The role of basic amino acid surface clusters on the collagenase activity of cathepsin K.

Cathepsin K is a highly potent collagenase in osteoclasts and is responsible for bone degradation. We have previously demonstrated that its unique collagenolytic activity is modulated by glycosaminoglycans that form high molecular weight complexes with the protease. However, mutational analysis of a specific glycosaminoglycan-cathepsin K binding site only led to a 60% reduction of the collagenolytic activity suggesting additional glycosaminoglycan binding sites or other determinants controlling this activity. We identified eight cathepsin K specific arginine/lysine residues that form three positively charged clusters at the bottom part of the protease opposing the active site. These residues are highly conserved among mammalian, avian, and reptilian cathepsin K orthologues and to a lesser degree in amphibian and fish specimens. Mutational analysis of these residues revealed an approximately 50% reduction of the collagenolytic activity when the basic amino acids in cluster 2 (K103, K106, R108, R111) were mutated into alanine residues and resulted in a 100% loss of this activity when the mutations were expanded into cluster 3 (K122, R127). Cluster 1 mutations (K77, R79) had no effect. A partial rescue effect was observed when the hexamutant variant was combined with three mutations in the previously identified glycosaminoglycan binding site (N190, K101, L195K) indicating the relevance of at least two independent interaction sites. Amino acid substitutions in all sites had no effect on the catalytic efficacy of the protease variants as reflected in their unaltered peptidolytic and gelatinolytic activities and their overall protein stabilities. This study suggests that the basic amino acid clusters in cathepsin K are involved in alternative glycoasaminoglycan binding sites, play other roles in the formation of collagenolytically active protease complexes, or contribute in a yet unknown manner to the specific binding to collagen.

A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter.

BACKGROUND: To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein. Direct chromogenic staining for α-sec-EAP activity is non-specific as its NBT/BCIP substrate cross-reacts with cellular phosphatases which can be inhibited with Levulinic acid. However, the parental E(P) strain only exports detectable levels of α-sec-EAP at 69 hours and not within the 36 hour period post-seeding required for effective screening with the consequent absence of a reference for secretion. We substituted the endogenous cellular phosphatase activity as a comparative reference for secretion rate and levels as well as for colony alignment while elevating specificity and sensitivity of detection of the exported protein with other innovative modifications of the immuno-chromogenic staining application for screening protein export mutants. RESULTS: Raising the specificity and utility of staining for α-sec-EAP activity required 5 modifications including some to published methods. These included, exploitation of endogenous phosphatase activity, reduction of the cell/protein burden, establishment of the direct relation between concentrations of transcriptional inducer and exported membrane immobilized protein and concentrations of protein exported into growth media, amplification of immuno-specificity and sensitivity of detection of α-sec-EAP reporter enzyme signal and restriction of staining to optimal concentrations of antisera and time periods. The resultant immuno-chromogenic screen allows for the detection of early secretion and as little as 1.3 fold over-secretion of α-sec-EAP reporter protein by E(M) mutants in the presence of 10 fold -216 fold higher concentrations of HSA. CONCLUSIONS: The modified immuno-chromogenic screen is sensitive, specific and has led to the isolation of mutants E(M) over-secreting the α-sec-EAP reporter protein by a minimum of 50 fold higher levels than that exported by non-mutagenized E(P) parental strains. Unselected proteins were also over-secreted.

Structure-activity analysis of cathepsin K/chondroitin 4-sulfate interactions.

In the presence of oligomeric chondroitin 4-sulfate (C4-S), cathepsin K (catK) forms a specific complex that was shown to be the source of the major collagenolytic activity in bone osteoclasts. C4-S forms multiple contacts with amino acid residues on the backside of the catK molecule that help to facilitate complex formation. As cathepsin L does not exhibit a significant collagenase activity in the presence or in the absence of C4-S, we substituted the C4-S interacting residues in catK with those of cathepsin L. Variants revealed altered collagenolytic activities with the largest inhibitory effect shown by the hexavariant M5. None of the variants showed a reduction in their gelatinolytic and peptidolytic activities when compared with wild-type catK, indicating no structural alteration within their active sites. However, the crystal structure of the M5 variant in the presence of oligomeric C4-S revealed a different binding of chondroitin 4-sulfate. C4-S is not continuously ordered as it is in the wild-type catK·C4-S complex. The orientation and the direction of the hexasaccharide on the catK surface have changed, so that the hexasaccharide is positioned between two symmetry-related molecules. Only one M5 variant molecule of the dimer that is present in the asymmetric unit interacts with C4-S. These substitutions have changed the mode of catK binding to C4-S and, as a result, have likely affected the collagenolytic potential of the variant. The data presented here support our hypothesis that distinct catK/C4-S interactions are necessary for the collagenolytic activity of the enzyme.

Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex.

Genome stability requires a set of RecQ-Top3 DNA helicase-topoisomerase complexes whose sole budding yeast homolog is encoded by SGS1-TOP3. RMI1/NCE4 was identified as a potential intermediate in the SGS1-TOP3 pathway, based on the observation that strains lacking any one of these genes require MUS81 and MMS4 for viability. This idea was tested by confirming that sgs1 and rmi1 mutants display the same spectrum of synthetic lethal interactions, including the requirements for SLX1, SLX4, SLX5, and SLX8, and by demonstrating that rmi1 mus81 synthetic lethality is dependent on homologous recombination. On their own, mutations in RMI1 result in phenotypes that mimic those of sgs1 or top3 strains including slow growth, hyperrecombination, DNA damage sensitivity, and reduced sporulation. And like top3 strains, most rmi1 phenotypes are suppressed by mutations in SGS1. We show that Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast and that these proteins interact directly in a recombinant system. The Rmi1-Top3 complex is stable in the absence of the Sgs1 helicase, but the loss of either Rmi1 or Top3 in yeast compromises its partner's interaction with Sgs1. Biochemical studies demonstrate that recombinant Rmi1 is a structure-specific DNA binding protein with a preference for cruciform structures. We propose that the DNA binding specificity of Rmi1 plays a role in targeting Sgs1-Top3 to appropriate substrates.

Production and activation of recombinant papain-like cysteine proteases.

Papain-like cysteine proteases are the most numerous family of the cysteine protease class. They are expressed throughout the animal and plant kingdoms as well as in viruses and bacteria. More recently, this protease family has drawn attention as a potential pharmaceutical drug target in diseases characterized by excessive extracellular matrix degradation such as in osteoporosis, arthritis, vascular diseases, and cancer. Moreover, papain-like cysteine proteases have been identified as critical components of the life cycle and invasive potential of various human and live stock pathogens as well as major allergens. Therefore, this protease class is rigorously studied and requires sufficient amounts of protease protein to analyze structure-activity relationships, their 3-D structures as well as to screen for and optimize potent and selective inhibitors. This review summarizes approaches to generate active papain-like cysteine proteases by heterologous expression in a variety of expression systems.

Resultados del Censo de Servicios de Hemodinamia y Cardiología Intervencionista 2000 en la República Mexicana / Results from the 2000-census of hemodynamic and interventional cardiology services in Mexico

Presentamos los resultados del Censo Nacional de Servicios de Hemodinamia y Cardiología Intervencionista realizado por el capítulo de cardiología intervencionista de la Sociedad Mexicana de Cardiología hasta el año 2000. En el presente documento se señalan el número total de servicios existentes, así como las características físicas de dichas salas y los recursos humanos con los que cuentan. Finalmente se hace un señalamiento sobre el número y tipo de procedimientos que se practican.

[Myocardial injury caused by reperfusion]. / Daño miocárdico por reperfusión.

Arterial coronary occlusion produces ischaemic changes, and alter the aerobic metabolism, creatinephosphate depletion and accumulation of anoxic metabolites in the ischaemic tissue, with an alteration in the calcium regulation. With the recovery of the blood flow, the myocardial ischaemic injury and infarct zone are diminished, leading to an improvement of survival. The adverse effect induced by the reperfusion of ischaemic cells with the production of free radicals and dearrangements in the glucose metabolism, fatty acids and intracellular calcium flow as well, has been proven. There are 4 kinds of reperfusion damage: stunned myocardium, reperfusion arrhythmia, no-reflow phenomena, and cellular death, all of them with a particular physiopathology. Nowadays, there are too many paraclinics in order to reach a diagnosis, and perhaps the only available treatment is still under research. This field has great expectancy in the future. In this article, some of the contemporary concepts are reviewed.

[Results of the Census of Hemodynamic Services and Interventional Cardiology 2000 in the Mexican Republic]. / Resultados del Censo de Servicios de Hemodinamia y Cardiología Intervencionista 2000 en la República Mexicana.

We present the results from the National Census of cardiac catheterization and interventionist Cardiology Services performed by the Chapter of interventional Cardiology of the Mexican Society of Cardiology up to the year 2000. The present document identifies the total number of existing services, their physical characteristics and available human resources, as well as the number and type of procedures performed in them.

[Detection of early occlusion of aortocoronary by-passes. Usefulness of a non-angiographic method]. / Detección de la oclusión temprana de puentes aortocoronarios. Utilidad de un método no angiográfico.

A total of 83 bypasses were studied. Angiographic results demonstrated occlusion in 3 of 24 bypass of internal mammary artery placed in the anterior descending artery, 2 in the right coronary artery, 1 in the posterolateral of the circumflex and 1 in the first diagonal branch, with a total occlusion average of 8.4% within the first 8 days. We found a good correlation between the coronarographic angiograms and the positivity or negativity of the echo-electrocardiographic tests, during atrial pacing. We believe that this simple method could be done routinely in all the patients after coronary surgery, to decide the need of a new coronary angiogram. Furthermore, this study shows that the occlusion of a single coronary bypass does not produce myocardial infarction, detectable by enzymatic measures or by resting EKG. This method also detects the early post-operatory sinus sick syndrome.

[An assessment of myocardial ischemia in women with the exercise test validated by coronary angiography]. / Evaluación de la isquemia miocárdica en mujeres con la prueba de esfuerzo validada mediante coronariografía.

Ischemic heart disease has been studied in men and women only as a group. We studied the sensitivity and specificity of the exercise stress testing in women. The exercise stress testing (EST) was validated with the coronary arteriography. We studied 72 women and 129 men, the mean age was 55 years for women and 51 years for men. We found in women 83.2% of estimated maximal heart rate; the rate pressure product was 2.4, no different from the values recorded in men (p greater than 0.06). The blood pressure response to exercise was higher in women, and the maximal work capacity was more elevated in men (p less than 0.006). We found normal coronary arteries in 41 women, 70.7% with a positive (EST) compared with a 87.07% in men. The sensitivity and specificity of the exercise stress testing for the diagnosis of ischemic heart disease is smaller in women.

[The clinical features of myocardial infarct via "normal" coronary angiography. The experience of the Instituto Nacional de Cardiología]. / Rasgos clínicos del infarto del miocardio con coronariografía "normal". Experiencia del Instituto Nacional de Cardiología.

The end-point of our study was to find clinical differences between patients with myocardial infarction and evident obstructions in coronary angiography, and patients without coronary obstructions. In a 10 years follow-up period, 48 patients with diagnosis of myocardial infarction and normal coronary arteries by angiography (group A) where admitted al hospital. We compared this group with 80 patients (group B), randomly selected from all patients with myocardial infarction and evident obstructions in coronarography. Patient age at the ischemic event, familiar history of ischemic heart disease or sudden death, obesity, drug habits, contraception drugs use, and history of migraine and Raynaud's phenomenon were pointed out. Thirty patients from group A were admitted with acute myocardial infarction, and we also analysed in these patients prodromal symptoms, precipitating factors, AMI localisation, serum enzymatic levels, rhythm and/or conduction disturbances, clinical condition, treatment and new ischemic events. Group A patients showed lower age-average, smoking habit, prodromal symptoms and serum CPK levels. They have better ventricular performance parameters and a greater incidence of women. The only different risk factor for ischemic heart disease between both groups was smoking.

[Exercise test in patients with permanent pacemakers]. / La prueba de esfuerzo en pacientes con marcapaso permanente.

From June 1988 to June 1990 we studied fifty patients who had implantation of a pacemaker. (31 females and 19 males). All of them underwent stress test with Bruce's protocol. Patients were divided in two groups; pacemaker-independent (PI) and pacemaker-dependent (PD). Over 50% of the patients inhibited the pacemaker with their own rhythm, most of them had sinus dysfunction. Complete A-V block was predominant in PD. The group of PI achieved more mets and had more oxygen consumption. Blood pressure response was similar in both groups.

[Value of the exercise test in asymptomatic myocardial ischemia]. / El valor de la prueba de esfuerzo en la isquemia miocárdica asintomática.

To evaluate the predictive value of ischemic ST segment depression without associated chest pain during exercise testing, data were analyzed from 7305 studies. Two hundred thirty six patients were included in this study and were separated in 2 groups. Group A consisted of 169 patients without chest pain who, during exercise testing, showed a positive ST segment response (at least 1.5 mm of horizontal or downward ST segment depression for at least 0.08 second, compared with the resting baseline value), and Group B consisted of 67 patients who had both chest pain and a positive ST segment response. Selective coronary angiogram was performed on all patients. Each Group was separated into 3 sub-group according to the Cohn criteria: sub-group I (asymptomatic persons 8.3 vs 19.4%); sub-group II (patients with history of Myocardial Infarction 36.7% vs 19.4%); sub-group III (patients with chronic angina 55% vs 61.2%). The clinical characteristics, coronary risk factors, distribution of coronary artery disease, and exercise test response were similar in both groups. During treadmill exercise, the mean heart rate was 140.6 +/- 22 in group A versus 127.1 +/- 23 in the group B. The pressure-rate product was 2.4 +/- 0.8 versus 1.9 +/- 0.5, respectively (P less than or equal to 0.05). The predictive value for severe coronary artery disease of an exercise test in patients with asymptomatic ischemia was 77.5% as compared with 89.6% in the group with angina. This study confirms the high frequency of asymptomatic myocardial ischemia during exercise testing, compared with patients who had angina during exercise testing, with high percentage of prediction (77.5%) for coronary artery disease.

[Asymptomatic myocardial ischemia]. / Isquemia miocárdica asintomática.

The concept, physiopathology, epidemiology, diagnostic procedures, prognosis and treatment of asymptomatic myocardial ischemia are reviewed. Hypotheses given to explain the absence of pain in the presence of myocardial ischemia are analyzed; Cohn's classification of asymptomatic myocardial ischemia is described and complemented with other clinical entities of painless myocardial ischemia. Prevalence of asymptomatic myocardial ischemia in different groups of patients is also discussed as well as the most important characteristics of diagnostic procedures. Finally the prognosis of asymptomatic myocardial ischemia is analyzed and the present therapeutic possibilities are discussed.

[Diagnosis of ischemic heart disease by infusion of isopropylarterenol: comparison with ergometry and validation by coronary arteriography]. / Diagnóstico de la cardiopatía isquémica con infusión de isopropilarterenol: comparación con ergometría y validación mediante coronariografía.

In order to evaluate isopropylarterenol infusion (ISO) as a diagnostic procedure in ischemic heart disease, we performed a clinical study in 54 patients controlled with exercise stress test (ERGO) and validated by coronary arteriography. Eighteen patients had normal coronary arteriographic findings and 36 had coronary artery disease. In both groups the hemodynamic response was similar in either test used (ERGO or ISO), when similar heart rates were reached. The sensitivity of ERGO was 80.55% and 86.11% for ISO and specificity of 77.72% and 72.26% respectively in the diagnosis of coronary artery disease. When both tests are associated their sensitivity is 83.30% and their specificity is 75.0% (Table V). The ISO test was not accompanied with adverse effects and could be considered as a useful method in the diagnosis of coronary heart disease, similar to ERGO. Additionally ISO could be applied to understand some physiopathological mechanisms of ischemic heart disease.

[Continuous electrocardiographic recording during a first parachute jump]. / Registro electrocardiográfico continuo durante el primer salto en paracaídas.

The behavior of the cardiac rhythm under intense stress was studied with continuous electrocardiographic recording during the first jump with an automatic parachute in 13 members of the Universidad Nacional Autónoma de México sky diving club. There were 12 male (92.3%) and one female (7.68) with an average age of 22.8 years. A complete clinical history, 12 lead electrocardiogram and a treadmill stress test were performed two weeks before jumping. A two-channel Holter recording system was hooked up 30 min. before boarding the airplane and was turned off one hour later. The heart rate values were compared two weeks prior (64.5 beats/min), before (112.8 beats/min), during (170 beats/min) and after the jump (122.8 beats/min). The mean difference between each phase was statistically significant with p less than 0.001 values. The observed cardiac rhythm was sinus tachycardia in each case. In six cases (43.6%) 22 episodes of sudden decrease of the heart rate were seen and there were no major rhythm or conduction disturbances. The urinary catecholamines were similar in nine cases (69.2%) after the jump (x 51.2 micrograms/100 ml) and two weeks later in four control cases (x 10.3 micrograms/100 ml). We concluded there were no significant rhythm disturbances in the presence of an intense but brief stress condition in young healthy people. This study was classified as longitudinally, descriptive, experimental and projective.