Impact of TGF-ß family-related growth factors on chondrogenic differentiation of adipose-derived stem cells isolated from lipoaspirates and infrapatellar fat pads of osteoarthritic patients.

The success of cell-based approaches for the treatment of cartilage defects requires an optimal autologous cell source with chondrogenic differentiation ability that maintains its differentiated properties and stability following implantation. The objective of this study was to compare the chondrogenic capacity of mesenchymal stem cells (MSCs) isolated from lipoaspirates (ASCs) and the infrapatellar fat pad (IFPSCs) of osteoarthritic patients and treated with transforming growth factor (TGF)-ß family-related growth factors. Cells were cultured for 6 weeks in a 3D pellet culture system with the chimeric activin A/bone morphogenic protein (BMP)-2 ligand (AB235), the chimeric nodal/BMP-2 ligand (NB260) or BMP-2. To investigate the stability of the new cartilage, ASCs-treated pellets were transplanted subcutaneously into severe combined immunodeficiency (SCID) mice. Histological and immunohistochemical assessment confirmed that the growth factors induced cartilage differentiation in both isolated cell types. However, reverse transcription-quantitative PCR results showed that ASCs presented a higher chondrogenic potential than IFPSCs. In vivo results revealed that AB235-treated ASCs pellets were larger in size and could form stable cartilage-like tissue as compared to NB260-treated pellets, while BMP-2-treated pellets underwent calcification. The chondrogenic induction of ASCs by AB235 treatment was mediated by SMAD2/3 activation, as proved by immunofluorescence analysis. The results of this study indicated that the combination of ASCs and AB235 might lead to a cell-based cartilage regeneration treatment.

Activin A/BMP2 chimera AB235 drives efficient redifferentiation of long term cultured autologous chondrocytes.

Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. Here we have developed a reproducible and efficient chondrogenic protocol to redifferentiate chondrocytes isolated from osteoarthritis (OA) patients. We used morphological, histological and immunological analysis together with a RT-PCR detection of collagen I and collagen II gene expression to show that chondrocytes isolated from articular cartilage biopsies of patients and subjected to long-term culture undergo dedifferentiation and that these cells can be redifferentiated following treatment with the chimeric Activin A/BMP2 ligand AB235. Examination of AB235-treated cell pellets in both in vitro and in vivo experiments revealed that redifferentiated chondrocytes synthesized a cartilage-specific extracellular matrix (ECM), primarily consisting of vertically-orientated collagen fibres and cartilage-specific proteoglycans. AB235-treated cell pellets also integrated into the surrounding subcutaneous tissue following transplantation in mice as demonstrated by their dramatic increase in size while non-treated control pellets disintegrated upon transplantation. Thus, our findings describe an effective protocol for the promotion of redifferentiation of autologous chondrocytes obtained from OA patients and the formation of a cartilage-like ECM that can integrate into the surrounding tissue in vivo.

Structure of 4-diphosphocytidyl-2-C- methylerythritol synthetase involved in mevalonate- independent isoprenoid biosynthesis.

The YgbP protein of Escherichia coli encodes the enzyme 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) synthetase, a member of the cytidyltransferase family of enzymes. CDP-ME is an intermediate in the mevalonate-independent pathway for isoprenoid biosynthesis in a number of prokaryotic organisms, algae, the plant plastids and the malaria parasite. Because vertebrates synthesize isoprenoid precursors using a mevalonate pathway, CDP-ME synthetase and other enzymes of the mevalonate-independent pathway for isoprenoid production represent attractive targets for the structure-based design of selective antibacterial, herbicidal and antimalarial drugs. The high-resolution structures of E. coli CDP-ME synthetase in the apo form and complexed with both CTP-Mg2+ and CDP-ME-Mg2+ reveal the stereochemical principles underlying both substrate and product recognition as well as catalysis in CDP-ME synthetase. Moreover, these complexes represent the first experimental structures for any cytidyltransferase with both substrates and products bound.

Identification of a small-molecule binding site at the dimer interface of the HIV integrase catalytic domain.

Integration of the reverse-transcribed HIV cDNA into the host DNA is a required step in viral replication. The virus-encoded integrase protein catalyzes the initial DNA breaking and joining reactions that mediate cDNA integration. Here, the identification by X-ray crystallography of a small-molecule binding site on the integrase catalytic domain is reported. The small-molecule family studied consists of a core of arsenic or phosphorus surrounded by four aromatic groups. Two arsenic derivatives were visualized bound to integrase. In each case, two molecules bound at symmetry-related sites on the catalytic domain dimer interface. The first compound studied, tetraphenyl arsonium, did not inhibit integrase. However, a synthetic compound substituting a catechol for one of the phenyl rings, dihydroxyphenyltriphenylarsonium, bound to the same site and did inhibit the enzyme. Changes in the vicinity of the catalytic site were seen with the inhibitory compound only, potentially explaining its mechanism of action. Further substituting phosphonium for arsonium yielded a compound with an IC(50) in the low micromolar range. These findings may be useful in designing new inhibitors of integrase, which is at present the only one of the three HIV enzymes for which clinically useful inhibitors are not available.

Absolute value of the difference of Tl-201 uptake between redistribution and rest is a specific marker of myocardial viability.

Although Tl-201 rest redistribution SPECT is widely used to assess myocardial viability, there is no agreement on the best prognostic marker of left ventricle contraction improvement after revascularization. More recent data suggest that not only rest or redistribution uptake but also reverse redistribution patterns may serve to indicate the viability of myocardium. The aim of this study was to define criteria (which include reversibility and reverse redistribution) for viability testing and prediction of functional outcome in Tl-201 rest redistribution SPECT. Twenty-five patients with left ventricle dyssynergy were studied before and after revascularization with Tl-201 SPECT and echocardiography. Perfusion and contractility was assessed in a 16-segment model of the left ventricle. Out of 400 left ventricular segments, contraction disturbances of various degree of intensity (hypokinesis, akinesis and dyskinesis) were found by echocardiography in 107 segments. Revascularization was performed in 97 segments. In 57% of the segments, improvement of contraction was observed after PTCA or CABG. Perfusion was analysed in the segments between segments with and without contraction improvement. In discriminant analysis, only the modulus of difference between rest and redistribution study > or = 10% was the common parameter for hypo-, a- and dyskinetic segments to predict the functional recovery of left ventricle (LV) with the specificity of 93% and sensitivity of 78%. The modulus of segmental quantitative difference between redistribution and rest image is a new parameter adding specificity to Tl-201 rest redistribution SPECT in prediction of recovery of left ventricle function.

O-phospho-DL-threonine and O-phospho-L-threonine compared with their serine analogs.

In crystals of O-phospho-DL-threonine and O-phospho-L-threonine, the molecules are zwitterions HO3-POCH-(CH3)CH(NH3+)CO2H linked by three-dimensional networks of strong P-O-H...O = P, C-O-H...O = P, N-H...O = P and N-H...O = C hydrogen bonds with (O...O) = 2.55 (3) A and (N...O) = 2.84 (4) A. Both the molecular conformations and the nearest-neighbor hydrogen-bonded surroundings are very similar in the racemic and enantiomeric crystals of the threonine compounds, but earlier studies of crystals of the analogous serine compounds have shown that the serine zwitterions HO3-POCH2CH(NH3+)CO2H have different conformations about the C beta-O gamma-P phosphate ester bonds and different hydrogen-bonded surroundings.

Structural and electronic conditions for anticonvulsant activity of bicyclic hydantoin derivatives.

A series of bicyclic derivatives based on 5,5-diphenylhydantoin (DPH) and/or 5-arylidene-hydantoin skeletons (BZH) are discussed as potential anticonvulsants. In preliminary pharmacological tests a few of these agents showed some anticonvulsant activities, like the parent DPH. The electronic parameters (molecular electrostatic potential, MEP, and dipole moment orientation) for the DPH molecule used as a model differed significantly from those calculated for the bicyclic molecules. These parameters were derived from semiempirical quantum chemistry calculations applying the PM3 method.

Bicyclic [b]-heteroannulated pyridazine derivatives--II. Structure-activity relationships in the 6-aryltriazolo-[4,3-b]pyridazine ligands of the benzodiazepine receptor.

Electronic parameters (molecular electrostatic potential MEP, charge distribution on the nitrogen atoms, dipole moment mu and ionization potential IP) were calculated by semiempirical quantum chemistry methods for 2 sets (X = H and m-CF3, the syn- and anti-rotamers of the latter being considered separately) of the 6-aryl-3-substituted-triazolo[4,3-b]pyridazine ligands of the benzodiazepine receptors (Figure 1; for X and Y c.f. Table 1). The calculations located the deepest MEP minimum near the = N-N = fragment of the triazole ring (Figure 2). Activity of the investigated compounds (1 microM), expressed as % inhibition of in vitro 3H-diazepam (1.5 nM) binding, revealed a significant dependence on IP, which combined in correlation studies with the hydrophobic constants pi X and pi Y and the Swain-Lupton field constant FY gave a 100% explanation of variance (Equations 1-3). However, extrapolation pointed to a compound with excessive hydrophobicity. The dipole moment orientation, roughly consistent with the C(6)-aryl main molecular axis, was considered as another factor controlling the docking of the investigated triazolopyridazine ligands to the benzodiazepine receptor (Figure 3). A model of the triazolopyridazine-benzodiazepine receptor interaction was proposed (Figure 4).

Use of molecular electrostatic potentials for analysis of anticonvulsant activities of phenylsuccinimides.

The present study was performed on a group of 27 derivatives of phenylsuccinimides, of which only 12 were active against maximal electrical shock in spite of the structural similarities of these compounds. The work consisted of four main parts: 1. crystallographic investigations of a subset of chosen compounds; 2. conformational analysis of characteristic molecules from the investigated series, performed by means of molecular mechanics calculations; 3. molecular orbital optimization of all the molecules using the MNDO method starting with conformations obtained in 2; 4. molecular electrostatic potential (MEP) analysis which was performed on the semiempirical (MNDO) and ab initio levels. This research showed that MEP maps provide a signature that distinguishes between active and inactive compounds. There are MEP minima close to the two carbonyl oxygens of the imide ring, and although the magnitude of the difference between the two minima is approximately constant, the sign of the difference provides an activity index. The initial orientations of phenylsuccinimide molecules in relation to the receptor are not equivalent and they depend on the potential distribution around both the succinimide molecules and around the receptor. In the active compounds the negative potential difference at the discussed points most probably influences the initial set-up of the molecules in relation to the receptor and results in a considerably higher probability of the molecules being bound at the right place on the receptor.

Structure of an anticonvulsant N-methyl-m-bromophenylsuccinimide.

3-(3-Bromophenyl)-1-methyl-2,5-pyrrolidinedione, C11H10BrNO2, Mr = 268.11, triclinic P1, a = 11.606 (2), b = 11.832 (2), c = 10.370 (2) A, alpha = 101.84 (1), beta = 107.76 (1), gamma = 118.28 (2) degrees, V = 1085.7 (5) A3, Z = 4, Dx = 1.640 Mg m-3, lambda(Cu K alpha) = 1.54178 A, mu = 5.04 mm-1, F(000) = 536, room temperature, final R = 0.061 for 2971 observed reflections with I greater than 3 sigma(I) (of 3743 unique data). The only slight differences in conformation of the two independent molecules are in the inclination of the phenyl and five-membered rings. The torsion angle C4-C1-C11-C12 is -115.4 (4) degrees in molecule I and -131.6 (4) degrees in molecule II. The five-membered imide ring has the open-envelope conformation in molecule I [the deviation of C11 from the plane N11-C41-C21-C31 is -0.117 (3) A] and the twist conformation in molecule II [the deviations of C12 and C22 from the plane N12-C42-C32 are 0.117 (1) and -0.064 (1) respectively].

Structures of three N-pyridyl-2-phenylsuccinimides and structural evidence for substituent effects on anticonvulsant properties.

T = 295 K, Mo K alpha with lambda = 0.70930 A. Compound (I-6): C15H12N2O2, Mr = 252.26, monoclinic, P2(1)/c, a = 8.441 (3), b = 15.269 (1), c = 9.745 (2) A, beta = 92.34 (2) degrees, V = 1254.9 (19) A3, Z = 4, Dx = 1.335 g cm-3, mu = 0.85 cm-1, F(000) = 528, R = 0.0345 for 1682 observed reflections. Compound (I-10): C16H14N2O2, Mr = 266.30, monoclinic, P2(1)/n, a = 11.637 (1), b = 5.793 (1), c = 20.778 (2) A, beta = 105.26 (1) degrees, V = 1351.3 (25) A3, Z = 4, Dx = 1.309 g cm-3, mu = 0.82 cm-1, F(000) = 560, R = 0.0379 for 1840 observed reflections. Compound (I-11): C16H13C1N2O2, Mr = 300.74, triclinic, P1, a = 9.076 (3), b = 9.366 (1), c = 10.477 (3) A, alpha = 118.27 (2), beta = 93.85 (2), gamma = 105.26 (1) degree, V = 737.2 (15) A3, Z = 2, Dx = 1.350 g cm-3, mu = 2.61 cm-1, F(000) = 312, R = 0.0528 for 2018 observed reflections. The three N-pyridyl-2-phenylsuccinimides [N-(3-methyl-2-pyridyl)-2-p-chlorophenylsuccinimide (I-11); N-(3-methyl-2-pyridyl)-2-phenylsuccinimide (I-10) and N-(3-pyridyl)-2-phenylsuccinimide (I-6)], examined by means of X-ray structure analysis, have been previously subjected to extensive pharmacological screening, with regard to their anticonvulsive activity. Pharmacological properties of the compounds examined are clearly connected with the conformation of the molecules. The conformation of the molecules of biologically active derivatives (I-10) and (I-11) differs from the conformation of the inactive molecule of (I-6). This difference involves relative positioning of the pyridyl ring and the succinimide moiety. The Cl atom in (I-11) has only a minor effect on the conformation and geometry of the molecule in comparison with (I-10).

[Intravenous treatment with streptokinase of acute myocardial infarction. II. Effect of patency of the artery supplying the infarction area on the infarction size and post-infarction impairement of left-ventricular function]. / Dozylne leczenie streptokinaza swiezego zawalu serca. II. Wplyw droznosci tetnicy zaopatrujacej obszar zawalu na mase zawalu i pozawalowy ubytek czynnosci lewej komory.

153 patients with a first acute myocardial infarction underwent the study. 90 of them received 1.000.000 units of streptokinase intravenously, followed by intravenous heparin administration for 5-7 days. The control group consisted of 63 remaining. In all patients serum CK-MB activity was determined every 4 hours for 72 hours: the infarct mass was calculated from the obtained curves. In 118 patients selective coronarography and left ventriculography was performed in the 2-nd or 3-rd week of hospitalisation. Left ventricular ejection fraction (E.F.) and dyssynergy index were calculated from ventriculographic data. Coronarography revealed a patent infarct-related artery in 76.7% of patients treated with streptokinase and in 44.4% of the control group (p less than 0.001). Among patients with a patent infarct-related artery an early peak of serum CK-MB activity (suggesting early recanalisation) occurred in 72.2% of streptokinase patients but in only 42.1% of the control group. Patients with a patent infarct-related artery had a significantly lower infarct mass (45 +/- 28 g vs 56 +/- 30 g), a lower left ventricular dyssynergy index (229 +/- 243 vs 348 +/- 247) and a significantly higher E.F. (63 +/- 12% vs 54 +/- 15%) compared with patients with an occluded artery.

[Intravenous streptokinase treatment of acute myocardial infarction. I. Effect of the treatment on the course of infarction and the patency of the artery supplying the infarction area]. / Dozylne leczenie streptokinaza swiezego zawalu serca. I. wplyw leczenia na przegieg zawalu i droznosc tetnicy zaopatrujacej obszar zawalowy.

128 consecutive patients with a first myocardial infarction, admitted within 4 hours after the onset of an angina pain, were divided into two groups according to the history of the peptic ulcer. Group I (with a negative history, n = 72) received intravenously 1,000,000 units of streptokinase followed by intravenous heparin infusion for 5-7 days. Group II (with a positive history, n = 56) was the control one. In hospital mortality was 2.8% in group I and 5.3% in group II (N. S.). Coronarography performed during second or third week of hospitalization revealed the patency of a coronary artery supplying an infarcted region twice as frequent in group I than in group II (78% vs 41%, p less than 0.001). Percentage of patients with the early serum peak of CKMB activity (from 8 to 12 hours from the start of therapy) suggesting early recanalization of a coronary artery supplying an infarcted region was significantly higher in group I (44.7% and 70.1%) than in group II (7.8% and 19.5%). Both differences between groups were significant (p less than 0.001). Early serum peak of CKMB activity (from 8 to 12 hours from start of treatment) was stated respectively in 46.5% and 81.4% of patients of group I in which subsequent coronarography revealed the patency of a coronary artery supplying the infarcted region.