Perianal metastases from lobular breast carcinoma.

Breast cancer gastrointestinal and soft tissue metastases are extremely rare. We present the case of a woman with perianal metastases from a primary lobular breast carcinoma 11 years after mastectomy and local radiotherapy.

Purification and catalytic properties of human caspase family members.

Members of the caspase family of cysteine proteases are known to be key mediators of mammalian inflammation and apoptosis. To better understand the catalytic properties of these enzymes, and to facilitate the identification of selective inhibitors, we have systematically purified and biochemically characterized ten homologues of human origin (caspases 1 - 10). The method used for production of most of these enzymes involves folding of active enzymes from their constituent subunits which are expressed separately in E. coli, followed by ion exchange chromatography. In cases where it was not possible to use this method (caspase-6 and -10), the enzymes were instead expressed as soluble proteins in E. coli, and partially purified by ion exchange chromatography. Based on the optimal tetrapeptide recognition motif for each enzyme, substrates with the general structure Ac-XEXD-AMC were used to develop continuous fluorometric assays. In some cases, enzymes with virtually identical tetrapeptide specificities have kcat/Km values for fluorogenic substrates that differ by more than 1000-fold. Using these assays, we have investigated the effects of a variety of environmental factors (e.g. pH, NaCl, Ca2+) on the activities of these enzymes. Some of these variables have a profound effect on the rate of catalysis, a finding that may have important biological implications.

The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1.

BACKGROUND: Granzyme B, one of the most abundant granzymes in cytotoxic T-lymphocyte (CTL) granules, and members of the caspase (cysteine aspartyl proteinases) family have a unique cleavage specificity for aspartic acid in P1 and play critical roles in the biochemical events that culminate in cell death. RESULTS: We have determined the three-dimensional structure of the complex of the human granzyme B with a potent tetrapeptide aldehyde inhibitor. The Asp-specific S1 subsite of human granzyme B is significantly larger and less charged than the corresponding Asp-specific site in the apoptosis-promoting caspases, and also larger than the corresponding subsite in rat granzyme B. CONCLUSIONS: The above differences account for the variation in substrate specificity among granzyme B, other serine proteases and the caspases, and enable the design of specific inhibitors that can probe the physiological functions of these proteins and the disease states with which they are associated.

Sensitive procedures for measuring chloride fluxes mediated by the purified glycine receptor incorporated into phospholipid vesicles.

Two novel methods have been developed to directly measure chloride influx into purified glycine receptor-containing phospholipid vesicles. Using a method based on the fluorescence quenching of a chloride-sensitive probe entrapped into the vesicles, a chloride influx was detected which could be enhanced by glycine and completely abolished by the antagonist strychnine. In addition, by tracing the 36Cl- influx into the proteoliposomes, a stimulatory effect of both glycine and beta-alanine could be seen, which can be inhibited by strychnine and other glycine antagonists. These data, together with a previous report demonstrating ligand-mediated iodide fluxes in the same preparation (Biochemistry, 28 (1989) 6405-6409), clearly demonstrate the utility of the reconstituted receptor preparation to investigate some ion channel and pharmacological properties of the glycine receptor.

[Mucoprotein secretion in calculous gallbladder]. / Secrección de mucoproteínas en la vesícula biliar con cálculos.

Secretion of mucoproteins or mucine (MP) have been studied as possible markers in several pathological conditions of the digestive tract, such us colonic polyposis or gastric dysplasia. In the gallbladder (VB) it has been established that form the core of crystalization for the calculi. A study in 100 gallbladders have been made based on the utility of the analysis of the qualitative and quantitative modifications of MP in lithogenesis. It was been determined by histochemical techniques the three main types of MP (neutral, low and high sulphated acid) to evaluate the alterations in the process of lithiasis. Results show a high production of the MP in VB with lithiasis, presenting in 97% a mixed composition of MP (48.9% of 2 types, and 3 types in 46%), without a predominating type in this pathology.

[An adenocarcinoid tumor of the vermiform appendix]. / Tumor adenocarcinoide de apéndice vermiforme.

Goblet cell carcinoids of the vermiform appendix are uncommon. A comprehensive review of the literature revealed about 100 reported cases. Clinical presentation included: asymptomatic patients, acute appendicitis, and/or chronic intermittent lower abdominal pain with or without a palpable mass. We report the case of a 57 year-old woman with lower abdominal pain, nausea and vomiting. Normal blood tests. X-ray of the abdomen showed dilated small bowel loops with fluid levels. Initially, conservative treatment was started. After three days with no clinical improvement, the patient was operated on. An appendicocecal inflammation was found and a terminal ileum plus cecal resection with an end-to-side anastomosis was performed. The pathological diagnosis was goblet cell carcinoid. The patient shows no evidence of recurrence after 1 year follow-up.

[Correlation between dysplasia and malignization risk of Crohn disease]. / Correlación entre displasia y riesgo de malignización de la enfermedad de Crohn.

The malignancy risk in Crohn's disease is less than that of ulcerative colitis, although it has not been so well studied. The possibility of an early detection of malignant changes during the long term follow-up of the patients induces to look for parameters which might be helpful in this respect. In the second surgical clinic of the Hospital Clinico in Madrid, the pathological findings of 11 patients with the diagnosis of Crohn's disease have been analyzed and correlated with the clinical findings. The evaluation of the degree of dysplasia was done on 15 samples obtained by endoscopy and compared with the findings of the surgical specimens in 6 operated patients. The degree of dysplasia increases with age of the patients and years of evolution of the disease with a significant correlation (p less than 0.001) between negative dysplasia and positive dysplasia grade I.

[Peutz-Jeghers syndrome with malignization of hamartomatous polyp]. / Síndrome de Peutz-Jeghers con malignización de un pólipo hamartomatoso.

Peutz-Jeghers syndrome is characterized by hamartomatous polyps in the gastrointestinal tract associated with mucocutaneous melanin spots; the condition is inherited in an autosomic dominant fashion. Although the hamartomatous polyps are not regarded as malignant, there is an association between the Peutz-Jeghers syndrome and the development of carcinomas of the gastrointestinal tract. Similarly, there is an increasing evidence suggesting that hamartomas can give rise to adenocarcinomas. We present a patient with Peutz-Jeghers syndrome who developed an adenocarcinoma in an hamartomatous jejunal polyp.

[Resolution with steroid therapy of an idiopathic esophageal fistula in a patient with AIDS]. / Resolución con tratamiento esteroideo de una fístula esofágica idiopática en un paciente con SIDA.

We present a case of AIDS with idiopathic esophagic fistula which had a positive response to steroid therapy. We discuss the diagnostic potential and the therapeutic attitude in these cases.

Attorneys' attitudes towards alcohol and drug use: a prospective study (1983-1988).

Legal professionals come into close contact with problems arising from the consumption of alcoholic beverages and other abused drugs. The present study was designed to analyse the possible changes in attitude towards this situation, and specifically to analyse how social change has affected the legal profession. A group of lawyers practising and residing in Granada, Spain was studied. A questionnaire was used to gather data about habits of alcohol consumption and attitudes toward consumption-related problems. The data was analysed statistically with the BMDP. Cultural factors and age seem to act as moderating influence regarding attitudes toward the consumption of alcohol.

Characterization of the solubilized charybdotoxin receptor from bovine aortic smooth muscle.

Monoiodotyrosine ([125I]ChTX) binds with high affinity to a single class of receptors present in bovine aortic smooth muscle sarcolemmal membranes that are functionally associated with the high-conductance Ca(2+)-activated K+ channel [maxi-K channel; Vázquez, J., et al. (1989) J. Biol. Chem. 265, 20902-20909]. Cross-linking experiments carried out with this preparation in the presence of [125I]ChTX and disuccinimidyl suberate indicate specific incorporation of radioactivity into a protein of Mr 35,000. The smooth muscle ChTX receptor can be solubilized in active form in the presence of selected detergents. Treatment of membranes with digitonin releases about 50% of the ChTX binding sites. The solubilized receptor retains the same biochemical and pharmacological properties that are characteristic of toxin interaction with membrane-bound receptors. The solubilized receptor binds specifically to wheat germ agglutinin-Sepharose resin, suggesting that it is a glycoprotein. Functional ChTX binding sites can also be solubilized in 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS). Sucrose density gradient centrifugation of either digitonin or CHAPS extracts indicates that the ChTX receptor has a high apparent sedimentation coefficient (s20,w = 23 and 18 S, respectively). Cross-linking experiments indicate that the appearance of the 35-kDa membrane protein correlates with ChTX binding activity after both wheat germ agglutinin-Sepharose and sucrose density gradient centrifugation steps. Given the high apparent sedimentation coefficient of the ChTX receptor, the 35-kDa membrane protein may be a subunit of a higher molecular weight complex which forms the maxi-K channel in smooth muscle sarcolemma.

Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor.

Amino acid residues that participate in antagonist binding to the strychnine-sensitive glycine receptor (GlyR) have been identified by selectively modifying functional groups with chemical reagents. Moreover, a region directly involved with strychnine binding has been localized in the 48-kDa subunit of this receptor by covalent labeling and proteolytic mapping. Modification of tyrosyl or arginyl residues promotes a marked decrease of specific [3H]strychnine binding either to rat spinal cord plasma membranes or to the purified GlyR incorporated into phospholipid vesicles. Occupancy of the receptor by strychnine, but not by glycine, completely protects from the inhibition caused by chemical reagents. Furthermore, these tyrosine- or arginine-specific reagents decrease the number of binding sites (Bmax) for [3H]strychnine binding without affecting the affinity for the ligand (Kd). These observations strongly suggest that such residues are present at, or very close to, the antagonist binding site. In order to localize the strychnine binding domain within the GlyR, purified and reconstituted receptor preparations were photoaffinity labeled with [3H]strychnine. The radiolabeled 48-kDa subunit was then digested with specific chemical proteolytic reagents, and the peptides containing the covalently bound radioligand were identified by fluorography after gel electrophoresis. N-Chlorosuccinimide treatment of [3H]strychnine-labeled 48K polypeptide yielded a single labeled peptide of Mr approximately 7300, and cyanogen bromide gave a labeled peptide of Mr 6200.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of bilirubin with the synaptosomal plasma membrane.

The interaction of the neurotoxic pigment bilirubin with synaptosomal plasma membrane vesicles (SPMV) isolated from rat brain was investigated. The interaction seems to involve three steps: (a) a rapid formation of an electrostatic complex between bilirubin and polar lipid head groups; (b) a slow inclusion of the pigment into the hydrophobic core of the membrane; and (c) a SPMV-induced bilirubin aggregation, observed when membrane capacity for bilirubin is exceeded. The association constant of the initial complex increased markedly when pH was lowered below 7.4, particularly in SPMV isolated from newborn rats. A preferential binding of bilirubin to pure gangliosides and sphingomyelin was observed, thus suggesting a role for these lipids as first targets of the pigment in the synaptic membrane. The inclusion of bilirubin into the membranes was gradually enhanced when decreasing the pH or the age of the rats from which SPMV were isolated. In addition, membranes from 2-day-old rats have a higher capacity for bilirubin incorporation compared to those from adult rats. Experiments with reconstituted liposomes of varying protein and cholesterol contents suggest that the effect of age may be related to changes in synaptosomal membrane fluidity during development. Our results support the hypothesis that the interaction of bilirubin with the synaptic membrane plays an important role in the molecular mechanisms of bilirubin neurotoxicity.

Subunit composition of the high conductance calcium-activated potassium channel from smooth muscle, a representative of the mSlo and slowpoke family of potassium channels.

High conductance Ca(2+)-activated K+ (maxi-K) channels from bovine tracheal and aortic smooth muscle membranes have been purified employing monoiodotyrosine charybdotoxin binding as a marker for the channel and conventional chromatographic techniques. This K+ channel is composed of two subunits, alpha and beta, of 62 and 31 kDa, respectively. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electroeluted tracheal smooth muscle alpha-subunit was subjected to tryptic cleavage and a number of fragments were isolated by microbore C18 high performance liquid chromatography. Several of these peptides were microsequenced using Edman degradation techniques. Amino acid sequence information obtained from these fragments reveals the existence of very high sequence homology with the recently cloned mSlo maxi-K channel (Butler, A., Tsunoda, S., McCobb, D. P., Wei, A., and Salkoff, L. (1993) Science 261, 221-224). A specific anti-peptide antibody directed against the amino acid sequence of one of the fragments of the alpha-subunit is capable of specifically immunoprecipitating not only the denatured 125I-Bolton-Hunter-labeled alpha-subunit, but also, under nondenaturing conditions, the complex of alpha and beta subunits, demonstrating specific noncovalent association of both subunits. Thus, our results indicate that the alpha-subunit of the purified tracheal smooth muscle maxi-K channel is a member of the mSlo family of K+ channels and forms a noncovalent complex with a beta-subunit. It is concluded that the extensive biochemical information acquired to date on smooth muscle charybdotoxin receptors is pertinent to the structure of native maxi-K channels.

Functional reconstitution of the glycine receptor.

The functional reconstitution of the chloride channel coupled glycine receptor is described. Glycine receptors were purified from the cholate extract of rat spinal cord membranes by affinity chromatography and incorporated into phospholipid vesicles by the addition of phosphatidylcholine and removal of detergent by gel filtration. The reconstituted vesicles showed the same polypeptide composition as the purified receptor (proteins of Mr 48,000 and 58,000). The pharmacological characteristics of the glycine receptor were also preserved in the proteoliposomes, as demonstrated by the displacement of [3H]strychnine binding by several glycinergic ligands and by photoaffinity labeling experiments. In order to observe functional responses (i.e., specific agonist-induced anion translocation), we have developed an assay based on the fluorescence quenching of an anion-sensitive entrapped probe, SPQ [6-methoxy-N-(3-sulfopropyl)quinolinium]. Reconstituted vesicles were loaded with the fluorescent probe during a freeze-thaw-sonication cycle in the presence of added liposomes containing cholesterol. In such a reconstituted system, glycine receptor agonists are able to increase the rate of anion influx into the vesicles. The action of agonists is blocked by the simultaneous presence of strychnine or other glycine antagonists. Our results show that the purified 48,000- and 58,000-dalton polypeptides reconstituted into phospholipid vesicles can bind ligands and promote specific ion translocation in a way similar to the glycine receptor in its native environment.

Purification and characterization of three inhibitors of voltage-dependent K+ channels from Leiurus quinquestriatus var. hebraeus venom.

Three new toxins from the venom of the scorpion Leiurus quinquestriatus var. hebraeus have been identified on the basis of their ability to block the Shaker K+ channel. These toxins have been purified using HPLC techniques and characterized as 38 amino acid peptides by mass spectroscopy, amino acid analysis, and sequence determination. Their chemical identity was confirmed by producing fully functional synthetic toxins using recombinant methods. These peptides are potent inhibitors of the Shaker K+ channel (Kd < 1 nM) as well as the mammalian homologues of Shaker. They are related to other previously described K+ channel toxins, but form a new subclass within the larger family of K+ channel inhibitors derived from scorpion venom. We have named these toxins agitoxin 1, 2, and 3, respectively.