Purification and secondary structural analysis of tissue inhibitor of metalloproteinases-1.

In connective tissue diseases such as rheumatoid arthritis, the matrix metalloproteinases are the primary enzymes involved in tissue degradation. Tissue inhibitor metalloproteinases-1 (TIMP-1) is a specific inhibitor of these enzymes, which is thought to regulate their action in vivo. The structure and function of TIMP-1 may therefore be important as the basis for the rational design of therapeutic agents. This paper describes a simple and effective method for the purification of sufficient quantities of TIMP-1 for spectroscopic studies. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy have, together, showed TIMP-1 to be mostly in a beta-sheet conformation, with significant amounts of alpha-helix and beta-turn. Two-dimensional nuclear magnetic resonance spectroscopy indicated a correspondingly high proportion of beta-sheet. CD and FTIR have also shown TIMP-1 to have high thermostability.

Crystallization and preliminary X-ray analysis of porcine synovial collagenase.

Crystals of porcine synovial collagenase suitable for an X-ray structure analysis have been obtained. The crystals belong to space group I4, with unit cell dimensions a = b = 160.0 A, c = 53.1 A, with one molecule in the asymmetric unit. Diffraction extends beyond 3 A perpendicular to the c axis but along the 4-fold axis, the intensities are measurable only to 4 A.

Obesity, sodium intake, and blood pressure in adolescents.

It has been postulated that increased dietary sodium associated with greater food intake by obese people is a mechanism for the relationship between obesity and blood pressure (BP). We have evaluated this hypothesis by exploring the interrelationships of measures of obesity, sodium intake, and BP in 248 "normal" adolescents, 16 to 17 years of age. As an index of sodium intake, the sodium excretion in three overnight urine collections was used. As a more specific index of saltiness of diet, we used a ratio of sodium excretion to calorie intake, with calories estimated from 3-day diet records and dietary interview. Body weight and other measures of obesity showed a positive relationship with systolic blood pressure (SBP), but not with diastolic (K5) blood pressure (DBP5). Measures of overnight sodium excretion were positively correlated with body weight and calculated body fat percentage, suggesting that heavier people indeed ingest more sodium. This may result not from increased intake of food per se, but from increased saltiness of diet, since calorie intake did not increase with body weight. No significant relationships were found between BP and concurrent measures of sodium excretion or diet saltiness.

Anorexia nervosa and affective disorders: a controlled family history study.

Analysis of family history information from a prospectively studied group of 40 young female patients with anorexia nervosa and 23 normal control female subjects of similar age showed more depression and substance use disorders in first- and second-degree relatives of anorexia nervosa patients. Further, the pedigrees of the patients differed significantly from those of the control subjects in the higher frequency of depression and substance use disorders in consecutive generations and in the family "loading" of these disorders. These findings, consistent with previous reports, add to the growing evidence of an association between anorexia nervosa and familial risk for affective and related disorders.

Platelet MAO activity in anorexia nervosa patients with and without a major depressive disorder.

Platelet MAO activity was determined in 33 anorexia nervosa patients. A subgroup of 15 patients who met Research Diagnostic Criteria for a concomitant major depressive disorder were found to have, both initially and after 5 weeks of treatment, significantly lower mean platelet monoamine oxidase (MAO) activity than 28 matched normal control subjects. In contrast, mean platelet MAO activity in the patients who did not meet criteria for major depressive disorder was similar to values in control subjects. The authors found that significantly more depressed patients had low MAO activity compared with nondepressed patients and controls. Platelet MAO activity may be useful in discriminating among subtypes of anorexia nervosa patients.

Synthesis of novel modified dipeptide inhibitors of human collagenase: beta-mercapto carboxylic acid derivatives.

The synthesis of a series of thiol-containing, modified dipeptide inhibitors (8) of human collagenase, which incorporate various carboxylic acid derivatives at the presumed P1 position, beta to the thiol group, is described. The compounds were evaluated, in vitro, for their ability to inhibit the degradation of rat skin type 1 collagen by purified human lung fibroblast collagenase, and structure-activity relationship studies are described. Optimum potency (IC50 values in the nanomolar range) was achieved by incorporating methyl (compounds 43a, 56a, and 57ab) or benzyl esters (44a) at the P1 position. Small amides were also accommodated (e.g. primary amide 47a), but in general, increasing the size of the P1 amide substituent lowered potency. PheNHMe, TrpNHMe, and Tyr(Me)NHMe substituents were found to be approximately equipotent P2'-residues. The results of testing all four diastereoisomers 56a-d of the compound with (S)-TrpNHMe at the P2' position indicated that the S,S,S diastereoisomer 56a possessed highest potency (IC50 2.5 nM) and that the second most potent diastereoisomer was 56d (IC50 12 nM) with the R,R,S configuration. It appeared that the orientation of the P1' and the thiol-bearing centers to each other is a more critical influence on potency than any absolute stereochemical requirements. It is suggested that the high potency of the beta-mercapto carboxylic acid derivatives may be a consequence of bidentate coordination of the thiol and carbonyl groups to the active-site zinc ion in the collagenase enzyme.

Synthesis of novel N-phosphonoalkyl dipeptide inhibitors of human collagenase.

The synthesis of a series of N-phosphonalkyl dipeptides 6 is described. Syntheses were devised that allowed the preparation of single diastereoisomers and the assignment of stereochemistry. The compounds were evaluated in vitro for their ability to inhibit the degradation of radiolabeled collagen by purified human lung fibroblast collagenase. Several of the compounds were potent collagenase inhibitors and were at least 10-fold more potent than their corresponding N-carboxyalkyl analogues. Activity was lost when the phosphonic acid group P(O)(OH)2 was replaced by the phosphinic acid groups P(O)(H)(OH) and P(O)(Me)(OH). At the P1 position, (R)- or (S)-alkyl groups, especially ethyl and methyl (e.g., 12a,b, 52a,b, and 53a,b), or an (R)-phenethyl moiety (55a) conferred high potency (IC50 values in the range 0.23-0.47 microM). (S)-Stereochemistry was preferred for the P1' isobutyl side chain. Structure-activity relationships were also investigated at the P2' site, and interestingly, compounds with basic side chains, such as the guanidine 57a, were equipotent with more lipophilic compounds, such as 52a. As with other series of collagenase inhibitors, potency was enhanced by introducing bicyclic aromatic P2' substituents. The most potent phosphonic acid of the series was the bicyclic aromatic P2' tryptophan analogue 59a (IC50 0.05 microM).

Urinary mhpg in anorexia nervosa patients with and without a concomitant major depressive disorder.

Twenty-four hour urinary MHPG excretion was measured in a group of anorexia nervosa patients before and after five weeks of treatment and in matched normal control subjects. A sub-group of anorexia nervosa patients who met research diagnostic criteria (RDC) for a concomitant major depressive disorder (AN-RDC +) was found to have, both initially and after treatment, significantly lower mean urinary MHPG levels than the normal control subjects. In contrast, mean urinary MHPG levels in anorexia nervosa patients who did not meet criteria for major depressive disorder (AN-RDC +) were similar to values in normal controls. Utilizing the median value of all urinary MHPG samples as the cut-off point, it was found that significantly more AN-RDC + patients excreted low MHPG compared with AN-RDC-patients and normal control subjects. The manifestation of a major depressive disorder according to RDC was found to be more important than body size variables in predicting the variance of MHPG. It is suggested that urinary MHPG levels may be useful in discriminating between sub-types of anorexia nervosa patients.

Collagen degradation within subcutaneous air pouches in vivo: the effects of proteinase inhibitors.

A straightforward in vivo model of collagen degradation is described that can be used to measure the effects of different classes of proteinase inhibitors. Air pouches, formed subcutaneously in the dorsal thoracic region of rats, were inflamed 6 to 8 days later by injecting lambda-type carrageenan. 14C-Collagen was injected into the air pouches either 1 day before or 1 day after lambda-carrageenan-induced inflammation: in the latter case, the inflammatory exudate fluid was drained from the air pouches immediately prior to administering 14C-collagen. Ninety percent of the 14C-collagen was degraded and cleared within 3 days from pre-inflamed air pouches, but degradation was much slower from the post-inflamed or non-inflamed air pouches. Proteinase inhibitors injected simultaneously with the 14C-collagen, and again 6 hr later, reduced the extent of 14C-collagen degradation from air pouches measured after 24 hr. Forty-two percent of the degradation of 14C-collagen could be inhibited by a mixture of enzyme inhibitors (leupeptin, alpha 1-anti-proteinase, aprotinin, and pepstatin) injected together with 1,10 phenanthroline, the zinc metalloenzyme inhibitor. The 1,10 phenanthroline alone caused a 33% inhibition of 14C-collagen degradation, and the inhibitor mixture given alone inhibited 14C-collagen loss by 25%. Approximately 60% of the degradation of 14C-collagen in this model was mediated by mechanisms resistant to this combination of proteinase inhibitors, which may indicate the significant involvement of non-enzymic modalities, or degradation in intracellular compartments inaccessible to extracellular agents.

The purification of nerve growth factor from bovine seminal plasma. Biochemical characterization and partial amino acid sequence.

Nerve growth factor (NGF), a protein regulating the development and function of certain neural crest derivatives, has been purified from bovine seminal plasma, and extremely rich source of the protein. Around 10 mg of pure NGF (yield, 10-20%) can be isolated from 10 g of lyophilized seminal plasma (around 100 ml of semen). The behavior during purification indicates that, like the NGF in the mouse submandibular gland, bovine NGF exists as a high molecular weight complex that dissociates at extremes of pH to reveal a smaller subunit having NGF biological activity. The isolated low molecular weight form of bovine NGF is a dimer of noncovalently linked polypeptide chains (Mr approximately 15,000 on sodium dodecyl sulfate-polyacrylamide gels), with an isoelectric point of 9.5-10. These properties differ from those of low molecular weight (beta-subunit) mouse NGF, which comprises two noncovalently linked peptide chains of Mr = 13,256 (from sequence studies), and which has an isoelectric point of 9.3. The amino acid sequence of the NH2-terminal 26 residues of bovine NGF has been determined and found to be similar to, but not identical with, that of mouse NGF. Thus, residues 3, 9, and 18, which are threonine, methionine, and valine, respectively, in mouse NGF, are serine, arginine, and isoleucine in bovine NGF.

The International Spinal Research Trust's strategic approach to the development of treatments for the repair of spinal cord injury.

The International Spinal Research Trust (ISRT) has selected a sub-set of the key molecular and cellular events underlying spinal injury and nerve regeneration, to be focus of their funding and other means of research support. These priority targets are (i) to understand and to minimise the damage caused by spinal injury and the resulting inflammatory and fibrotic events, in order to prevent the establishment of a post-acute situation that is ill-placed for regeneration; and (ii) to understand and then to manipulate the integrated environment for regrowth that is created by the interplay of soluble and matrix- or membrane-associated factors, both trophic and inhibitory. Investigation and, ultimately, exploitation of these targets requires the development of standardised and representative animal models and the application of quantitative methods for assessing functional re-innervation. The ISRT will also sponsor the networking of different disciplines and technologies to apply the most productive skills to spinal repair.

Progress in spinal cord research - a refined strategy for the International Spinal Research Trust.

Achieving regeneration in the central nervous system represents one of the greatest intellectual and practical challenges in neurobiology, and yet it is an absolute requirement if spinal cord injury patients are to have any hope of recovery. The mission of the International Spinal Research Trust (ISRT), established in 1980, is to raise money specifically for spinal research, with a view to ending the permanence of paralysis caused by spinal cord injury. This review summarises some of the major steps forward made in recent years in understanding the mechanisms involved in spinal cord injury and where these discoveries fit in with the ISRT's overall objectives. We review approaches aimed at (1) preventing immediate adverse reactions to injury such as neuronal death and scar formation; (2) minimising inhibitory properties of the CNS environment and maximising the growth potential of damaged neurons; (3) understanding axonal guidance systems that will be required for directed outgrowth and functional reconnection; and (4) optimising the function of surviving systems. We also discuss 'infrastructural' prerequisites for applying knowledge gained through spinal research to the clinical condition, including basic scientific issues such as developing representative animal models of spinal cord injury and sensitive quantitative methods for assessing growth and functional restoration. In addition, we point out the importance of communication. The need to share knowledge between research groups is vital for advancing our understanding of injury and repair mechanisms. Equally important is the need for communication between basic scientists and clinicians which will be essential for what is the ultimate goal of the ISRT, the development of relevant treatment strategies that will prove beneficial to the spinal injured patient.

Inhibition of arachidonic acid-induced ear oedema as a model for assessing topical anti-inflammatory compounds.

We have found that mouse ear oedema induced by the topical application of arachidonic acid is not a specific screen for compounds inhibiting the lipoxygenase or cyclo-oxygenase pathways of arachidonic acid metabolism. Although such compounds are able to reduce the oedema substantially, pharmacological agents such as histamine antagonists, phosphodiesterase inhibitors, free radical scavengers, and also various compounds not normally considered to have anti-inflammatory properties, can equally effectively reduce the oedema. A mutual potentiation of the effects of prostaglandins, leukotrienes and mast cell-derived histamine would allow many, but not all, of the active agents to be rationalised. The ability of compounds not influencing these three types of inflammatory mediators to reduce the oedematous response means the model is of limited value for directed screening.

In vivo model of cartilage degradation--effects of a matrix metalloproteinase inhibitor.

OBJECTIVES: To develop a model of cartilage degradation that (i) enables the testing of synthetic, small molecular weight matrix metalloproteinase (MMP) inhibitors as agents to prevent cartilage erosion, (ii) permits the direct assay of the principal constituents of the extracellular matrix (collagen and proteoglycan) in both the non-calcified articular cartilage and the calcified cartilage compartments, and (iii) is mediated by a chronic, granulomatous tissue that closely apposes intact articular cartilage, and in this respect resembles the pannus-cartilage junction of rheumatoid arthritis. METHODS: Femoral head cartilage was obtained from donor rats, wrapped in cotton and implanted subcutaneously into recipient animals. After a two stage papain digestion procedure, the proteoglycan and collagen contents were measured by assaying for glycosaminoglycans and hydroxyproline, respectively, in both the non-calcified cartilage that comprises the articular surface layer and the calcified cartilage compartment. The incorporation in vitro of [35S]-sulphate into glycosaminoglycans was assayed as a measure of proteoglycan biosynthesis. An osmotic minipump was cannulated to the implanted femoral head cartilage and synthetic MMP inhibitors (MI-1 and MI-2) were infused continuously over a 14 day period. RESULTS: The implanted, cotton wrapped femoral head cartilages provoked a granulomatous response that resulted in the removal of collagen and proteoglycan from the cartilage matrix. The removal of proteoglycan and collagen was exclusively from the non-calcified articular cartilage, whereas the proteoglycan and collagen content of the calcified compartment increased during the experiments. MI-1 reproducibly reduced the degradation of proteoglycan and collagen in implanted femoral head cartilage. CONCLUSIONS: We have described an in vivo model of cartilage degradation that permits the measurement of proteoglycan and collagen in both non-calcified articular cartilage and calcified cartilage compartments. The model can be used to test the effects of agents of unknown systemic bioavailability and pharmacokinetic profile by infusing them directly to the site of cartilage degradation. The removal of cartilage extracellular matrix by granulomatous tissue was inhibited by an MMP inhibitor, thus proving the involvement of this family of proteinases in cartilage catabolism in this model.

A synthetic peptide metalloproteinase inhibitor, but not TIMP, prevents the breakdown of proteoglycan within articular cartilage in vitro.

IL1-stimulated pig articular cartilage fragments were cultured in the and absence of various metalloproteinase inhibitors. Tissue inhibitor of metalloproteinases (TIMP) was unable to stop the release of proteoglycan from the cartilage. Incubation of cartilage with a potent synthetic metalloproteinase inhibitor inhibited the release of proteoglycan in a dose-dependent fashion. The results suggest that low-M(r) metalloproteinase inhibitors may have therapeutic potential in limiting connective tissue breakdown in conditions such as rheumatoid arthritis.

A simple in vivo model of collagen degradation using collagen-gelled cotton buds: the effects of collagenase inhibitors and other agents.

A simple in vivo model of collagen degradation has been developed, and the effects of various agents have been tested. Type I collagen was prepared from rat skin and acetylated with either [3H]- or [14C] acetic anhydride. The radiolabelled collagen was added to sterile cotton buds and incubated at 37 degrees C to allow the collagen to form native fibrils that were firmly adsorbed to the cotton matrix. After subcutaneous implantation of the collagen-gelled cotton buds into rats, the radiolabelled collagen was progressively removed over a period of weeks by an infiltrating granuloma. Of the agents that were administered directly into the cotton buds using subcutaneously implanted osmotic mini-pumps, only the synthetic collagenase inhibitors CI-A (containing a hydroxamate moiety as a zinc ligand) and CI-C (containing a thiol moiety as a zinc ligand) were able to prevent the removal of collagen: their efficacy correlated with the level of collagenase inhibitory activity assayed in the exudate fluid sequestered within the cotton bud granuloma. Of the agents that were administered systemically, including anti-inflammatory drugs and other compounds used as therapies for arthritis, only hydrocortisone was able to inhibit the removal of radiolabelled collagen. These results suggest that, in this model, interstitial collagenase, a member of the matrix metalloproteinase family, comprised the major degradative pathway for collagen. The collagen-gelled cotton bud model is a useful test system for delineating those processes that result in collagen catabolism. In addition, the model can be used for testing agents, including those of limited or unknown systemic bioavailability, in order to discover novel therapeutic agents for preventing collagen degradation in connective tissue diseases such as arthritis.

Visualisation of subchondral erosion in rat monoarticular arthritis by magnetic resonance imaging.

High-resolution magnetic resonance imaging (MRI) was used to investigate antigen-induced monoarticular arthritis (AIMA) in the rat. In sagittal, spin-echo images of the knee, characteristic parallel bands, in the order dark-light-dark, were consistently observed 5-8 days after arthritis induction; the bands ran concentric with, and just beneath, the femoral and tibial articular surfaces. Concurrent radiology, histology and MRI (chemical shift-selective imaging and contrast enhancement with magnetisation transfer and gadolinium) established that the phenomenon reflected subchondral erosion, not artefact. The outer hypointense band corresponded to calcified cartilage underlying the articular surface. The central hyperintense band reflected inflammatory matrix displacing normal haematopoietic tissue immediately subchondrally; here, trabecular bone had mostly disappeared, but adjacent articular cartilage, although under attack and lacking proteoglycan, appeared structurally normal. The inner hypointense band reflected deeper, truncated trabeculae within inflammatory matrix, layered with pallisading osteoblast-like cells. This study exemplifies the power of MRI for revealing localised joint pathology non-invasively, and shows that rat AIMA shares many pathological features with arthritis in human beings.