The nature of anterior knee pain following injection of hypertonic saline into the infrapatellar fat pad

The infrapatellar fat pad has been implicated as a possible source of anterior knee pain. This study examined the nature, distribution and time-course of experimentally induced pain in the infrapatellar fat pad. Hypertonic saline (5%) was injected into the medial fat pad of 11 healthy individuals with no history of knee pain. Severity of pain was assessed at rest and during activity using an 11 point numerical rating scale (NRS) at regular intervals over 15-30 min following injection. Participants described the size of the pain region from a series of different sized circles while the area and type of pain was established from a body chart and the McGill pain questionnaire. The effect of pain on temperature-pain threshold and sensory thresholds of the anterior knee was assessed. Participants generally reported a deep aching pain that peaked in severity around 3 min and gradually declined over 15 min. Pain levels were not altered by clinical manoeuvres designed to impinge the fat pad. The size of the pain region was related to pain intensity. Pain was most commonly felt in the region of the fat pad medial to the patella, although some individuals reported proximal referred pain as far as the groin region. Thermal and sensory thresholds were not altered at a region close to the injection site during the experimental pain. These results suggest that nociceptive stimulation of the infrapatellar fat pad may cause anterior knee pain that is not necessarily confined locally particularly if pain is severe. This has implications for the investigation of pathological structures in patients presenting clinically with anterior knee pain and provides an experimental model of anterior knee pain. (C) 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Embracing ligands. A synthetic strategy towards new nitrogen-thioether multidentate ligands and characterization of the cobalt(III) complexes

The synthesis of the hexadentate ligand 2,2,9,9-tetra(methyleneamine)-4,7-dithiadecane (EtN(4)S(2)amp) is reported. The ligand is of a type in which bifurcations of the chain occur at atoms other than donor atoms. The cobalt(III) complex [Co(EtN(4)S(2)amp)](3+) (1) was isolated and characterized. The synthetic methodology also results in a number of by-products, notably 2,9,9-tris(methyleneamine)-9-methylenehydroxy-4,7-dithiadecane (Et(HO)N(3)S(2)amp) and an eleven-membered pendant arm macrocyclic ligand 6,10-dimethyl-6,10-bis(methyleneamine)-1,4-dithia-8-azaacycloundec-7- ene (dmatue). The complexes [Co(Et(HO)N(3)S(2)amp)](3+) (2), in which the alcohol is coordinated to the metal ion, and [Co(dmatue)Cl](2+) (4) were isolated and characterized. Et(HO)N(3)S(2)amp also undergoes complexation with cobalt(III) to produce two isomers endo-[Co(Et(HO) N(3)S(2)amp)Cl](2+) (endo-3) and exo-[Co(Et(HO) N(3)S(2)amp)Cl](2+) (exo-3), both with an uncoordinated alcohol group. endo- 3 has the alcohol positioned cis, and exo-3 trans, to the sixth metal coordination site. Reaction of 1 with isobutyraldehyde, paraformaldehyde and base in dimethylformamide results in the encapsulated complex [Co(1,5,5,9,13,13-hexamethyl-18,21-dithia-3,7,11,15-tetraazabicyclo[7.7.6]docosa- 3,14-diene)](ClO4)(3) . 2H(2)O ([Co(Me(6)docosadieneN(4)S(2))](3+) ( 5). All complexes have been characterized by single crystal X-ray study. The low-temperature (11 K) absorption spectrum of 1 has been measured in Nafion films with spin-allowed (1)A(1g) --> T-1(1g) and (1)A(1g) --> T-1(2g) and spin forbidden (1)A(1g) --> T-3(1g) and (1)A(1g) --> T-3(2g) bands observed. The octahedral ligand-field parameters were determined (10Dq = 22570 cm(-1), B = 551 cm(-1); C = 3500 cm(-1)). For 5 10Dq and B were determined (20580 cm(-1); 516 cm(-1), respectively) and compared with those for similar expanded cavity complexes [Co(Me(8)tricosatrieneN(6))](3+) and [Co(Me(5)tricosatrieneN(6))](3+).

Catheter effects in organ perfusion experiments

In a typical isolated organ perfusion experiment, a substance is injected upstream of an organ and then collected at some distance downstream. To reach the organ from the injection site, and then from the organ to the collector, a solute passes through catheters, usually tubes with circular cross-sections. Catheters cause distortion to the concentration-time profile of the perfusion. In this paper, we analyse catheter distribution kinetics from a mathematical point of view, develop the function most suitable for modeling this distribution and successfully apply this function to experimental data. (C) 2002 Academic Press.

Topographic plasticity in primary visual cortex is mediated by local corticocortical connections

The placement of monocular laser lesions in the adult cat retina produces a lesion projection zone (LPZ) in primary visual cortex (V1) in which the majority of neurons have a normally located receptive field (RF) for stimulation of the intact eye and an ectopically located RF ( displaced to intact retina at the edge of the lesion) for stimulation of the lesioned eye. Animals that had such lesions for 14 - 85 d were studied under halothane and nitrous oxide anesthesia with conventional neurophysiological recording techniques and stimulation of moving light bars. Previous work suggested that a candidate source of input, which could account for the development of the ectopic RFs, was long-range horizontal connections within V1. The critical contribution of such input was examined by placing a pipette containing the neurotoxin kainic acid at a site in the normal V1 visual representation that overlapped with the ectopic RF recorded at a site within the LPZ. Continuation of well defined responses to stimulation of the intact eye served as a control against direct effects of the kainic acid at the LPZ recording site. In six of seven cases examined, kainic acid deactivation of neurons at the injection site blocked responsiveness to lesioned-eye stimulation at the ectopic RF for the LPZ recording site. We therefore conclude that long-range horizontal projections contribute to the dominant input underlying the capacity for retinal lesion-induced plasticity in V1.

Tumour necrosis factor inhibitors

The cytokine, tumour necrosis factor-alpha (TNF-alpha) plays a key role in the pathogenesis of many chronic inflammatory and rheumatic diseases, in particular, Crohn's disease, rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis. Controlled trials have shown that the TNF inhibitors (etanercept, infliximab and adalimumab) significantly reduce symptoms and signs, improve function and quality of life, and reduce radiologically evident damage in patients with rheumatoid diseases. For reasons that are not entirely clear, etanercept does not work in Crohn's disease. Injection site and intravenous reactions and increased risk of infection (in particular, reactivation of tuberculosis) are associated with the use of these agents. Increased risk of lymphoproliferative disease, the development of lupus-like syndromes and demyelination, including optic neuritis and reactivation of multiple sclerosis, are under evaluation in long-term follow-up studies. The TNF inhibitors are expensive (about $18000 per year), and in some patients need to be given continuously to maintain benefit, even in the presence of other immunosuppressive therapy.

A phase I biological and pharmacologic study of the heparanase inhibitor PI-88 in patients with advanced solid tumors

Purpose: PI-88 is a mixture of highly sulfated oligosaccharides that inhibits heparanase, an extracellular matrix endoglycosidase, and the binding of angiogenic growth factors to heparan sulfate. This agent showed potent inhibition of placental blood vessel angiogenesis as well as growth inhibition in multiple xenograft models, thus forming the basis for this study. Experimental Design: This study evaluated the toxicity and pharmacokinetics of PI-88 (80-315 mg) when administered s.c. daily for 4 consecutive days bimonthly (part 1) or weekly (part 2). Results: Forty-two patients [median age, 53 years (range, 19-78 years); median performance status, 1] with a range of advanced solid tumors received a total of 232 courses. The maximum tolerated dose was 250 mg/d. Dose-limiting toxicity consisted of thrombocytopenia and pulmonary embolism. Other toxicity was generally mild and included prolongation of the activated partial thromboplastin time and injection site echymosis. The pharmacokinetics were linear with dose. Intrapatient variability was low and interpatient variability was moderate. Both AUC and C-max correlated with the percent increase in activated partial thromboplastin time, showing that this pharmacodynamic end point can be used as a surrogate for drug exposure, No association between PI-88 administration and vascular endothelial growth factor or basic fibroblast growth factor levels was observed. One patient with melanoma had a partial response, which was maintained for >50 months, and 9 patients had stable disease for >= 6 months. Conclusion: The recommended dose of PI-88 administered for 4 consecutive days bimonthly or weekly is 250 mg/d. PI-88 was generally well tolerated. Evidence of efficacy in melanoma supports further evaluation of PI-88 in phase II trials.

The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and Hop is located in the dimerization domain of hsp90

Structurally related tetratricopeptide repeat motifs in steroid receptor-associated immunophilins and the STI1 homolog, Hop, mediate the interaction with a common cellular target, hsp90, We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA library. All isolated clones encoded the intact carboxyl terminus of hsp90 and overlapped with a common region corresponding to amino acids 558-724 of murine hsp84, The interaction was confirmed in vitro with bacterially expressed CyP40 and deletion mutants of hsp90 beta and was delineated further to a 124-residue COOH-terminal segment of hsp90, Deletion of the conserved MEEVD sequence at the extreme carboxyl terminus of hsp90 precludes interaction with CyP40, signifying an important role for this motif in hsp90 function. We show that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90 COOH-terminal region. Our results are consistent with a common tetratricopeptide repeat interaction site for Hop and steroid receptor associated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.

Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: Site-directed mutagenesis and kinetic studies

Sulfonation is an important metabolic process involved in the excretion and in some cases activation of various endogenous compounds and xenobiotics. This reaction is catalyzed by a family of enzymes named sulfotransferases. The cytosolic human sulfotransferases SULT1A1 and SULT1A3 have overlapping yet distinct substrate specificities. SULT1A1 favors simple phenolic substrates such as p-nitrophenol, whereas SULT1A3 prefers monoamine substrates such as dopamine. In this study we have used a variety of phenolic substrates to functionally characterize the role of the amino acid at position 146 in SULT1A1 and SULT1A3. First, the mutation A146E in SULT1A1 yielded a SULT1A3-like protein with respect to the Michaelis constant for simple phenols. The mutation E146A in SULT1A3 resulted in a SULT1A1-like protein with respect to the Michaelis constant for both simple phenols and monoamine compounds. When comparing the specificity of SULT1A3 toward tyramine with that for p-ethylphenol (which differs from tyramine in having no amine group on the carbon side chain), we saw a 200-fold preference for tyramine. The kinetic data obtained with the E146A mutant of SULT1A3 for these two substrates clearly showed that this protein preferred substrates without an amine group attached. Second, changing the glutamic acid at position 146 of SULT1A3 to a glutamine, thereby neutralizing the negative charge at this position, resulted in a 360-fold decrease in the specificity constant for dopamine. The results provide strong evidence that residue 146 is crucial in determining the substrate specificity of both SULT1A1 and SULT1A3 and suggest that there is a direct interaction between glutamic acid 146 in SULT1A3 and monoamine substrates.

A new small molecule C5a receptor antagonist inhibits the reverse-passive arthus reaction and endotoxic shock in rats

C5a is implicated as a pathogenic factor in a wide range of immunoinflammatory diseases, including sepsis and immune complex disease, Agents that antagonize the effects of C5a could be useful in these diseases. We have developed some novel C5a antagonists and have determined the acute anti-inflammatory properties of a new small molecule C5a receptor antagonist against C5a- and LPS-induced neutrophil adhesion and cytokine expression, as well as against some hallmarks of the reverse Arthus reaction in rats. We found that a single i.v. dose (1 mg/kg) of this antagonist inhibited both C5a- and LPS-induced neutropenia and elevated levels of circulating TNF-alpha, as well as polymorphonuclear leukocyte migration, increased TNF-alpha levels and vascular leakage at the site of immune complex deposition. These results indicate potent anti-inflammatory activities of a new C5a receptor antagonist and provide more evidence for a key early role for C5a in sepsis and the reverse Arthus reaction. The results support a role for antagonists of C5a receptors in the therapeutic intervention of immunoinflammatory disease states such as sepsis and immune complex disease.

The uncharged surface features surrounding the active site of Escherichia coli DsbA are conserved and are implicated in peptide binding

DsbA is a protein-folding catalyst from the periplasm of Escherichia coli that interacts with newly translocated polypeptide substrate and catalyzes the formation of disulfide bonds in these secreted proteins. The precise nature of the interaction between DsbA and unfolded substrate is not known. Here, we give a detailed analysis of the DsbA crystal structure, now refined to 1.7 Angstrom, and present a proposal for its interaction with peptide. The crystal structure of DsbA implies flexibility between the thioredoxin and helical domains that may be an important feature for the disulfide transfer reaction. A hinge point for domain motion is identified-the typo IV beta-turn Phe 63-Met 64-Gly 65-Gly 66, which connects the two domains. Three unique features on the active site surface of the DsbA molecule-a groove, hydrophobic pocket, and hydrophobic patch-form an extensive uncharged surface surrounding the active-sits disulfide. Residues that contribute to these surface features are shown to be generally conserved in eight DsbA homologues. Furthermore, the residues immediately surrounding the active-site disulfide are uncharged in all nine DsbA proteins. A model for DsbA-peptide interaction has been derived from the structure of a human thioredoxin:peptide complex. This shows that peptide could interact with DsbA in a manner similar to that with thioredoxin. The active-site disulfide and all three surrounding uncharged surface features of DsbA could, in principle, participate in the binding or stabilization of peptide.

Molecular orbital theory calculations of the H2O-carbon reaction

Carbon gasification with steam to produce H-2 and CO is an important reaction widely used in industry for hydrogen generation. Although the literature is vast, the. mechanism for the formation of H-2 is still unclear. In particular, little has, been done to investigate the potential of molecular orbital theory to distinguish different mechanism possibilities. In this work, we used molecular orbital theory to demonstrate a favorable energetic pathway where H2O is first physically adsorbed on the virgin graphite surface with negligible change in molecular structure. Chemisorption occurs via O approaching the carbon edge site with one H atom stretching away from the O in the transition state. This is followed by a local minimum. state in which the stretching H is further disconnected from the O atoms and the remaining OH group is still on the carbon edge site. The disconnected H then pivot around the OH group to bond with the H of the OH group and forms H-2. The O atom remaining on the carbon edge site is subsequently desorbed as CO. The reverse occurs when H-2 reacts with the surface oxygen to produce H2O.

Development and evaluation of a species diagnostic polymerase chain reaction-restriction fragment-length polymorphism procedure for cryptic members of the Culex sitiens (Diptera : Culicidae) subgroup in Australia and the southwest Pacific

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

Synthesis, crystal structure and herbicidal activity of mimics of intermediates of the KARI reaction

Two mimics of the intermediate in the reaction catalyzed by ketol-acid reductoisomerase (KARI) were synthesized. Their structures were established on the basis of elemental analyses, IR, H-1 NMR and GC/mass detector. The crystal structure of compound 2 was found to be a substituted dioxane, formed by the condensation of two molecules. The two compounds showed some herbicidal activity on the basis of tests using rape root and barnyard grass growth inhibition. However, the herbicidal effect was weaker in greenhouse tests. (c) 2004 Society of Chemical Industry.

Market reaction to takeover rumour in internet discussion sites

We examine the market reaction to takeover rumour postings in the Hotcopper Internet Discussion Site (IDS). Results from the interday analysis show abnormal returns and trading volumes on the day before and the day of the posting. Results of the intraday analysis show abnormal returns and trading volumes during the 10 min posting interval and abnormal trading volume during the 10 min interval immediately preceding it. Sensitivity analyses indicate that the results are robust to concerns regarding potential confounds, credibility and bid–ask spread bias. Taken together, these findings are consistent with the market reacting to the posting of takeover rumours in IDS.