Contribution of IL-12A and IL-12B polymorphisms to Chlamydia trachomatis-specific cell-mediated immune responses.

Inherited variance in the IL-12B gene is associated with susceptibility to Chlamydia trachomatis-induced tubal factor infertility and disease severity. In this study, our aim was to discover how polymorphisms in IL-12-coding genes influence C. trachomatis-induced immune responses and IL-12 production. The study population consisted of 240 women. IL-12A and IL-12B single nucleotide polymorphisms (SNPs) were determined from isolated DNA using the Sequenom system with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. We studied lymphocyte proliferative (LP) responses to C. trachomatis strains E and F elementary bodies (EBs) and recombinant chlamydial heat-shock protein 60 (CHSP60) antigen. IL-12p40 and IL-12p70 levels were measured using the BD Flex Set method. We found a statistically significant association between the C. trachomatis EB antigen-specific LP response and the rs2853694 SNP (P = 0.02). Our study demonstrates that the IL-12 cytokine family is involved in C. trachomatis-specific immune responses. Moreover, C. trachomatis-induced IL-12 production and the IL-12B rs2853694 SNP partially explain individual variation in the C. trachomatis LP response.

Effect of IL12A and IL12B polymorphisms on the risk of Chlamydia trachomatis-induced tubal factor infertility and disease severity.

BACKGROUND: Interleukin-12 (IL-12) and related cytokines induce activation and differentiation of T cells. Our aim was to investigate the associations between genetic differences in IL-12-family cytokines and the pathogenesis of chlamydial disease. METHODS: The final study population consisted of 100 women with Chlamydia trachomatis-induced tubal factor infertility (TFI) and 125 pregnant women as controls. Three single nucleotide polymorphisms (SNPs) of IL12A and seven SNPs of IL12B genes were determined from isolated DNA using the Sequenom system with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. RESULTS: We found that the IL12B SNP rs3212227 was associated with both susceptibility and severity of TFI. The minor allele C was rare and only one CC homozygote was found among the controls. AC heterozygotes were more common among TFI cases than among controls (P = 0.009) and were associated with increased risk of TFI [odds ratios (OR) = 2.44, 95% confidence intervals (CI) = 1.23-4.87]. Carrying the minor allele C was also associated with disease severity (P for trend = 0.008) and moderate (OR = 2.51, 95% CI = 1.06-5.95) and severe tubal damage (OR = 2.73, 95% CI = 1.15-6.52). CONCLUSIONS: The results suggest that variation in the IL12B gene partly explains inter-individual differences in disease susceptibility and severity.

Induction of long-term H-Y-specific tolerance in female mice given male lymphoid cells while transiently depleted of CD4+ or CD8+ T cells.

Rejection of H-Y-bearing primary skin grafts and generation of H-Y-specific cytolytic T cells by female mice requires the participation of both CD4+ and CD8+ T lymphocytes. Studies were conducted to investigate long-term tolerance of H-Y antigen induced in female mice by transiently depleting them of CD4+ and/or CD8+ T cells and, at the same time, giving them an injection of male lymphoid cells. We confirmed that after recovery of CD4+ to normal levels, female mice that had been transiently depleted of CD4+ cells and concurrently given an injection of male spleen cells were unable to generate H-Y-specific cytolytic T cells. Tolerance was also manifest by greatly extended survival (probably permanent in most cases) of male skin grafts. Further investigations revealed that female mice transiently depleted of CD8+ cells, and concurrently given an injection of male spleen cells, were similarly tolerant of H-Y antigen later when numbers of CD8+ T cells returned to normal. Moreover, small numbers of male cells were detectable in spleen and lymph nodes of tolerant females long after they had been given an injection of male cells and depleted of either CD4+ or CD8+ T cells, whereas no male cells were detected in (nontolerant) females given male cells and control antibodies. These findings show that tolerance of the relatively weak transplantation antigen, H-Y, can be achieved simply by giving male antigen-bearing spleen cells to the host while it is transiently depleted of a type of cell it needs in order to reject those cells, thus allowing the male cells to persist in the host. Furthermore, depletion of helper cells is not obligatory to achieve tolerance. It has been hypothesized that tolerance of H-Y antigen in females given male lymphoid cells while temporarily depleted of CD4+ lymphocytes results from unresponsiveness (anergy) induced in H-Y-specific CD8+ cells that are exposed to H-Y antigen in the absence of help from CD4+ cells. Interpretations of our findings are discussed in relation to this hypothesis.

In situ assays demonstrate that interferon-gamma suppresses infection-stimulated hepatic fibrin deposition by promoting fibrinolysis.

BACKGROUND: Inflammatory cytokines potently impact hemostatic pathways during infection, but the tissue-specific regulation of coagulation and fibrinolysis complicates studies of the underlying mechanisms. METHODS AND RESULTS: Here, we describe assays that quantitatively measuring prothrombinase (PTase), protein C-ase (PCase) and plasminogen activator (PA) activities in situ, thereby facilitating studies of tissue-specific hemostasis. Using these assays, we investigate the mechanisms regulating hepatic fibrin deposition during murine toxoplasmosis and the means by which interferon-gamma (IFN-gamma) suppresses infection-stimulated fibrin deposition. We demonstrate that Toxoplasma infection upregulates hepatic PTase, PCase, and PA activity. Wild type and gene-targeted IFN-gamma-deficient mice exhibit similar levels of infection-stimulated PTase activity. By contrast, IFN-gamma-deficiency is associated with increased PCase activity and reduced PA activity during infection. Parallel analyses of hepatic gene expression reveal that IFN-gamma-deficiency is associated with increased expression of thrombomodulin (TM), a key component of the PCase, increased expression of thrombin-activatable fibrinolysis inhibitor (TAFI), a PC substrate, and reduced expression of urokinase PA (u-PA). CONCLUSIONS: These findings suggest that IFN-gamma suppresses infection-stimulated hepatic fibrin deposition by suppressing TM-mediated activation of TAFI, thereby destabilizing fibrin deposits, and concomitantly increasing hepatic u-PA activity, thereby promoting fibrinolysis. We anticipate that further application of these in situ assays will improve our understanding of tissue-specific hemostasis, its regulation by cytokines, and its dysregulation during coagulopathy.

X-ray structure of a hydroxamate inhibitor complex of stromelysin catalytic domain and its comparison with members of the zinc metalloproteinase superfamily.

BACKGROUND: Stromelysin belongs to a family of zinc-dependent endopeptidases referred to as matrix metalloproteinases (MMPs, matrixins) because of their capacity for selective degradation of various components of the extracellular matrix. Matrixins play key roles in diseases as diverse as arthritis and cancer and hence are important targets for therapeutic intervention. RESULTS: The crystal structure of the stromelysin catalytic domain (SCD) with bound hydroxamate inhibitor, solved by multiple isomorphous replacement, shows deep S1' specificity pocket which explains differences in inhibitors binding between the collagenases and stromelysin. The binding of calcium ions by loops at the two ends of a beta-strand which marks the boundary of the active site provides a structural rationale for the importance of these cations for stability and catalytic activity. Major differences between the matrixins are clustered in two regions forming the entrance to the active site and hence may be determinants of substrate selectivity. CONCLUSIONS: Structural comparisons of SCD with representative members of the metalloproteinase superfamily clearly highlight the conservation of key secondary structural elements, in spite of major variations in the sequences including insertions and deletions of functional domains. However, the three-dimensional structure of SCD, which is generally closely related to the collagenases, shows significant differences not only in the peripheral regions but also in the specificity pockets; these latter differences should facilitate the rational design of specific inhibitors.

Peak regional acceleration: a method to identify subtle regional ventricular dysfunction from gated blood pool scans at rest in patients with coronary artery disease.

Peak regional acceleration images were obtained from gated blood pool scans at rest in 10 normal subjects, 16 patients who underwent cardiac catheterization for unstable angina or nontransmural infarction and were found to have normal ejection fraction and wall motion and 10 patients with prior infarction and regional dyssynergy. The second derivative of the time-activity curve of each pixel was generated and the maximal systolic value of the derivative for each pixel was displayed as a functional image (peak regional acceleration). Anterior and left anterior oblique views were evaluated for abnormalities and the presence and location of defects were correlated with the coronary anatomy. The scans from the 10 normal subjects were used to establish the normal range for regional second derivative values. Both gated blood pool scans and second derivative images showed regional abnormalities in all 10 patients with prior transmural infarction. Regional abnormalities were present in the second derivative images in the distribution of 17 of the 20 coronary arteries with greater than 50% stenosis; there were no regional abnormalities in the distribution of 7 of the 8 arteries with less than 50% stenosis. In addition, regional second derivative image abnormalities were present in 15 of the 16 patients with unstable angina and normal wall motion and global ejection fraction. These 16 patients showed regional abnormalities on second derivative images in the distribution of 19 of the 23 coronary arteries with significant stenosis and no regional abnormalities in the distribution of 21 of the 23 coronary arteries without significant stenosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of infarct size measured from antimyosin single-photon emission computed tomographic scans on left ventricular remodeling.

To evaluate the effect of infarct size on left ventricular volumes and geometric remodeling, 26 patients with a first acute Q wave myocardial infarction (anterior in 14, inferior in 12) had the infarct sized from single-photon emission computed tomographic (SPECT) imaging of indium-111 antimyosin. All patients underwent gated blood pool scintigraphy before hospital discharge for determination of ejection fraction and end-diastolic and end-systolic volume indexes. Infarct size was quantitated from indium-111 antimyosin uptake in coronal slices with use of a threshold technique for edge detection. Nineteen of 26 patients had additional simultaneous acquisitions of indium-111 and thallium-201 uptake and the infarct was expressed as a percent of the total left ventricle. Infarct size was larger (59 +/- 16 vs. 33 +/- 16 g), predischarge ejection fraction lower (35 +/- 5% vs. 60 +/- 9%) and end-systolic volume index higher (57 +/- 13 vs. 36 +/- 10 ml/m2) in the group with anterior infarction. Despite these differences, predischarge end-diastolic volume index was not significantly different between the group with anterior (88 +/- 17 ml/m2) versus inferior (89 +/- 14 ml/m2) infarction. There was a significant inverse correlation between percent infarct size and ejection fraction for patients with dual isotope imaging (r = -0.90) and a significant direct correlation between infarct size and end-systolic volume index (r = 0.79, p less than 0.01). Fourteen patients without subsequent myocardial infarction or coronary artery bypass grafting had a repeat gated blood pool study late (26 +/- 15 months) after infarction.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase analysis of gated blood pool scintigraphic images to localize bypass tracts in Wolff-Parkinson-White syndrome.

The ability of radionuclide techniques to localize bypass tracts in patients with Wolff-Parkinson-White syndrome to sites around the atrioventricular (AV) ring using a three view triangulation method was investigated. In 17 patients with Wolff-Parkinson-White syndrome, phase images were generated from gated blood pool scans using the first Fourier harmonic of the time-activity curve of each pixel. In addition, the difference between left and right ventricular mean phase angles was calculated for each patient and for 13 control subjects. Bypass tracts were localized to one or more sites on a 10 site grid schematically superimposed on the AV ring (Duke grid) by electrophysiologic study in all patients and by intraoperative mapping in 7 of the 17 patients. These same 10 anatomic sites were projected onto three scintigraphic views and the site of earliest ventricular phase angle was located in each view. The 10 sites around the AV ring were divided into two anatomic groups: free wall and septal/paraseptal. Phase image locations correlated with electrophysiologic locations within one grid site in 11 of 11 patients with free wall tracts and were confirmed at surgery in 5 of the 11. In five of six patients with septal/paraseptal tracts, electrophysiologic study could not localize the bypass tract to one site, whereas phase images localized two of the five as free wall adjacent to the septum, one as paraseptal and two as true posteroseptal. One posteroseptal site was confirmed at surgery. In one patient, in whom phase image analysis and electrophysiologic study showed different sites, existence of both tracts was confirmed at surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimyosin imaging in acute transmural myocardial infarctions: results of a multicenter clinical trial.

Murine monoclonal antimyosin antibody has been shown experimentally to bind selectively to irreversibly damaged myocytes. To evaluate the safety and efficacy of monoclonal antimyosin for identifying acute transmural infarction, 50 patients with acute Q wave myocardial infarction were entered into a phase I/II multicenter trial involving three clinical sites. Indium-111 antimyosin was prepared from an instant kit formulation containing 0.5 mg of diethylene triamine pentaacetic acid (DTPA)-coupled Fab fragment (R11D10) and 1.2 to 2.4 mCi of indium-111. Average labeling efficiency was 92%. Antimyosin was injected 27 +/- 16 h after the onset of chest pain. Planar or tomographic imaging was performed 27 +/- 9 h after injection in all patients, and repeat imaging was done 24 h later in 39 patients. Of the 50 patients entered, 46 showed myocardial uptake of antimyosin (sensitivity 92%). Thirty-one of 39 planar scans performed at 24 h were diagnostic; 8 showed persistent blood pool activity that cleared by 48 h. Focal myocardial uptake of antimyosin corresponded to electrocardiographic infarct localization. No patient had an adverse reaction to antimyosin. In addition, 125 serum samples, including 21 collected greater than 42 days after injection, were tested for human antimouse antibodies, and all samples were assessed as having undetectable titers. Intensity of antimyosin uptake was correlated with infarct location and the presence or absence of collateral vessels. There was a significant correlation between faint uptake and inferoposterior infarct location. In 21 patients who had coronary angiography close to the time of antimyosin injection, there was a significant correlation between faint tracer uptake and closed infarct-related vessel with absent collateral flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Exercise-induced myocardial ischemia in patients with coronary artery disease: lack of evidence for platelet activation or fibrin formation in peripheral venous blood.

The hypothesis that exercise-induced myocardial ischemia is associated with abnormal platelet activation and fibrin formation or dissolution was tested in patients with coronary artery disease undergoing upright bicycle stress testing. In vivo platelet activation was assessed by radioimmunoassay of platelet factor 4, beta-thrombo-globulin and thromboxane B2. In vivo fibrin formation was assessed by radioimmunoassay of fibrinopeptide A, and fibrinolysis was assessed by radioimmunoassay of thrombin-increasable fibrinopeptide B which reflects plasmin cleavage of fibrin I. Peripheral venous concentrations of these substances were measured in 10 normal subjects and 13 patients with coronary artery disease at rest and during symptom-limited peak exercise. Platelet factor 4, beta-thromboglobulin and thromboxane B2 concentrations were correlated with rest and exercise catecholamine concentrations to determine if exercise-induced elevation of norepinephrine and epinephrine enhances platelet activation. Left ventricular end-diastolic and end-systolic volumes, ejection fraction and segmental wall motion were measured at rest and during peak exercise by first pass radionuclide angiography. All patients with coronary artery disease had documented exercise-induced myocardial ischemia manifested by angina pectoris, ischemic electrocardiographic changes, left ventricular segmental dyssynergy and a reduction in ejection fraction. Rest and peak exercise plasma concentrations were not significantly different for platelet factor 4, beta-thromboglobulin, thromboxane B2, fibrinopeptide A and thrombin-increasable fibrinopeptide B. Peripheral venous concentrations of norepinephrine and epinephrine increased significantly (p less than 0.001) in both groups of patients. The elevated catecholamine levels did not lead to detectable platelet activation. This study demonstrates that enhanced platelet activation, thromboxane release and fibrin formation or dissolution are not detectable in peripheral venous blood of patients with coronary disease during exercise-induced myocardial ischemia.

Postischemic stunning can affect left ventricular ejection fraction and regional wall motion on post-stress gated sestamibi tomograms.

OBJECTIVES: This study was designed to investigate whether left ventricular ejection fraction (LVEF) calculated from post-stress single-photon emission computed tomography (SPECT) reflects the basal value for LVEF or whether post-stress LVEF is reduced in some patients with stress-induced ischemia. BACKGROUND: Automated programs are now commercially available for assessing global left ventricular (LV) function from post-stress technetium-99m sestamibi gated SPECT performed >15 min after completion of exercise. METHODS: Eighty-one sequential patients who underwent a 2-day stress/rest sestamibi imaging protocol and showed perfusion defects on the post-stress tomogram underwent gated acquisition of the second-day rest tomogram. The post-stress and rest tomographic images were read for presence, location, severity and reversibility of defects by consensus of two to three experienced observers with the aid of circumferential count displays. Defects were scored as mild, moderate or severe and as completely or partially reversible or fixed, and a summed defect severity score was calculated. Of these 81 scans, 20 showed nonreversible perfusion defects (group 3), whereas 61 showed reversible perfusion defects. Post-stress and rest LVEF was calculated from the processed gated SPECT data. From 15 additional patients who underwent rest gated SPECT studies on separate days, serial reproducibility of LVEF values calculated from the gated SPECT data was determined to be +/-5.2%. Coronary angiography was performed within 3 months of the scan without intervening events in 47 of 81 patients, including 39 of 61 with reversible perfusion defects. RESULTS: In 22 (36%) of 61 patients with reversible perfusion defects, post-stress LVEF was >5% lower than that at rest (group 2), whereas in the remaining 39 patients, post-stress LVEF was either +/-5% or greater than that at rest (group 1). Segmental chordal shortening analysis performed in group 2 studies showed that differences in chordal shortening between rest and post-stress were significantly greater in the reversible perfusion defect territories than in the nonischemic perfusion defect territories ([mean +/- SD] 0.14 +/- 0.14 vs. 0.02 +/- 0.09, respectively, p < 0.0001). There were no significant differences among groups for any of the following variables: age, gender, previous myocardial infarction and type of stress. Time to imaging and stress and scan variables were correlated with the change in LVEF by univariate analysis, and the two variables that correlated significantly were the summed defect reversibility score on the scan and a left anterior descending coronary artery location of the scan defect. Only summed defect reversibility score was significant on multivariate analysis. CONCLUSIONS: When the only gated sestamibi scan is the post-stress scan, global and regional LV function will not represent basal LV function in all patients with stress-induced ischemia.

Effects of propranolol and timolol on left ventricular volumes during exercise in patients with coronary artery disease.

The hemodynamic effects of beta-receptor blocking agents on the ejection fraction of patients with coronary artery disease during exercise have been studied previously using radionuclide techniques. Left ventricular volume measurements and the peak systolic pressure/end-systolic volume (PSP/ESV) index have been shown to be variables of left ventricular function that are less influenced by preload and afterload than is ejection fraction. Left ventricular volumes and PSP/ESV were therefore measured in 18 patients with proven coronary artery disease in the control state and after 2 weeks of daily maintenance therapy with either 240 mg propranolol or 60 mg timolol. Values at rest and during symptom-limited upright exercise were compared using the first pass technique and a multicrystal scintillation camera. Left ventricular volumes were measured by the area-length method. Because there was no difference between the propranolol and timolol groups, the results for both groups were combined. The ejection fraction at rest after beta-receptor blocker treatment was not significantly different from pretreatment measurements because of an increase in both end-diastolic and end-systolic volumes (p less than 0.01). However, the value for peak systolic pressure/end-systolic volume (PSP/ESV) index at rest was lower after treatment. The exercise ejection fraction was greater after treatment (p less than 0.01), owing to an increase in end-diastolic volume and unchanged end-systolic volume. In addition, there was a significant improvement in the directional change in the PSP/ESV ratio between rest and exercise from pretreatment to treatment (-1.1 +/- 2.5 to +0.2 +/- 1.2, p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Imaging of cardiac allograft rejection in dogs using indium-111 monoclonal antimyosin Fab.

The acute rejection of cardiac allografts is currently diagnosed by the presence of myocyte necrosis on endomyocardial biopsy. We evaluated the efficacy of noninvasive scintigraphic imaging with indium-111-labeled anticardiac myosin Fab fragments (indium-111 antimyosin) to detect and quantify cardiac allograft rejection. Six dogs that had intrathoracic heterotopic cardiac allograft transplantation were injected with indium-111 antimyosin and planar and single photon emission computed tomographic (SPECT) images were obtained in various stages of acute and subacute rejection. Four dogs had an allograft older than 8 months and had been on long-term immunosuppressive therapy; two dogs had an allograft less than 2 weeks old and were not on immunosuppressive therapy. Count ratios comparing heterotopic with native hearts were calculated from both SPECT images and in vitro scans of excised and sectioned hearts and were compared with the degree of rejection scored by an independent histopathologic review. Indium-111 antimyosin uptake was not visible in planar or SPECT images of native hearts. Faint diffuse uptake was apparent in cardiac allografts during long-term immunosuppression and intense radioactivity was present in hearts with electrocardiographic evidence of rejection. The heterotopic to native heart count ratios in SPECT images correlated significantly with the count ratios in the excised hearts (r = 0.93) and with the histopathologic rejection score (r = 0.97). The distribution of indium-111 antimyosin activity in right and left ventricles corresponded to areas of histopathologic abnormalities. Immunoperoxidase studies showed deposition of indium-111 antimyosin only in areas of myocyte necrosis. The results demonstrate that indium-111 antimyosin imaging can noninvasively detect the presence, location and severity of canine cardiac allograft rejection.

Matrix metalloproteinases.

Matrix metalloproteinases are a family of highly regulated peptidases that are collectively responsible for the degradation of extracellular matrix during tissue remodeling. Dysregulated activity has long been implicated in the pathologies of cancer and arthritis, and the number of diseases more recently associated with these enzymes has been increasing. In the past year, new transgenic models of matrix metalloproteinase knockouts have been described, allowing the direct assessment of specific enzyme activity in particular disease models. In addition, more selective inhibitors with improved pharmacokinetic profiles have entered clinical trials, allowing the assessment of the safety and efficacy of this strategy.

Bacteriophage lambda N-dependent transcription antitermination. Competition for an RNA site may regulate antitermination.

Bacteriophage lambda controls the expression of its early genes in a temporal manner by a series of transcription termination and antitermination events. This antitermination requires the lambda N protein as well as host proteins called Nus, and cis-acting sites called nut. Following transcription of the nut site, N and Nus proteins bind to the nut RNA and modify the transcription complex to a termination-resistant form. The nut site is a composite of at least two components; one is the boxB hairpin structure which interacts with N. The other is boxA, a nine-nucleotide sequence upstream of boxB. To understand more about the formation of the antitermination complex, we have characterized the effect of point mutations in and deletions of boxA on antitermination. Point mutations in boxA were found to either enhance or reduce N-mediated antitermination. Several boxA deletions, on the other hand, had little effect on antitermination other than to eliminate the requirement for the NusB host protein. To explain these observations, we propose that at least two factors compete to interact with boxA, NusB and an inhibitor of the antitermination reaction. In addition, we propose that NusB is required to prevent the inhibitor from binding at boxA. The results with various nusB and boxA mutations can be explained by this model of competition between NusB and an inhibitor for boxA RNA.

No association between alcohol supplementation and autoantibodies to DNA damage in postmenopausal women in a controlled feeding study.

Alcohol consumption is linked to increased breast cancer risk. Since oestrogens increase breast cancer risk, possibly through oxidative damage, and we have shown that alcohol consumption increases serum oestrogens, we tested whether moderate alcohol supplementation increased oxidative DNA damage among healthy postmenopausal women not on hormone replacement therapy in a randomized controlled crossover study. We used serum 5-hydroxymethyl-2-deoxyuridine (5-HMdU) autoantibodies (aAbs) as a marker of oxidative DNA damage. The results showed no evidence for increased or decreased levels of oxidative DNA damage among women who consumed 15 g or 30 g alcohol per day for 8 weeks compared with women in the 0 g alcohol group. We conclude that among healthy women, it is possible that an 8-week trial of moderate alcohol supplementation might be too short to make enough 5-HMdU aAbs to compare differences by alcohol dose. In future studies, a panel of biomarkers for DNA damage should be used.

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface.

X-ray structure of human stromelysin catalytic domain complexed with nonpeptide inhibitors: implications for inhibitor selectivity.

Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue-degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavailability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin-1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 A. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S'1 pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S'1 pocket. The largest differences occur in the loop forming the "top" of this pocket. The occupation of these nonpeptidic inhibitors in the S'1 pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.

Distribution and regulation of galanin-like peptide (GALP) in the hypothalamus of the mouse.

Galanin-like peptide (GALP) is a newly discovered molecule whose expression in the brain is confined to the arcuate nucleus and median eminence. In the rat, cellular levels of GALP mRNA are reduced by fasting and reversed by peripheral administration of leptin. The purpose of this investigation was 1) to clone and map the distribution of GALP mRNA in the brain of the mouse; 2) to compare the pattern and magnitude of GALP mRNA expression in the leptin-deficient obese (ob/ob) mouse with that of wild-type controls; and 3) to examine the effects of leptin delivered into the brain on the expression of GALP mRNA in the ob/ob mouse. We report the sequence of a mouse GALP cDNA and show that GALP mRNA is expressed in the arcuate nucleus, median eminence, infundibular stalk, and the neurohypophysis of this species. The expression of GALP mRNA in the brain was markedly reduced in the ob/ob mice, compared with wild-type animals. Intracerebroventricular infusion of leptin to ob/ob mice increased both the number of GALP mRNA-expressing neurons and their content of GALP mRNA, compared with vehicle-treated controls. These observations demonstrate that GALP mRNA is induced by leptin through a direct action on the brain.