Tumour necrosis factor alpha stimulated rheumatoid synovial microvascular endothelial cells exhibit increased shear rate dependent leucocyte adhesion in vitro.

OBJECTIVE: To investigate endothelial cell adhesion molecule expression and leucocyte adhesion to endothelial cells isolated from the microvasculature of rheumatoid arthritic synovial tissue (SMEC) in comparison with similar cells isolated from healthy subcutaneous adipose tissue (ADMEC) or from umbilical veins (HUVEC). METHODS: Cultured endothelial cells were treated with tumour necrosis factor alpha (TNFalpha) for 2-24 hours before the assessment of cell surface E-selectin, vascular (VCAM-1) or intercellular cell adhesion molecule-I (ICAM-1) expression. Neutrophil and T lymphocyte adhesion to TNFalpha treated endothelial cells was assessed using static and shear dependent assay systems. RESULTS: VCAM-1 expression by SMEC was significantly less sensitive to TNFalpha stimulation than HUVEC or ADMEC. E-selectin expression by SMEC appeared to be more sensitive to TNFalpha stimulation and maximal expression was about 30% greater in comparison with HUVEC or ADMEC. Sensitivity to TNFalpha induction and maximal ICAM-1 expression was similar in all three endothelial cell types. Static neutrophil adhesion to TNFalpha stimulated SMEC was significantly increased in comparison with HUVEC, however this phenomenon was dependent on the presence of neutralising antibodies to ICAM-1. At shear rates in excess of 2.4 dynes/cm(2) significantly more neutrophils and, predominantly CD45RO+, T lymphocytes adhered to TNFalpha stimulated SMEC than HUVEC. CONCLUSION: Rheumatoid synovial endothelial cells differentially regulate E-selectin and VCAM-1. The increased ability of TNFalpha stimulated synovial endothelial cells to support leucocyte adhesion may help to explain the leucocyte, in particular CD45RO+ T-lymphocyte, recruitment observed in the rheumatoid synovium.

2-nitroimidazol-5-ylmethyl as a potential bioreductively activated prodrug system: reductively triggered release of the PARP inhibitor 5-bromoisoquinolinone.

5-Chloromethyl-1-methyl-2-nitroimidazole reacted efficiently with the anion derived from 5-bromoisoquinolin-1-one to give 5-bromo-2-((1-methyl-2-nitroimidazol-5-yl)methyl)isoquinolin -1-one. Biomimetic reduction effected release of the 5-bromoisoquinolin-1-one. The 2-nitroimidazol-5-ylmethyl unit thus has potential for development as a general prodrug system for selective drug delivery to hypoxic tissues.

Synthesis of thiophenecarboxamides, thieno[3,4-c]pyridin-4(5H)-ones and thieno[3,4-d]pyrimidin-4(3H)-ones and preliminary evaluation as inhibitors of poly(ADP-ribose)polymerase (PARP).

Inhibitors of poly(ADP-ribose)polymerase (PARP) inhibit repair of damaged DNA and thus potentiate radiotherapy and chemotherapy of cancer. Treatment of 3-cyanothiophene with potassium nitrate and concentrated sulphuric acid gave 5-nitrothiophene-3-carboxamide. 4-Nitrothiophene-2-carboxamide and 5-nitrothiophene-2-carboxamide were formed similarly from 2-cyanothiophene. Reduction with tin(II) chloride gave the corresponding aminothiophenecarboxamide salts which were isolated via their N-Cbz derivatives. Lithiation of 3,4-dibromothiophene at -116 degrees C and quenching with alkyl chloroformates gave 4-bromothiophene-3-carboxylates, which were hydrolysed to 4-bromothiophene-3-carboxylic acid. Hurtley reactions with the enolates of pentane-2,4-dione and of 1-phenylbutane-1,3-dione, followed by acyl cleavage, led to 4-(2-oxopropyl)thiophene-3-carboxylic acid and 4-phenacylthiophene-3-carboxylic acid, respectively. Condensation with ammonia in acetic acid gave 6-methyl- and 6-phenylthieno[3,4-c]pyridin-4-ones, which were selectively nitrated at the 1- and 7-positions or were dinitrated. Ethyl 4-acetamido- and 4-benzamido-thiophene-3-carboxylates were cyclised to 2-methyl- and 2-phenyl-thieno[3,4-d][1,3]oxazin-4-ones, respectively. Ring-opening with ammonia and recyclisation led to 2-substituted thieno[3,4-d]pyrimidin-4-ones. The aminothiophenecarboxamides are analogues of 3-aminobenzamide, a selective inhibitor of poly(ADP-ribose)polymerase (PARP); the thienopyridinones and the thienopyrimidinones are analogues of isoquinolin-1-ones and quinazolin-4-ones, respectively, which inhibit this enzyme. In preliminary assays, several thienopyridinones and thienopyrimidinones showed potent inhibitory activity against PARP.

Synthesis of 3-substituted benzamides and 5-substituted isoquinolin-1(2H)-ones and preliminary evaluation as inhibitors of poly(ADP-ribose)polymerase (PARP).

Inhibitors of poly(ADP-ribose)polymerase (PARP) inhibit repair of damaged DNA and thus potentiate radiotherapy and chemotherapy of cancer. 3-Substituted benzamides and 5-substituted isoquinolin-1-ones have been synthesised and evaluated for inhibition of PARP. Reduction of 3-(bromoacetyl)benzamide, followed by treatment with base, gave RS-3-oxiranylbenzamide. Reduction of 3-(hydroxyacetyl)benzonitrile with bakers' yeast gave the R-diol which was converted to R-3-(1,2-dihydroxyethyl)benzamide. Similar reduction of 3-(acetoxyacetyl)benzonitrile led towards the S-diol which was converted to its cyclic acetonide. E-2-(2,6-Dicyanophenyl)-N,N-dimethylethenamine was formed by condensation of 2,6-dicyanotoluene with dimethylformamide dimethyl acetal (DMFDMA); cyclisation under acidic conditions afforded 5-cyanoisoquinolin-1-one. Heck coupling of 5-iodoisoquinolin-1-one with propenoic acid formed E-3-(1-oxoisoquinolin-5-yl)propenoic acid. 3-Oxiranylbenzamide, 5-bromoisoquinolin-1-one and 5-iodoisoquinolin-1-one were among the most potent inhibitors of PARP activity in a preliminary screen in vitro.

A model for the initial phase of cell/surface interactions based on ligand binding phenomena.

The interaction between mammalian cells and solid surfaces plays an important role in a number of biological phenomena. Of particular clinical importance is the migration of cells suspended in blood to the wall of a blood vessel in the event of tissue damage. While the resultant inflammation often represents a desirable response to an external challenge, responses of this type can also lead to adverse consequences. Although the cell migration phenomenon is well known, a plausible mechanism for controlling the critical 'rolling' stages of adhesion has yet to be proposed. In this report we suggest how a simple consideration of ligand/receptor binding interactions can be used to explain a switch between a situation where a cell population is almost entirely in free suspension, to one where a significant fraction is attached to the solid surface.

The distribution of calmodulin/calmodulin binding proteins in the rat tapeworm, Hymenolepis diminuta.

Live tapeworms have been fixed to retain antigenicity of their proteins, and subsequently prepared for electron microscopy. Thin sections of tapeworms were prepared from resin blocks. Sections were immunocytochemically labelled using a colloidal gold probe and viewed using transmission electron microscopy. Calmodulin was detected associated with cellular structures to which calmodulin has previously been linked in other higher eukaryotes. Calmodulin would appear to have a similar role of importance in tapeworms, as it does in higher eukaryotes although tapeworms are prevalently a syncitium.

In vitro poly-(ADP-ribosyl)ation of chromatin proteins in the rat tapeworm, Hymenolepis diminuta.

1. (ADP-ribose)-transferase activity in crude chromatin of H. diminuta was demonstrated. 2. Chromatin proteins were ADP-ribosylated in vitro and selectively extracted. 60, 12 and 18% of the (ADP-ribose)n of chromatin proteins was associated with total histones, histone H1 and histone H2B, respectively. 3. The extent of oligo-(ADP-ribose) compared to total (ADP-ribose)n in the chromatin fraction, in the histone fraction, the histone H1 fraction and the histone H2B fraction was 45, 60, 26 and 49%, with an average chain length of 2.8, 2.1, 1.8 and 2.6, respectively. 4. Analysis of (ADP-ribosyl)n-ated proteins by acetic acid/urea polyacrylamide gel electrophoresis demonstrated that histone H1, histone H2B and a 35 kDa non-histone protein were major (ADP-ribose)n acceptors.