Normal cellular senescence and cancer susceptibility in mice genetically deficient in Ras-induced senescence-1 (Ris1).

Oncogenic Ras triggers a permanent cell-cycle arrest known as oncogene-induced senescence (OIS) that constitutes a relevant tumor suppressor mechanism. Ris1 (Ras-induced senescence-1) is a novel gene that was identified in a screen as specifically upregulated during Ras-induced senescence, and that is located at a chromosomal region, 3p21.3, frequently lost in human cancer. Moreover, Ris1 is highly conserved in vertebrates, does not present paralogs, and its sequence does not reveal similarities with other proteins or domains. To analyse the physiological function of Ris1 and test its putative role as a tumor suppressor gene, we have generated mutant mice deficient for this gene. Ris1-null mice are viable, fertile, develop normally and do not display any obvious abnormalities. Of relevance, Ris1-deficient mice had a normal lifespan and did not exhibit predisposition to spontaneous tumors or to tumors induced by chemical carcinogens. Finally, Ris1-deficient embryonic fibroblasts were indistinguishable from wild-type cells regarding their proliferation properties, immortalization, senescence and oncogenic transformation. These findings do not support a role of Ris1 in tumor suppression or in OIS.

Trauma care systems in Spain.

Trauma care systems in Spain are provided by the Nacional Health Service in a decentralized way by the seventeen autonomous communities whose process of decentralization was completed in January 2002. Its organisation is similar in all of them. Public sector companies of sanitary emergencies look after the health of citizens in relation to medical and trauma emergencies with a wide range of up to date resources both technical and human. In the following piece there is a description of the emergency response teams divided into ground and air that are responsible for the on site care of the patients in coordination with other public services. They also elaborate the prehospital clinical history that is going to be a valuable piece of information for the teams that receive the patient in the Emergency Hospital Unit (EHU). From 1980 to 1996 the mortality rate per 10.000 vehicles and the deaths per 1.000 accidents dropped significantly: in 1980 6.4 and 96.19% and in 1996, 2.8 and 64.06% respectively. In the intrahospital organisation there are two differentiated areas to receive trauma patients the casualty department and the EHU. In the EHU the severe and multiple injured patients are treated by the emergency hospital doctors; first in the triage or resuscitation areas and after when stabilised they are passed too the observation area or to the Intensive Care Unit (ICU) and from there the EHU or ICU doctors call the appropriate specialists. There is a close collaboration and coordination between the orthopaedic surgeon the EHU doctors and the other specialists surgeons in order to comply with treatment prioritization protocols. Once the patient has been transferred an entire process of assistance continuity is developed based on interdisciplinary teams formed in the hospital from the services areas involved in trauma assistance and usually coordinated by the ICU doctors. There is also mentioned the assistance registry of trauma patients, the ICU professional training in the ATLS and the future guidelines for trauma care in the ICU based on epidemiological studies carried out in both the North Spanish Group and the Southern one to promote development and improvement in several areas.

Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP).

Matrix metalloproteinase (MMP)-19 and MMP-20 (enamelysin) are two recently discovered members of the MMP family. These enzymes are involved in the degradation of the various components of the extracellular matrix (ECM) during development, haemostasis and pathological conditions. Whereas MMP-19 mRNA is found widely expressed in body tissues, including the synovium of normal and rheumatoid arthritic patients, MMP-20 expression is restricted to the enamel organ. In this study we investigated the ability of MMP-19 and MMP-20 to cleave two of the macromolecules characterising the cartilage ECM, namely aggrecan and the cartilage oligomeric matrix protein (COMP). Both MMPs hydrolysed aggrecan efficiently at the well-described MMP cleavage site between residues Asn(341) and Phe(342), as shown by Western blotting using neo-epitope antibodies. Furthermore, the two enzymes cleaved COMP in a distinctive manner, generating a major proteolytic product of 60 kDa. Our results suggest that MMP-19 may participate in the degradation of aggrecan and COMP in arthritic disease, whereas MMP-20, due to its unique expression pattern, may primarily be involved in the turnover of these molecules during tooth development.

Dm1-MMP, a matrix metalloproteinase from Drosophila with a potential role in extracellular matrix remodeling during neural development.

We have cloned and characterized a cDNA encoding Dm1-MMP, the first matrix metalloproteinase (MMP) identified in Drosophila melanogaster. The isolated cDNA encodes a protein of 541 residues that has a domain organization identical to that of most vertebrate MMPs including a signal sequence, a prodomain with the activation locus, a catalytic domain with a zinc-binding site, and a COOH-terminal hemopexin domain. Northern blot analysis of Dm1-MMP expression in embryonic and larval adult tissues revealed a strong expression level in the developing embryo at 10-22 h, declining thereafter and being undetectable in adults. Western blot analysis confirmed the presence of pro- and active forms of Dm1-MMP in vivo during larval development. In situ hybridization experiments demonstrated that Dm1-MMP is expressed in a segmented pattern in cell clusters at the midline during embryonic stage 12-13, when neurons of the central nervous system start to arise. Recombinant Dm1-MMP produced in Escherichia coli exhibits a potent proteolytic activity against synthetic peptides used for analysis of vertebrate MMPs. This activity is inhibited by tissue inhibitors of metalloproteinases and by synthetic MMP inhibitors such as BB-94. Furthermore, Dm1-MMP is able to degrade the extracellular matrix and basement membrane proteins fibronectin and type IV collagen. On the basis of these data, together with the predominant expression of Dm1-MMP in embryonic neural cells, we propose that this enzyme may be involved in the extracellular matrix remodeling taking place during the development of the central nervous system in Drosophila.

[Economic study on asthma in Mexico]. / Un estudio económico sobre asma en México.

OBJECTIVE: The objective of this study is to establish to cost of bronchial asthma in the budget for a Clinic-Hospital with a closed population and to learn about our achievements in controlling that illness, as well as our weaknesses and areas of opportunity. Another objective is to compare our results with similar studies in other countries. METHOD: In 1998, the Clinic Nova of Monterrey had a fixed population of 28,394 persons who receive the complete range of medical services, including hospitalization and drugs from the pharmacy. The prevalence of asthma in our clinic is 2.6%, direct and indirect costs were obtained according to the Biostatistics Department, Operative Control and Emergencies. RESULTS: In 1998, the total cost of bronchial asthma was: 1,624,765.00 pesos. The direct cost of asthma was 1,582,735.00 pesos (97% of the total) and out of this, the cost of medications was 1,131,575.00 pesos (71% of the direct costs) and 42,030.00 pesos in indirect costs only represented 3% of the total. CONCLUSION: Our results indicate that the greatest cost lies in the purchase of medication for ambulatory patients. The costs for hospitalization and visits to the emergency room only represented 10% of the total and, for that reason, our most important conclusion is that the physicians at Clinica Nova are using the preventive anti-inflammatory schemes recommended at present. This has helped to reduce the most important direct cost, hospitalization.

One-step sandwich enzyme immunoassay using monoclonal antibodies for detection of human enamelysin (MMP-20).

A one-step sandwich enzyme immunoassay (EIA) system for human matrix metalloproteinase 20 (MMP-20, enamelysin) was established by use of a solid-phase monoclonal antibody and a separate peroxidase-labeled monoclonal antibody. The EIA system was shown to be sensitive and quantitative for the detection of MMP-20. As little as 1.0 ng/ml (50 pg/assay) of MMP-20 protein could be reliably detected. The EIA system was linear over a range of 2.5-160 ng/ml (125-8,000 pg/assay), and the EIA system was versatile in that it was capable of detecting with equal sensitivity proMMP-20, active MMP-20, and MMP-20 with COOH-terminal deletions. The EIA system was validated by the successful detection of MMP-20 in the culture medium of Chinese hamster ovary cells (CHO-K1) that were transfected with an MMP-20 expression vector. No MMP-20 was detected in normal human serum, normal saliva, or in selected tumors. However, when recombinant human MMP-20 was added to human saliva, the EIA system did detect quantifiable amounts of the MMP-20, indicating that the system will work within the framework of complex in biological fluids.

Identification and characterization of human MT5-MMP, a new membrane-bound activator of progelatinase a overexpressed in brain tumors.

A cDNA encoding a new member of the membrane-type (MT) matrix metalloproteinase (MMP) family has been identified and cloned from a human brain cDNA library. The isolated cDNA encodes a polypeptide of 645 amino acids that displays a similar domain organization as other MMPs, including a predomain with the activation locus, a zinc-binding site, and a hemopexin domain. The deduced amino acid sequence contains a COOH-terminal extension, rich in hydrophobic residues and similar in size to the equivalent domains identified in MT-MMPs. Immunofluorescence and Western blot analysis of COS-7 cells transfected with the isolated cDNA revealed that the encoded protein is localized in the plasma membrane. On the basis of these features, this novel human MMP has been called MT5-MMP because it represents the fifth member of the MT-MMP subfamily of MMPs. Fluorescent in situ hybridization experiments showed that the human MT5-MMP gene (MMP-24) maps to 20q11.2, a region frequently amplified in tumors from diverse sources. Northern blot analysis demonstrated that MT5-MMP is predominantly expressed in brain, kidney, pancreas, and lung. In addition, MT5-MMP transcripts were detected at high levels compared to normal brain tissue in a series of brain tumors, including astrocytomas and glioblastomas. The catalytic domain of MT5-MMP, produced in Escherichia coli as a fusion protein with glutathione S-transferase, exhibits a potent proteolytic activity against progelatinase A, leading to the generation of the Mr 62,000 active form of this enzyme. These data suggest that MT5-MMP may contribute to the activation of progelatinase A in tumor tissues, in which it is overexpressed, thereby facilitating tumor progression.

Structural analysis and promoter characterization of the human collagenase-3 gene (MMP13).

Human collagenase-3 (MMP13) is a recently identified member of the matrix metalloproteinase (MMP) family that is expressed in breast carcinomas and in articular cartilage from arthritic patients. In this work we have isolated and characterized genomic clones coding for human collagenase-3. This gene is composed of 10 exons and 9 introns and spans over 12.5 kb. The overall organization of the collagenase-3 gene is similar to that of other MMP genes clustered at chromosome 11q22, including fibroblast collagenase (MMP-1), matrilysin (MMP-7), and macrophage metalloelastase (MMP-12), but is more distantly related to genes coding for stromelysin-3 (MMP-11), gelatinase-A (MMP-2), and gelatinase-B (MMP-9), which map outside of this gene cluster. Nucleotide sequence analysis of about 1 kb of the 5'-flanking region of the collagenase-3 gene revealed the presence of a TATA box, an AP-1 motif, a PEA-3 consensus sequence, an osteoblast specific element (OSE-2), and a TGF-beta inhibitory element. Transient transfection experiments in HeLa and COS-1 cells with chloramphenicol acetyltransferase (CAT)-containing constructs showed that the AP-1 site is functional and responsible for the observed inducibility of the reporter gene by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). However, and in contrast to other MMP genes, no significative synergistic effect on CAT activity between the AP-1 and PEA-3 elements found in the collagenase-3 gene promoter was found. DNA binding analysis with nuclear extracts from HeLa cells revealed the formation of specific complexes between collagenase-3 promoter sequences containing the AP-1 site and nuclear proteins. The presence of this AP-1 functional site, which is able to confer responsiveness to a variety of tumor promoters and oncogene products, amy contribute to explaining the high-level expression of collagenase-3 in breast carcinomas and degenerative joint diseases.

Identification and characterization of a novel human matrix metalloproteinase with unique structural characteristics, chromosomal location, and tissue distribution.

We have cloned a novel member of the matrix metalloproteinase (MMP) family of proteins from a human liver cDNA library. The isolated cDNA contains an open reading frame coding for a polypeptide of 508 amino acids, which has been tentatively called MMP-19. This protein exhibits the domain structure characteristic of previously described MMPs, including a signal sequence, a prodomain with the cysteine residue essential for maintaining the latency of these enzymes, an activation locus with the zinc-binding site, and a COOH-terminal fragment with sequence similarity to hemopexin. However, it lacks a series of structural features distinctive of the diverse MMP subclasses, including the Asp, Tyr, and Gly residues located close to the zinc-binding site in collagenases, the fibronectin-like domain of gelatinases, the transmembrane domain of membrane-type (MT) MMPs, and the furin-activation sequence common to stromelysin-3 and MT-MMPs. In addition, the 9-residue insertion rich in hydrophobic amino acids present at the hinge region in stromelysins is replaced in MMP-19 by a longer insertion very rich in acidic residues. On the basis of these structural characteristics, we propose that MMP-19 does not belong to any of the previously defined MMP-subclasses and may represent the first member of a new MMP subfamily. Chromosomal location of the MMP-19 gene revealed that it maps to chromosome 12q14, which is also a unique location for any MMPs mapped to date. The cDNA encoding a full-length MMP-19 was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade synthetic substrates for MMPs. MMP-19 proteolytic activity was abolished by TIMP-2 and EDTA, thus providing additional evidence that the isolated cDNA codes for an authentic MMP. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues revealed that MMP-19 is mainly expressed in placenta, lung, pancreas, ovary, spleen, and intestine, suggesting that it may play a specialized role in these tissues.

Identification and structural and functional characterization of human enamelysin (MMP-20).

A cDNA encoding a new human matrix metalloproteinase (MMP) has been cloned from RNA prepared from odontoblastic cells. The open reading frame of the cloned cDNA codes for a polypeptide of 483 amino acids and is extensively similar to the sequence of recently described porcine enamelysin, suggesting that the isolated cDNA codes for the human homologue of this enzyme. Human enamelysin (MMP-20) has a domain organization similar to other MMPs, including a signal peptide, a prodomain with the conserved motif PRCGVPD involved in maintaining enzyme latency, a catalytic domain with a Zn-binding site, and a COOH-terminal fragment similar to the sequence of hemopexin. The calculated molecular mass of human enamelysin is about 54 kDa, which is similar to that of collagenases or stromelysins. However, this human MMP lacks a series of structural features distinctive of these subfamilies of MMPs. The full-length human enamelysin cDNA has been expressed in Escherichia coli, and the purified and refolded recombinant protein is able to degrade synthetic peptides used as substrates of MMPs, confirming that human enamelysin belongs to this family of proteases. Furthermore, the recombinant human enamelysin is able to degrade amelogenin, the major protein component of the enamel matrix. On the basis of its degrading activity on amelogenin, and its highly restricted expression to dental tissues, we suggest that human enamelysin plays a central role in the process of tooth enamel formation. Finally, we have found that the human enamelysin gene (MMP-20) maps to chromosome 11q22, clustered to at least seven other members of the MMP gene family.

Gene characterization, promoter analysis, and chromosomal localization of human bleomycin hydrolase.

The human gene encoding bleomycin hydrolase, a cysteine proteinase involved in chemotherapy resistance, has been cloned, and its overall organization has been established. The gene is composed of 12 coding exons and 11 introns and spans more than 30 kilobases. The number and distribution of exons and introns differ from those reported for other human cysteine proteinases, indicating that these genes are only distantly related. Nucleotide sequence analysis of about 1.2 kilobases of the 5'-flanking region of the human bleomycin hydrolase gene revealed a high GC content, a ratio of CpG/GpC close to unity, and the absence of consensus transcriptional sequences such as TATA box or CCAAT box. All these features are characteristic of housekeeping genes and provide an explanation for the widespread expression of bleomycin hydrolase in human tissues. The 5'-flanking region of the gene also contains a polymorphic CCG trinucleotide repeat that may be a target of genetic instability events and affect its transcriptional activity. Chromosomal localization of the human bleomycin hydrolase gene revealed that it maps to chromosome 17q11.2, very close to the locus of the neurofibromatosis type 1 gene. This location is unique for any cysteine proteinase mapped to date. Finally, Southern blot analysis of DNA from leukocytes and autologous breast tumors has shown that the bleomycin hydrolase gene is not a frequent target of amplification in human breast carcinomas.

Molecular cloning of a novel membrane-type matrix metalloproteinase from a human breast carcinoma.

A new member of the matrix metalloproteinase (MMP) family of enzymes has been cloned from a human breast carcinoma cDNA library. The isolated cDNA contains an open reading frame 1554 bp long, encoding a polypeptide of 518 amino acids. The predicted amino acid sequence displays a similar domain organization as the remaining MMPs, including a prodomain with the activation locus, the zinc-binding site, and the hemopexin domain. In addition, it contains a C-terminal extension, rich in hydrophobic residues and similar in size to those present in the different membrane-type MMPs (MT-MMPs) identified to date. On the basis of these structural characteristics, this novel MMP has been tentatively called MT4-MMP, because it represents the fourth member of this subclass of MMPs characterized mainly by the occurrence of putative transmembrane domain in their amino acid sequences. MT4-MMP also contains a nine-residue insertion between the propeptide and the catalytic domain, which is a common feature of MT-MMPs and stromelysin-3. This amino acid sequence insertion ends with the consensus sequence R-X-R/K-R, which seems to be essential in the activation of these proteinases by furin. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues revealed that the MT4-MMP gene (MMP-17) is expressed mainly in the brain, leukocytes, the colon, the ovary, and the testis. The expression of MT4-MMP in leukocytes together with its putative membrane localization suggest that this enzyme could be involved in the activation of membrane-bound precursors of growth factors or inflammatory mediators such as tumor necrosis factor-alpha. In addition, MT4-MMP transcripts were detected in all breast carcinomas, as well as in all breast cancer cell lines analyzed in the present work. On the basis of these expression data in breast tumors, a potential role for human MT4-MMP in the tumoral process is also suggested.

Irrigación de los dedos en el bovino / Irrigation of the digits in the bovine

Se procura lograr un mejor conocimiento de la irrigación de los dedos en el bovino, dada la importancia que ésta adquiere frente a los cuadros clínicos abordados por la patología quirúrgica. Se examinaron 10 manos de ejemplares adultos y 5 fetos bovinos, previamente inyectados con celuloide coloreado. En los bovinos adultos la aplicación del celuloide se realizó a partir del tercio proximal de los matacarpianos, en tanto qu en los fetos fue en la arteria axilar. También se realiza inyección de solución de contraste para obtención de placas radiográficas. Los resultados revelan el comportamiento de la arteria metacarpiana dorsal III, en el 100 por ciento de los casos, originada a partir de la red dorsal del carpo hasta la formación del rodete coronario y la formación del arco terminal.

[The use of microcomputers in the processing of complex hemodynamic data]. / El uso de una microcomputadora en el procesamiento de datos hemodinámicos complejos.

The use of a low-cost microcomputer in the clinical study of left ventricular diastolic function is described. Both left ventricular filling and compliance were studied by means of the simultaneous recording of left ventricular pressure and an echocardiogram displaying ventricular dimensions. A program was written to calculate ventricular volume every 40 msec and a volume-time diagram was obtained. The raw curve was smoothed with a second degree polinomial. The rate of change of ventricular volume (dV/dt) was obtained. Simultaneous left ventricular volume and pressure were plotted. The diastolic, exponential portion of the pressure-volume loop was used to calculate an index of ventricular stiffness at zero volume. It is concluded that a simple microcomputer, when conveniently programed, is able to perform the time-consuming mathematical operations involved in the clinical assessment of left ventricular diastolic function.