[Spa therapy in osteoarthritis: experience in Cervia]. / La terapia termale nell'osteoartrosi: esperienza di Cervia.

AIMS: Mud-bath treatment has a relevant therapeutic activity in reducing symptoms and disabilities in rheumatic diseases, as studies on inflammation process and clinical symptomatology have shown. Objective of this study is to evaluate the efficacy of mud therapy by natural limans of Cervia on clinical parameters in patients with lumbar spine osteoarthrosis (OA). MATERIALS AND METHODS: 30 patients (20 females and 10 males) suffering from lumbar OA, diagnosed by clinical symptoms and X-Ray findings (osteophytosis, morphological changes of vertrebral somes, tightness of intervertebral spaces, alterated lumbar physiologic lordosis) were studied. All patients were examined first when recruited (T0) and after two weeks of therapy (T1). Interviews were submitted in order to estimate functional restrictions and pain. Statistical analysis was performed via t test for paired data. RESULTS: On the basis of the statistical analysis a very significant difference was demonstrated between the average values of the tests before and after the treatment. These results confirm the improvement of the symptoms concerning the pain, the articular functionality and the quality of life. CONCLUSIONS: The results of the study demonstrate the efficacy of the mud-bath treatment with natural limans of Cervia on the patients affected by lumbar spine osteoarthrosis (OA).

Structure of eicosapentaenoic and linoleic acids in the cyclooxygenase site of prostaglandin endoperoxide H synthase-1.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) can oxygenate 18-22 carbon polyunsaturated fatty acids, albeit with varying efficiencies. Here we report the crystal structures of eicosapentaenoic acid (EPA, 20:5 n-3) and linoleic acid (LA, 18:2 n-6) bound in the cyclooxygenase active site of Co(3+) protoporphyrin IX-reconstituted ovine PGHS-1 (Co(3+)-oPGHS-1) and compare the effects of active site substitutions on the rates of oxygenation of EPA, LA, and arachidonic acid (AA). Both EPA and LA bind in the active site with orientations similar to those seen previously with AA and dihomo-gamma-linolenic acid (DHLA). For EPA, the presence of an additional double bond (C-17/C-18) causes this substrate to bind in a "strained" conformation in which C-13 is misaligned with respect to Tyr-385, the residue that abstracts hydrogen from substrate fatty acids. Presumably, this misalignment is responsible for the low rate of EPA oxygenation. For LA, the carboxyl half binds in a more extended configuration than AA, which results in positioning C-11 next to Tyr-385. Val-349 and Ser-530, recently identified as important determinants for efficient oxygenation of DHLA by PGHS-1, play similar roles in the oxygenation of EPA and LA. Approximately 750- and 175-fold reductions in the oxygenation efficiency of EPA and LA were observed with V349A oPGHS-1, compared with a 2-fold change for AA. Val-349 contacts C-2 and C-3 of EPA and C-4 of LA orienting the carboxyl halves of these substrates so that the omega-ends are aligned properly for hydrogen abstraction. An S530T substitution decreases the V(max)/K(m) of EPA and LA by 375- and 140-fold. Ser-530 makes six contacts with EPA and four with LA involving C-8 through C-16; these interactions influence the alignment of the substrate for hydrogen abstraction. Interestingly, replacement of Phe-205 increases the volume of the cyclooxygenase site allowing EPA to be oxygenated more efficiently than with native oPGHS-1.

Inhibition of angiogenesis and tumor growth by SCH221153, a dual alpha(v)beta3 and alpha(v)beta5 integrin receptor antagonist.

New blood vessel formation is essential for tumor growth and metastatic spread. Integrins alpha(v)beta3 and alpha(v)beta5 are arginine-glycine-aspartic acid-dependent adhesion receptors that play a critical role in angiogenesis. Hence, selective dual alpha(v)beta3 and alpha(v)beta5 antagonists may represent a novel class of angiogenesis and tumor-growth inhibitors. Here, an arginine-glycine-aspartic acid-based peptidomimetic library was screened to identify alpha(v)beta3 antagonists. Selected compounds were then modified to generate potent and selective dual inhibitors of alpha(v)beta3 and alpha(v)beta5 receptors. One of these compounds, SCH 221153, inhibited the binding of echistatin to alpha(v)beta3 (IC50 = 3.2 nM) and alpha(v)beta5 (IC50 = 1.7 nM) with similar potency. Its IC50 values for related alpha(IIb)beta3 and alpha5beta1 receptors were 1294 nM and 421 nM, respectively, indicating that SCH 221153 is highly selective for alpha(v)beta3 and alpha(v)beta5 receptors. In cell-based assays, SCH 221153 inhibited the binding of echistatin to alpha(v)beta3- and alpha(v)beta5-expressing 293 cells and blocked the adhesion of endothelial cells to immobilized vitronectin and fibroblast growth factor 2 (FGF2). SCH 221153, but not the inactive analogue SCH 216687, was effective in inhibiting FGF2 and vascular endothelial growth factor-induced endothelial cell proliferation in vitro with an IC50 equal to 3-10 microM. Angiogenesis induced by FGF2 in the chick chorioallantoic membrane assay was also inhibited by SCH 221153. Finally, SCH 221153 exerted a significant inhibition on tumor growth induced by intradermal or s.c. injection of human melanoma LOX cells in severe combined immunodeficient mice.

Mutational and X-ray crystallographic analysis of the interaction of dihomo-gamma -linolenic acid with prostaglandin endoperoxide H synthases.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the committed step in prostaglandin biosynthesis. Both isozymes can oxygenate a variety of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-gamma-linolenic acid (DHLA) in the cyclooxygenase site of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with arachidonic acid (AA). DHLA is bound within the cyclooxygenase site in the same overall L-shaped conformation as AA. C-1 and C-11 through C-20 are in the same positions for both substrates, but the positions of C-2 through C-10 differ by up to 1.74 A. In general, substitutions of active site residues caused parallel changes in the oxygenation of both AA and DHLA. Two significant exceptions were Val-349 and Ser-530. A V349A substitution caused an 800-fold decrease in the V(max)/K(m) for DHLA but less than a 2-fold change with AA; kinetic evidence indicates that C-13 of DHLA is improperly positioned with respect to Tyr-385 in the V349A mutant thereby preventing efficient hydrogen abstraction. Val-349 contacts C-5 of DHLA and appears to serve as a structural bumper positioning the carboxyl half of DHLA, which, in turn, positions properly the omega-half of this substrate. A V349A substitution in PGHS-2 has similar, minor effects on the rates of oxygenation of AA and DHLA. Thus, Val-349 is a major determinant of substrate specificity for PGHS-1 but not for PGHS-2. Ser-530 also influences the substrate specificity of PGHS-1; an S530T substitution causes 40- and 750-fold decreases in oxygenation efficiencies for AA and DHLA, respectively.

Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid.

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.

The productive conformation of arachidonic acid bound to prostaglandin synthase.

Prostaglandin H synthase-1 and -2 (PGHS-1 and -2) catalyze the committed step in prostaglandin synthesis and are targets for nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin. We have determined the structure of PGHS-1 at 3 angstrom resolution with arachidonic acid (AA) bound in a chemically productive conformation. The fatty acid adopts an extended L-shaped conformation that positions the 13proS hydrogen of AA for abstraction by tyrosine-385, the likely radical donor. A space also exists for oxygen addition on the antarafacial surface of the carbon in the 11-position (C-11). While this conformation allows endoperoxide formation between C-11 and C-9, it also implies that a subsequent conformational rearrangement must occur to allow formation of the C-8/C-12 bond and to position C-15 for attack by a second molecule of oxygen.

Effect of general anesthesia on the severity of mitral regurgitation by transesophageal echocardiography.

The effect of general anesthesia on the severity of mitral regurgitation (MR) was examined in 43 patients with moderate or severe MR who underwent preoperative and intraoperative transesophageal echocardiography. Systolic blood pressure, mean arterial pressure, and left ventricular end-diastolic and end-systolic dimensions were significantly lower during the intraoperative study, reflecting altered loading conditions. The mean color Doppler jet area and mean vena contracta decreased and the mean pulmonary venous flow pattern changed from reversed to blunted, reflecting a significant reduction in the severity of MR. Overall, 22 of the 43 patients (51%) improved at least 1 MR severity grade when assessed under general anesthesia. Thus, intraoperative transesophageal echocardiography may significantly underestimate the severity of MR. A thorough preoperative assessment is preferable when deciding whether to perform mitral valve surgery.

The formation of stable fatty acid substrate complexes in prostaglandin H(2) synthase-1.

We have developed a protocol to purify apo-ovine (o) prostaglandin endoperoxide H(2) synthase-1 (PGHS-1) to homogeneity from ram seminal vesicles. The resulting apo enzyme can then be reconstituted with Co(3+)-protoporphyrin IX instead of Fe(3+)-protoporphyrin IX to produce a native-like, but functionally inert, enzyme suitable for the production of enzyme:fatty acid substrate complexes for biophysical characterization. Co(3+)-protoporphyrin IX reconstituted oPGHS-1 (Co(3+)-oPGHS-1) displays a Soret band at 426 nm that shifts to 406 nm upon reduction. This behavior is similar to that of cobalt-reconstituted horseradish peroxidase and myoglobin and suggests, along with resonance Raman spectroscopy, that the Co(3+)-protoporphyrin IX group is one in a six-coordinate, cobalt(III) state. However, Co(3+)-oPGHS-1 does not display cyclooxygenase or peroxidase activity, nor does the enzyme produce prostaglandin products when incubated with [1-(14)C]arachidonic acid. The cocrystallization of Co(3+)-oPGHS-1 and the substrate arachidonic acid (AA) has been achieved using sodium citrate as the precipitant in the presence of the nonionic detergent N-octyl-beta-d-glucopyranoside. Crystals are hexagonal, belonging to the space group P6(5)22, with cell dimensions of a = b = 181.69 A and c = 103.74 A, and a monomer in the asymmetric unit. GC-MS analysis of dissolved crystals indicates that unoxidized AA is bound within the crystals.

Combination therapy with the farnesyl protein transferase inhibitor SCH66336 and SCH58500 (p53 adenovirus) in preclinical cancer models.

SCH66336 is a p.o.-active, farnesyl protein transferase inhibitor. SCH66336 inhibits farnesylation of RAS and other proteins in tumor cells and suppresses tumor growth in human xenograft and transgenic mouse cancer models in vivo. SCH58500 is a replication-deficient, recombinant adenovirus, which expresses the human p53 tumor suppressor. In preclinical models, SCH58500 has therapeutic efficacy against a wide range of human tumor types containing nonfunctional p53 and enhanced activity in combination with many chemotherapeutic drugs. Here we report that combination therapy with SCH66336 and SCH58500 has synergistic or additive antiproliferative effects on a panel of tumor cells lines in vitro. The efficacy of the three-drug combination of SCH66336, SCH58500, and paclitaxel was also examined in vitro. Each two-drug interaction displayed such marked synergy, the addition of a third drug to the statistical model could only yield additivity. Greater combined efficacy for SCH66336 and SCH58500 was also observed in vivo in the DU-145 human prostate and wap-ras/F transgenic mouse cancer models.

Integrin alpha(v)beta(3)-mediated activation of apoptosis.

The alpha(v)beta(3) integrin mediates endothelial cell binding to the extracellular matrix and transduces an intracellular signal promoting survival of endothelial cells and various tumor cells. While the alpha(v)beta(3) integrin-mediated survival signal has been shown to be adhesion dependent, a thorough analysis has not been performed comparing the biochemical effects of antagonist binding to alpha(v)beta(3) integrin with the effects induced by the growth of cells in suspension. In this study we demonstrate that expression of alpha(v)beta(3) integrin in human embryonic kidney 293 cells transfers the alpha(v)beta(3) integrin survival pathway to an epithelial cell line. Furthermore, we show that alpha(v)beta(3) integrin-expressing cells respond differently to alpha(v)beta(3) integrin-specific antagonist treatment and growth in suspension conditions. Treatment with the alpha(v)beta(3) antagonist echistatin resulted in an apoptotic response occurring prior to cell detachment and was not observed in either suspended cells or antagonist-treated suspended cells. These data suggest that the death induced by antagonist binding to alpha(v)beta(3) integrin results in an apoptotic signal with different kinetics than the apoptotic signal induced by matrix detachment (anoikis). Since aberrant alpha(v)beta(3) integrin expression in tumor models is thought to play a role in tumor cell survival, these data have implications for the use of alpha(v)beta(3) antagonists as anti-tumor agents.

Effects of SCH 59228, an orally bioavailable farnesyl protein transferase inhibitor, on the growth of oncogene-transformed fibroblasts and a human colon carcinoma xenograft in nude mice.

The products of the Ha-, Ki-, and N-ras proto-oncogenes comprise a family of 21 kDa guanine nucleotide-binding proteins which play a crucial role in growth factor signal transduction and in the control of cellular proliferation and differentiation. Activating mutations in the ras oncogenes occur in a wide variety of human tumors. Ras proteins undergo a series of posttranslational processing events. The first modification is addition of the 15-carbon isoprene, farnesyl, to a Cys residue near the carboxy-terminus of Ras. Prenylation allows the Ras oncoprotein to localize to the plasma membrane where it can initiate downstream signalling events leading to cellular transformation. Inhibitors of the enzyme which catalyzes this step, farnesyl protein transferase (FPT), are a potential class of novel anticancer drugs which interfere with Ras function. SCH 59228 is a tricyclic FPT inhibitor which inhibits the farnesylation of purified Ha-Ras with an IC50 of 95 nM and blocks the processing of Ha-Ras in Cos cells with an IC50 of 0.6 microM. SCH 59228 has favorable pharmacokinetic properties upon oral dosing in nude mice. The in vivo efficacy of SCH 59228 was evaluated using a panel of tumor models grown in nude mice. These included several rodent fibroblast lines expressing mutationally-activated (val12) forms of the Ha-Ras oncogene. In some cases, these proteins contain their native C-terminal sequence (CVLS) which directs farnesylation. In one model, the C-terminal sequence was altered to CVLL, making the expressed protein a substrate for a distinct prenyl transferase, geranylgeranyl protein transferase-1. When dosed orally at 10 and 50 mg/kg (four times a day, 7 days a week) SCH 59228 significantly inhibited tumor growth of cells expressing farnesylated Ha-Ras in a dose-dependent manner; over 90% growth inhibition was observed at the 50 mg/kg dose. Tumor growth of cells expressing the geranylgeranylated form of Ha-Ras was less potently inhibited. Growth of tumors derived from a rodent fibroblast line expressing activated Ki-Ras containing its native C-terminal sequence (CVIM), which preferentially directs farnesylation, was also inhibited by SCH 59228. Inhibition in the Ki-Ras model was less than that observed in the Ha-Ras model. In contrast, tumors derived from cells transformed with the mos oncogene were not significantly inhibited even at the highest dose level. SCH 59228 also significantly and dose-dependently inhibited the growth of human colon adenocarcinoma DLD-1 xenografts (which express activated Ki-ras). These results indicate that SCH 59228 possesses in vivo antitumor activity upon oral dosing in tumor models expressing activated ras oncogenes. This is the first report of oral antitumor activity with an FPT inhibitor. These results are discussed in light of recent observations on alternative prenylation of some Ras isoforms.

Multiplane transesophageal echocardiographic identification of the involved scallop in patients with flail mitral valve leaflet: intraoperative correlation.

Although the role of multiplane transesophageal echocardiography in the diagnosis of flail mitral valve leaflet is well described, the accuracy of this modality in localizing the involved posterior leaflet scallop (medial, middle, or lateral) has never been validated. For 54 patients undergoing intraoperative transesophageal echocardiography for severe mitral regurgitation due to flail mitral valve leaflet, we assessed the accuracy of a systematic approach to localization of the flail mitral valve leaflet. Surgical confirmation was performed for all patients. At blinded review, a sensitivity of 78%, specificity of 92%, and overall diagnostic accuracy of 88% were achieved for correct localization of the flail posterior leaflet scallop. The middle scallop was most commonly affected in this series. The medial scallop was affected least often, and diagnosis of lesions in that area was least accurate. This diagnostic approach appears to be accurate and feasible and may assist in planning specific surgical therapy for this disorder.

Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice.

We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.

Minimally invasive direct coronary artery bypass: anesthetic, monitoring, and pain control considerations.

OBJECTIVE: Minimally invasive direct coronary artery bypass (MIDCAB) provides many anesthetic challenges including monitoring, managing myocardial ischemia, and pain control. The objective was to evaluate the monitoring requirements and the potential benefits of preischemic conditioning and intrathecal morphine sulfate in MIDCAB patients. DESIGN AND SETTING: This review was retrospective and unrandomized and was conducted at Allegheny University Hospitals, Allegheny General, Pittsburgh, PA. PARTICIPANTS: Sixty-four patients with single coronary artery lesions (> 70% obstruction) underwent attempted MIDCAB during a 1-year period between November 1995 and November 1996. Seven patients required conversion to conventional coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) and two patients required extended thoracotomy incisions. This report describes the remaining 55 patients who underwent MIDCAB. INTERVENTIONS: Some of the MIDCAB patients received intrathecal morphine before anesthetic induction. Ischemic preconditioning was assessed in a subset of patients. RESULTS: MIDCAB was performed in 55 of 64 patients. Transesophageal echocardiography (TEE) was used in all patients and a pulmonary artery catheter was used in 43% of patients. Esmolol was used in 25% of patients to reduce motion of the left ventricle (LV) during the left internal mammary artery (LIMA)-LAD anastomosis, but was used less often as the surgeons adapted to the use of a retractor that stabilized the ventricular wall adjacent to the site of the LIMA-LAD anastomosis. LAD occlusion caused reversible, regional systolic dysfunction by TEE in the anterior and apical LV segments. During LAD occlusion, nitroglycerin was used in 61% of patients and phenylephrine in 24%. Ischemic preconditioning did not prevent increases in systemic or pulmonary artery pressures during LAD occlusion. Most (85%) patients were extubated in the operating room. Intrathecal morphine decreased postoperative analgesic requirements. The mean hospital length of stay (LOS) was 4.0 +/- 1.7 days (range, 1 to 10 days). CONCLUSIONS: MIDCAB may reduce hospital LOS for patients with single vessel coronary artery lesions when compared with median sternotomy with a LIMA-LAD graft performed on cardiopulmonary bypass. Pharmacologic heart rate control during the LIMA-LAD anastomosis is not critical with the use of a surgical retractor which diminishes ventricular motion. A single 5-minute test LAD occlusion did not protect against subsequent regional ischemic dysfunction in our subset of patients with normal baseline function.

Transient ischemia does not limit subsequent ischemic regional dysfunction in humans: a transesophageal echocardiographic study during minimally invasive coronary artery bypass surgery.

OBJECTIVES: This study sought to assess the effects of sequential coronary artery occlusion during minimally invasive coronary artery bypass graft surgery (CABG) on hemodynamic variables and left ventricular systolic function by means of transesophageal echocardiography (TEE). BACKGROUND: Clinical and experimental studies suggest a protective effect of ischemic preconditioning in patients with acute coronary syndromes. However, the effect of repetitive myocardial ischemia on myocardial mechanical function in humans is not completely understood. METHODS: Seventeen patients with left anterior descending coronary artery (LAD) stenosis > or =70% and normal rest left ventricular systolic function referred for minimally invasive CABG underwent intraoperative TEE for assessment of regional left ventricular wall motion and measurement of hemodynamic variables at baseline (baseline 1), during a 5-min coronary occlusion (occlusion 1), after a 5-min reperfusion period (baseline 2) and a during a second coronary occlusion during bypass anastomosis (occlusion 2). RESULTS: Left ventricular wall motion score (LVWMS) increased significantly from baseline (16.0) to occlusion 1 (21.4+/-3.1 [mean +/- SD], p < 0.05) and occlusion 2 (21.8+/-3.1, p < 0.05). No difference in LVWMS was noted between occlusions 1 and 2. Pulmonary artery systolic pressure increased significantly from baseline (25+/-6 mm Hg) to occlusion 1 (32+/-7 mm Hg, p < 0.05) and occlusion 2 (33+/-6 mm Hg, p < 0.05). Pulmonary artery diastolic pressure also increased significantly from baseline (12+/-4 mm Hg) to occlusion 1 (16+/-4 mm Hg, p < 0.05) and occlusion 2 (16+/-4 mm Hg, p < 0.05). No significant differences in pulmonary artery pressures were noted between occlusions 1 and 2. CONCLUSIONS: Ischemic dysfunction was precipitated by the 5-min LAD occlusion, as shown by the increase in LVWMS and pulmonary artery pressure. However, a 5-min coronary occlusion and the resulting ischemia do not alter regional left ventricular systolic function during subsequent ischemia in humans.

The "Brisighella Terme" Project: effectiveness of preventive medicine in thermal ambient. Hematochemical parameters.

BACKGROUND: The main aims of the Brisighella Terme Project are: 1. evaluation of feasibility and effectiveness of preventive medicine intervention in thermal ambient; 2. identification of subjects with cardiovascular risk factors; 3. to give patients informations on risk factor correction. The data of this work concern the hematochemical parameters: CT, HDL-CT, LDL-CT, TG, ApoA1, ApoB, fibrinogen, GOT, GPT, CPK, glucose, uric acid. METHODS: CT, HDL-CT (after precipitation of the non-HDL fractions), TG, glucose, uric acid are evaluated by enzymatic-colorimetric reactions; GOT, GPT, CK by enzymatic method. ApoA1, ApoB, fibrinogen by immunoturbidimetric methods. LDL-CT is calculated by the Friedewald formula. RESULTS: 390 subjects spontaneously adhered to the Brisighella Terme Project. Of these subjects 38% requested laboratory service and we observed a 47% increment, from 1995 to 1996, with regard to this request. Females showed higher mean values than males of these parameters: CT, LDL-CT, HDL-CT, ApoA1, ApoB, fibrinogen, CPK. 55% of females had LDL-CT > 159 mg/dl, values considered high risk for cardiovascular diseases. 36% of males presented HDL-CT < 40 mg/dl, with 2 cases < 25 mg/dl. TG values > 399 mg/dl were found only in males (2%). We identified new cases of hypertriglyceridaemia, hypercholesterolaemia and hypoHD Laemia; these metabolic pathologies had not yet been diagnosed, whereas the patients already knew they were affected by hyperglicaemia or hyperuricaemia. CONCLUSIONS: People's interest in the Brisighella Terme Project, new case identification, the possibility of providing correct information about risk factors and healthy life style confirm the feasibility and effectiveness of preventive medicine in a thermal ambient.

Biochemical characterization of the binding of echistatin to integrin alphavbeta3 receptor.

Echistatin is a 49-amino-acid peptide belonging to the family of disintegrins that are derived from snake venoms and are potent inhibitors of platelet aggregation and cell adhesion. Integrin alphavbeta3 receptor plays a critical role in several physiological processes such as tumor-induced angiogenesis, tumor cell metastasis, osteoporosis and wound repair. In this study, we have characterized the binding of echistatin to purified integrin alphavbeta3 receptor and the form expressed on human embryonic kidney 293 cells. We show that both purified and membrane-bound integrin alphavbeta3 binds to echistatin with a high affinity, which can be competed efficiently by linear and cyclic peptides containing the RGD sequence. Previous studies have shown that alphavbeta3 binds to vitronectin in a nondissociable manner, whereas an RGD-containing peptide derived from vitronectin binds in a dissociable manner with a Kd of 9.4 x 10(-7) M. Our studies indicate that radiolabeled echistatin binds to alphavbeta3 in a nondissociable manner, similar to native echistatin. However, echistatin does not support the adhesion of 293 cells expressing alphavbeta3 receptor because of poor binding to plastic dishes and is a potent antagonist of the adhesion of these cells to vitronectin. These studies demonstrate that echistatin binding to alphavbeta3 is of high affinity and irreversible similar to vitronectin and provides an alternate ligand for high-throughput screening for alphavbeta3 antagonists.

Crystal structure of fibrinogen-Aalpha peptide 1-23 (F8Y) bound to bovine thrombin explains why the mutation of Phe-8 to tyrosine strongly inhibits normal cleavage at Arg-16.

A peptide containing residues 1-50 of the Aalpha-chain of fibrinogen, expressed as a fusion peptide with beta-galactosidase, is rapidly cleaved by thrombin at Arg-16, similarly to whole fibrinogen. When Phe-8, which is highly conserved, is replaced with tyrosine (F8Y), the cleavage is slowed drastically [Lord, Byrd, Hede, Wei and Colby (1990) J. Biol. Chem. 265, 838-843]. To examine the structural basis for this result, we have determined the crystal structure of bovine thrombin complexed with a synthetic peptide containing residues 1-23 of fibrinogen Aalpha and the F8Y mutation. The crystals are in space group P43212, with unit-cell dimensions of a = 88.3 A (1 A = 0.1 nm), c = 195.5 A and two complexes in the asymmetric unit. The final R factor is 0.183 for 2sigma data from 7.0 to 2.5 A resolution. There is continuous density for the five residues in the P3, P2, P1, P1' and P2' positions of the peptide (Gly-14f to Pro-18f) at the active site of thrombin, and isolated but well-defined density for Tyr-8f at position P9 in the hydrophobic pocket of thrombin. The tyrosine residue is shifted relative to phenylalanine in the native peptide because the phenol side chain is larger and makes a novel, intrapeptide hydrogen bond with Gly-14f. Adjacent peptide residues cannot form the hydrogen bonds that stabilize the secondary structure of the native peptide. Consequently, the 'reaction'geometry at the scissile bond, eight residues from the mutation, is perturbed and the peptide is mostly uncleaved in the crystal structure.