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.

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 (AU)

Enteritis eosinofílica complicada / Complicated eosinophilic enteritis

La enteritis eosinofílica transmural representa un cuadro clínico muy poco frecuente. Presentamos el caso de un paciente de 53 años de edad, con antecedentes personales de diabetes mellitus tipo 2, que fue intervenido quirúrgicamente con carácter urgente tras ingresar a causa de dolor abdominal de 24 h de evolución que se acompañaba de vómitos de carácter alimentario. Se realizó una laparotomía exploradora, evidenciándose un líquido libre de aspecto inflamatorio, así como una congestión de las asas yeyunales con estenosis importante de su luz; una vez resecada y analizada histológicamente la zona lesionada, se confirmó la presencia de enteritis eosinofílica con afección de todo el espesor de la pared intestinal. La evolución postoperatoria fue favorable, encontrándose el paciente asintomático en la actualidad (AU)

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.

Rotura intramural espontánea de la pared esofágica. Una entidad poco frecuente / Spontaneous rupture of the esophageal wall. A rare entity

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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.

Inhibition of human caspases by peptide-based and macromolecular inhibitors.

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.

Rustmicin, a potent antifungal agent, inhibits sphingolipid synthesis at inositol phosphoceramide synthase.

Rustmicin is a 14-membered macrolide previously identified as an inhibitor of plant pathogenic fungi by a mechanism that was not defined. We discovered that rustmicin inhibits inositol phosphoceramide synthase, resulting in the accumulation of ceramide and the loss of all of the complex sphingolipids. Rustmicin has potent fungicidal activity against clinically important human pathogens that is correlated with its sphingolipid inhibition. It is especially potent against Cryptococcus neoformans, where it inhibits growth and sphingolipid synthesis at concentrations <1 ng/ml and inhibits the enzyme with an IC50 of 70 pM. This inhibition of the membrane-bound enzyme is reversible; moreover, rustmicin is nearly equipotent against the solubilized enzyme. Rustmicin was efficacious in a mouse model for cryptococcosis, but it was less active than predicted from its in vitro potency against this pathogen. Stability and drug efflux were identified as two factors limiting rustmicin's activity. In the presence of serum, rustmicin rapidly epimerizes at the C-2 position and is converted to a gamma-lactone, a product that is devoid of activity. Rustmicin was also found to be a remarkably good substrate for the Saccharomyces cerevisiae multidrug efflux pump encoded by PDR5.

A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.

There is compelling evidence that members of the caspase (interleukin-1beta converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 and Caenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal.

Khafrefungin, a novel inhibitor of sphingolipid synthesis.

In the course of screening for antifungal agents we have discovered a novel compound isolated from an endophytic fungus that inhibits fungal sphingolipid synthesis. Khafrefungin, which is composed of aldonic acid linked via an ester to a C22 modified alkyl chain, has fungicidal activity against Candida albicans, Cryptococcus neoformans, and Saccharomyces cerevisiae. Sphingolipid synthesis is inhibited in these organisms at the step in which phosphoinositol is transferred to ceramide, resulting in accumulation of ceramide and loss of all of the complex sphingolipids. In vitro, khafrefungin inhibits the inositol phosphoceramide synthase of C. albicans with an IC50 of 0.6 nM. Khafrefungin does not inhibit the synthesis of mammalian sphingolipids thus making this the first reported compound that is specific for the fungal pathway.

Correction of hypertension by normalization of endothelial levels of fibroblast growth factor and nitric oxide synthase in spontaneously hypertensive rats.

Acidic and basic fibroblast growth factors (FGFs) share a wide range of diverse biological activities. To date, low levels of FGF have not been correlated with a pathophysiologic state. We report that blood vessels of spontaneously hypertensive rats are shown to be associated with a marked decrement in endothelial basic FGF content. This decrement correlates both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. Restoration of FGF to physiological levels in the vascular wall, either by systemic administration or by in vivo gene transfer, significantly augmented the number of endothelial cells with positive immunostaining for nitric oxide synthase, corrected hypertension, and ameliorated endothelial-dependent responses to vasoconstrictors. These results suggest an important role for FGFs in blood pressure homeostasis and open new avenues for the understanding of the etiology and treatment of hypertension.

Functional reconstitution of the large-conductance, calcium-activated potassium channel purified from bovine aortic smooth muscle.

The charybdotoxin (ChTX) receptor has been purified from bovine aortic smooth muscle using conventional chromatographic techniques and sucrose gradient centrifugation. Fractions from the final sucrose gradient purification were enriched in specific binding of monoiodinated ChTX (125i-ChTX) approximately 2000-fold over native sarcolemmal membranes. The ChTX binding activity correlated with the presence of two polypeptides of 65 (alpha) and 31 (beta) kDa. Using the cross-linking reagent, disuccinimidyl suberate, 125I-ChTX was specifically incorporated into a polypeptide of approximately 31 kDa. Cross-linking and binding of 125I-ChTX to the purified ChTX receptor was inhibited by ChTX, iberiotoxin (IbTX), and tetraethylammonium (TEA). Liposomes containing the purified ChTX receptor were incorporated into planar lipid bilayers. In symmetric 150 mM KC1, the channels observed were > 20-fold more selective for potassium over sodium and exhibited a large, single-channel conductance of 323 +/- 2.5 pS in charged lipids and 249 +/- 7 pS in neutral lipids. Depolarizing membrane potentials increased the open probability of the purified channels e-fold per 11.5 +/- 0.3 mV, while intracellular calcium increased the open probability according to a third power (2.9 +/- 0.2) relationship. Mean channel closed durations decreased while open times slightly increased as membrane potential and calcium concentration were elevated. The distributions of open and closed durations were well described by the sums of three and five to six exponential components, respectively. Purified maxi-K channels were blocked with micromolar affinity by external TEA and with nanomolar affinity by extracellular IbTX and ChTX. Kinetics of ChTX block of the purified channel revealed an equilibrium dissociation constant for toxin block 4.6 +/- 0.7 nM under conditions of physiological ionic strength. The purified maxi-K channel displays many of the biophysical and pharmacological properties of maxi-K channels derived from native tissue.

[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.

[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.

Primary sequence and immunological characterization of beta-subunit of high conductance Ca(2+)-activated K+ channel from smooth muscle.

The charybdotoxin receptor, purified from bovine tracheal smooth muscle, consists of two subunits (alpha and beta) and, when reconstituted into planar lipid bilayers, forms functional high conductance Ca(2+)-activated K+ channels. Amino acid sequence, obtained from proteolytic fragments of the beta-subunit, was used to design oligonucleotide probes with which cDNAs encoding this protein were isolated. The cDNAs encode a protein of 191 amino acids that contains two hydrophobic (putative transmembrane) domains and bears little sequence homology to subunits of other known ion channels. Site-directed antisera, raised against putative extracellular epitopes of this protein, specifically immunoprecipitated 125I-labeled Bolton-Hunter beta-subunit as well as [125I]charybdotoxin-cross-linked beta-subunit. Under nondenaturing conditions, however, these anti-beta sera immunoprecipitated a complex consisting of both the alpha- and beta-subunits. The data demonstrate that, in vivo, the high conductance Ca(2+)-activated K+ channel exists as a multimer containing both alpha- and beta-subunits, and this cDNA represents the first beta-subunit of a potassium channel cloned to date. Furthermore, we demonstrate that the cloned protein is the subunit to which charybdotoxin is specifically and covalently incorporated when cross-linked to the channel.

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.

[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.

Functional unit size of the charybdotoxin receptor in smooth muscle.

Target inactivation analysis was used to determine the functional size of the charybdotoxin (ChTX) receptor in aortic and tracheal sarcolemmal membrane vesicles. This receptor has previously been shown to be an integral component of the high-conductance Ca2+-activated K+ (Maxi-K) channel in these smooth muscles. Exposure of either bovine aortic or bovine tracheal sarcolemma to high-energy irradiation results in disappearance of 125I-labeled ChTX binding activity as a monoexponential function of radiation dose; from these functions molecular masses of 88 +/- 10 kDa and 89 +/- 6 kDa, respectively, can be calculated. Similar results were obtained from radiation inactivation studies with the detergent-solubilized ChTX receptor from aortic sarcolemmal membranes. The effect of radiation on 125I-labeled ChTX binding is to decrease the number of functional ChTX receptors without affecting the affinity of receptors for the toxin, indicating that radiation is destroying, rather than altering, the binding site. The validity of the radiation inactivation technique in these membrane preparations is supported by data obtained in parallel experiments in which target sizes of the alpha 1 subunit of the L-type Ca2+ channel and 5'-nucleotidase were measured. The molecular masses determined for these entities are in excellent agreement with those expected from previous studies. The present data are discussed in terms of the recently determined subunit composition of the smooth muscle Maxi-K channel. In light of the target size, a single alpha beta subunit heterodimer complex could serve as the ChTX receptor.