a mean particle diameter of less than 50 nm during rehydration of AlkCWK(18) DNA condensates prepared at DNA concentrations up to 200 microg/ml; however, higher DNA concentrations led to the formation of insoluble fibrous flocculates. Substitution of polyethylene glycol (PEG)-CWK(18) as a DNA condensing peptide eliminated the need for sucrose, resulting in peptide DNA condensates that retained particle size when rehydrated in water or normal saline at concentrations up to 5 mg/ml. The results suggest that sucrose functions primarily as a bulking agent during freeze-drying that only preserves the particle size of AlkCWK(18) DNA condensates up to a maximum concentration of 200 microg/ml. Alternatively, the steric layer created on the surface of PEG-CWK(18) DNA condensates provides far more efficient lyoprotection, preserving their particle size at a concentration of 5 mg/ml without a bulking agent.

Therapeutic thrombocytapheresis: a review of 132 patients.

Therapeutic thrombocytapheresis has been used is an attempt to obtain a rapid cytoreduction in thrombocythemic patients, in order to prevent or reduce thrombosis or bleeding complications. Long-term cytoreduction has to be performed with chemotherapy. We have treated with thrombocytapheresis 132 patients with increased platelet count (76 with thrombosis and/or bleeding) due to splenectomy, essential thrombocythemia and other myeloproliferative disorders: a comparative study has been performed to find a platelet count that could be helpful in preventing thrombotic and hemorrhagic complications and the role of diagnosis in predicting patient's risk.

[Methemoglobinemia; a description of a case of NADH methemoglobulin reductase deficiency]. / La metaemoglobinemia; Descrizione di un caso da deficit di NADH-metaemoglobin-reduttasi.

A case of NADH-methaemoglobin-reductase deficiency in a 64 year old man with marked cyanosis and without evidence of cardiac or pulmonary diseases is reported. The level of methaemoglobin was 36% and reached 25% after treatment with ascorbic acid. Erythrocyte NADH-methaemoglobin-reductase activity was only 5% of normal value. Some erythrocyte biochemical and enzymatic characteristics have been evaluated. A family study has also been carried out. Our patient has been considered homozygous for NADH-methaemoglobin-reductase deficiency.

Albumin/gentamicin microspheres produced by supercritical assisted atomization: optimization of size, drug loading and release.

In this work, the supercritical assisted atomization (SAA) is proposed, for the first time, not only as a micronization technology but also as a thermal coagulation process for the production of bovine serum albumin (BSA) microspheres charged with Gentamicin sulfate (GS). Particularly, different water solutions of BSA/GS were processed by SAA to produce protein microspheres with different size and antibiotic content. SAA precipitation temperature was selected in the range 100-130 °C to generate protein coagulation and to recover micronized BSA in form of hydrophobic aggregates; GS loading was varied between 10% and 50% (w/w) with an encapsulation efficiency which often reached 100%. In all cases, spherical and noncoalescing particles were successfully produced with a mean particle size of 2 µm and with a standard deviation of about ±1 µm. The microspheres also showed a good stability and constant water content after 60 days of storage. The release profiles of the entrapped drug were monitored using Franz cells to evaluate the possible application of the produced microspheres in wound dressing formulations. Particularly, the microspheres with a BSA/GS ratio of 4:1 after the first burst effect (of 40% of GS loaded) were able to release the GS continuously over 10 days.

Rhodamine phalloidin F-actin: critical concentration versus tensile strength.

The mechanic and elastic properties of rhodamine phalloidin F-actin were investigated as a function of the ionic strength and in the absence of Mg2+. By increasing ionic strength from 3 to 19 mM, critical concentration decreased from 146 to 36 nM and the yield strength increased from 5.6 pN to 28.6 pN. At the ionic strength of 12-13 mM, the elastic modulus by stretching increased by 330-430 kP. nm-1 up to the break point, where it was 38-44.2 MP. The work required to break the filament, 403-439 kJ.M-1 provides an estimate of the free energy of annealing of rhodamine phalloidin F-actin, the annealing constant being 2.8 x 1074 M-1.

Involvement of P1-purinoreceptors in the relaxing effect of adenosine in rat duodenum.

1. In isolated segments of rat duodenum, adenosine (50 microM-2 mM) caused a very rapid and short-lasting relaxation that was associated with a marked decrease in the amplitude of spontaneous contractions. 2. Theophylline (0.1-0.8 mM) and 8-phenyltheophylline (1-10 microM) antagonized, in a concentration-dependent manner, the effects of 0.3 mM adenosine on smooth muscle tension and spontaneous activity. 3. The concentration-response curves for adenosine (50 microM-2 mM) were progressively shifted to the right by increasing concentrations of theophylline and of 8-phenyltheophylline, with no change in the maximum effect. 4. 8-Phenyltheophylline (5 microM) did not affect relaxations induced by noradrenaline (0.5 microM) and by isoprenaline (5 nM). 5. These results indicate that the effects exerted by adenosine on rat duodenum are mediated by P1-purinoreceptors.

Correlation of the specific IgE in serum and nasal secretions with clinical symptoms in atopics.

In a unselected population of 133 young adults studied by prick testing to common allergens three groups were identified: eleven subjects with positive skin test responses and clinical symptoms of allergy; ten subjects only with psitive skin tests and the remainder with negative skin tests. All subjects with positive skin tests (with an without symptoms) were studied by RAST on the serum and nasal secretions. Specific and non-specific bronchial provocation tests (BPT) were also carried out. The serum RAST was positive in all subjects with positive skin tests, and there was good correlation between high levels of circulating specific IgE and the presence of clinical symptoms. The RAST of nasal secretions was negative in most symptom-free subjects and as a diagnostic test it was slightly better than the serum RAST. BPTs with extracts of the relevant allergens caused bronchospasm in every subject with a positive nasal secretion RAST. Only two subjects out of fifteen with a positive response were clinical asthmatics. Our results cast doubt on the clinical relevance of the BPT as it is usually conducted.

Erythrocyte lysis by monocytes: investigations on the mechanism and role of the target cell hydrogen peroxide catabolizing pathways.

The erythrocyte (RBC) lysis by human monocytes incubated with opsonized zymosan particles (OPZ), was inhibited by catalase, chloride-free medium, azide and hypochlorous acid (HOCl) scavengers (taurine, alanine). These findings suggest the requirement for the HOCl-generating myeloperoxidase-hydrogen peroxide-chloride system (MPO-H2O2-Cl- system). The HOCl-dependent lysis was increased by inhibiting RBC catalase with aminotriazole (AT). Conversely, the inhibition of RBC glutathione cycle with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) had no detectable effect. Moreover, the recovery of H2O2 and HOCl from OPZ-triggered monocytes was reduced by the presence of RBCs through a process almost completely preventable by pulsing RBCs with AT but not with BCNU. Thus, it appears that RBC targets protect themselves by consuming, primarily via catalase, significant amounts of monocyte-derived H2O2 with a consequent impairment of the HOCl generation. The results suggest a potential role of target cells in modulating the cytolysin production by monocytes.

Myofibrils of skeletal muscle: the activity coefficient of orthophosphate.

In the myofibrils of skeletal muscle, at 22 degrees C, pH 7.1 and at the physiological protein osmotic pressure of 1.8 x 10(5) dynes/cm2, orthophosphate behaves quite ideally, the activity coefficient being 0.85. Under the same conditions and at saturation, 2.67 mumoles of orthophosphate are bound per gram of dry myofibrils, with a dissociation constant of 7 x 10(-5) molal. Work is in progress to determine the activity coefficients of adenine nucleotide analogues. This work is needed to assess the actual value of the free energy of hydrolysis of ATP in muscle.