Isolation and structure of repressor-like proteins from the archaeon Sulfolobus solfataricus. Co-purification of RNase A with Sso7c.

The thermostable histone-like protein Sso7c (Sso for Sulfolobus solfataricus) from the archaeon Sulfolobus solfataricus was purified from the supernatant of acid-soluble cell lysates. Reverse phase HPLC of an apparently homogeneous Sso7c protein fraction from Mono S chromatography resulted in resolution of three further peaks. Sequence analysis revealed one of these components to be bovine RNase A, originating from the culture medium and explaining the RNA hydrolyzing activities of Sso7 preparations previously described. Sequence analysis of pure Sso7c showed an epsilon-Lys methylation pattern identical to that of Sso7d and a single Gln --> Glu mutational difference at position 13. The remaining two proteins obtained after HPLC separation were identified as homologues of bacterial repressor-like proteins. Thus, the existence of repressor-like proteins was demonstrated at the protein level in archaea, raising the question of structural and functional consequences of these proteins on the otherwise eukaryotic-like basal transcriptional machinery in archaea.

Full-length and N-terminally truncated chicken intestinal diazepam-binding inhibitor. Purification, structural characterization and influence on insulin release.

Two forms of diazepam-binding inhibitor (DBI) have been purified from chicken intestine and identified as the intact avian polypeptide (residues 1-86) and a truncated variant (residues 35-86). At 10 nM concentration, both the intact and the truncated peptide suppress in vitro-monitored glucose-induced insulin release by 50 (p < 0.02) and 64% (p < 0.01) respectively. The truncation starts at a segment. -Thr-Val-Gly-Asp-, that is strictly conserved between characterized DBI species, indicating special restrictions on the structure. However, overall DBI conservation appears to be complex. A number of differently bioactive fragments with separate processings and tissue distributions have been observed, suggesting multiple functions of DBI and its sub-segments.

Elastolisis de la dermis media: Comunicación de un caso clínico y revisión de la literatura / Mid dermal elastolysis: A case report and review

Describimos el cuadro clínico de un paciente, que presenta placas y pápulas con aspecto de piel arrugada en papel de cigarrillo, localizadas en abdomen. El estudio histopatológico muestra ausencia de fibras elásticas, en una banda que compromete la porción media de la dermis. La elastolisis de la dermis media es una afección infrecuente, adquirida, idiopática o secundaria a un proceso inflamatorio previo, caracterizada por la pérdida focal de tejido elástico en la dermis media y sin compromiso extra cutáneo.

We describe a patient presenting plaques and papules -like wrinkled cigarette paper-, located in abdomen. Histopathological study shows absence of elastic fibers in a band that compromised the middle portion of the dermis. Mid dermal elastolysis is a rare, acquired idiopathic condition characterized by focal loss of elastic tissue in the middle dermis without extra cutaneous involment.

Xantogranuloma juvenil del adulto: Reporte de un caso clínico y revisión de la literatura / Juvenile xanthogranuloma in adult: A case report and review

El xantoganuloma juvenil es una enfermedad benigna, poco frecuente en el adulto, forma parte del grupo de las histiocitosis de células no Langerhans, representando el 90 % de las mismas y el 15 % de éstas, se presentan en el adulto. El compromiso de otros órganos es excepcional. La evolución suele ser benigna con autoresolución de las lesiones. Presentamos el caso de una paciente de sexo femenino de 27 años de edad, que presentaba lesiones papulares de 15 días de evolución, en zona periocular, asintomáticas, las que fueron biopsiadas con diagnóstico histopatológico de xantogranuloma juvenil.

Juvenile Xanthogranuloma (JX) is an uncommon benign disease in adults, is part of the group of non-Langerhans cells histiocytosis, representing 90 % of them and 15% of these occur in adults. The involvement of other organs is exceptional. The evolution is usually benign with resolution of disease. We present a case of a female patient of 27 years who had papules with 15 days of evolution, in periocular area, asymptomatic, which were biopsied with histopathologic diagnosis of juvenile xanthogranuloma.

Leiomiosarcoma cutáneo ulcerado: Reporte de un caso clínico y revisión de la literatura / Ulcerated cutaneous leiomyosarcoma: A case report and literature review

Los leiomiosarcomas cutáneos son neoplasias malignas, originadas en el músculo liso, infrecuentes, tanto en las formas primarias como en el caso de las secundarias o metastásicas. Existen pocos casos publicados en la literatura. Presentamos el caso de un leiomiosarcoma cutáneo primario, en un hombre de 76 años de edad y realizamos una revisión de sus características clínicas y diagnóstico, así como, de las diferencias pronóstico-evolutivas según las distintas localizaciones de esta afección. Por último, debe señalarse que el tratamiento es fundamentalmente quirúrgico, con escisión y márgenes de seguridad, ya que, no existe una respuesta satisfactoria o significativa al tratamiento radiante y/o quimioterápico, siendo imprescindible efectuar un control evolutivo periódico de los pacientes, debido a la tasa de recurrencia local y la posibilidad de ocasionar metástasis, según la localización histológica del tumor.

Leiomyosarcomas located in superficial soft tissue are very rare or uncommon malignant neoplasms, both as primary tumors and as secondary or metastatic lesions. We present the case of a primary cutaneous leiomyosarcoma in a 76-year-old man, reviewing the clinical and diagnostic features, as well as the different prognoses depending on the site at this level. Treatment is basically surgical, involving excision with wide margins since the response to radiotherapy and/or chemotherapy is not satisfactory or significant. Given the high rate of local recurrence and the possibility of metastasis, depending on the histological location of the tumor, periodic follow-up of the patients is essential and indispensable.

Síndrome hipereosinofílico linfocítico: A propósito de dos casos / Lymphocytic hypereosinophilic syndrome: Two cases reported

El síndrome hipereosinofílico constituye un grupo de enfermedades, caracterizadas por sobreproducción de eosinófilos con el daño secundario de múltiples órganos, provocado por la infiltración eosinofílica y la liberación de mediadores de inflamación. Es infrecuente y su prevalencia desconocida. El principal factor pronóstico es el compromiso cardíaco.

The hypereosinofilic syndrome is a group of diseases characterized by overproduction of eosinophils with secondary multiple organ damage caused by the cells infiltration and release of mediators of inflammation. It is infrequent and its prevalence is unknown. The main prognostic factor is the cardiac involvement.

C-terminal sequence determination of modified peptides by MALDI MS.

Peptides, cleaved by a mixture of carboxypeptidases CPP and CPY, can be detected by MALDI MS and the amino acid sequence thereby determined by calculation of the differences between consecutive peaks. In the present study we have used derivatizations of Lys and Cys to facilitate identification of these residues. Since the mass values do not readily distinguish Lys from Gln, we have converted Lys to homoarginine by guanidination, allowing simple detection of Lys. To identify the Cys positions in peptides that contain cystine, cysteic acid, or carboxymethylcysteine is not possible using CPY and CPP because of the lack of proteolytic cleavage. Instead we find that identification of Cys residues within the sequence can be achieved after conversion to a basic derivative, 4-thialaminine (Thi), by trimethylaminoethylation.

Two alternative processing pathways for a preprohormone: a bioactive form of secretin.

An N-terminally 9-residue elongated form of secretin, secretin-(-9 to 27) amide, was isolated from porcine intestinal tissue and characterized. Current knowledge about peptide processing sites does not allow unambiguous prediction of the signal peptide cleavage site in preprosecretin but suggests cleavage in the region of residues -10 to -14 counted upstream from the N terminus of the hormone. However, the structure of the isolated peptide suggests that the cleavage between the signal peptide and the N-terminal propeptide occurs at the C-terminal side of residue -10. Moreover, the isolated peptide demonstrates that secretin can be fully processed C-terminally prior to the final N-terminal cleavage. The results from this report, and those from earlier studies, where C-terminally elongated variants were isolated, show that the processing of the secretin precursor may proceed by one of two alternative pathways, in which either of the two ends is processed first. The bioactivity of the N-terminally extended peptide on exocrine pancreatic secretion was lower than that of secretin, indicating the importance of the finally processed free N terminus of the hormone for interaction with secretin receptors.

C-terminal sequence analysis of peptides and proteins using carboxypeptidases and mass spectrometry after derivatization of Lys and Cys residues.

C-Terminal sequence analysis of peptides and proteins using carboxypeptidase digestion in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is convenient for protein and peptide characterization. After a short digestion, a sequence up to 20 residues can be identified, but the total number depends on the individual sequence. Due to the accuracy limits of the MALDI time-of-flight arrangement, the assignment of several residues with close mass values, including Lys/Glx, may remain ambiguous. We have used derivatization of lysine residues by guanidination to overcome the problem of Lys identification. The reaction is rapid and specific and results in full derivatization. In the case of Cys-containing peptides, problems arise from the fact that carboxypeptidases Y and P do not cleave peptides that contain nonderivatized cystine, cysteic acid, or (carboxymethyl)cysteine. Successful identification of Cys residues within the sequence is instead achieved by conversion of Cys to 4-thialaminine by (trimethylamino)-ethylation. The two derivatizations of Lys and Cys side chains provide opportunities for proton attachment and therefore facilitate the analysis by MALDI-MS. This C-terminal sequence analysis method is also useful for large proteins after fragmentation with specific enzymes.

Isolation and characterization of sulphated and nonsulphated forms of cholecystokinin-58 and their action on gallbladder contraction.

Cholecystokinin (CCK) exists in multiple molecular forms with different polypeptide lengths and the absence or presence of sulphation. We have isolated sulphated and nonsulphated forms of CCK-58 from porcine intestine and have determined their bioactivities in a guinea-pig gallbladder contraction assay. Both forms co-eluted in cation-exchange chromatography and in several rounds of reverse-phase (RP)-HPLC, but separated upon RP-HPLC using a water/acetonitrile system with heptafluorobutyric acid as counter ion. Nonsulphated CCK-58 was the form detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry because of desulphation in that process. The biological activity of CCK-58 and CCK-33 is equipotent, although the kinetics of the response differ. Sulphated CCK-58 was found to be 35 times more potent than nonsulphated CCK-58. In contrast, sulphated CCK-8 is 150 times more potent than nonsulphated CCK-8, and for sulphated and nonsulphated CCK-33, the activities differ by a factor of 100. This type of correlation indicates that the N-terminal end of CCK-58 partially compensates for the decrease in activity arising from the lack of sulphated tyrosine. Given its fairly high bioactivity, nonsulphated CCK-58 may have a physiological significance.

Spleen antibacterial peptides: high levels of PR-39 and presence of two forms of NK-lysin.

Antibacterial peptides were isolated from porcine spleen by acetic acid extraction, ion exchange chromatography and reverse-phase high-performance liquid chromatography. C-terminal ladder sequence analysis of a bioactive peptide with matrix-assisted laser desorption/ionization mass spectrometry after digestion with carboxypeptidases P and Y showed that it is identical to the antibacterial proline/arginine-rich intestinal peptide PR-39. It is present at high levels in granulocytes of the spleen, and peptides with C-terminal proline amide and internal adjacent Pro residues can be analyzed with this method. In addition, two forms of NK-lysin (NKL) were found. One, NKLi, is identical to that isolated from pig intestine, and the other, NKLbw, to a mature peptide deduced from a clone from a porcine bone marrow cDNA library.

Large-scale HPLC purification of calbindin D9k from porcine intestine.

Two efficient procedures for large-scale purification of calbindin D9k from porcine intestine by HPLC were developed. Both protocols start with heat treatment of the intestinal tissue followed by acetic acid extraction, a capture with alginic acid, NaCl precipitation of other proteins, and a concentration step on Amberlite XAD-2. In the first method, a single reverse-phase HPLC step completes the purification and results in milligram quantities of pure calbindin. In the second method, an additional ion exchange HPLC step was introduced, followed by a reverse-phase HPLC resulting in 100 milligram-scale preparations of homogeneous calbindin in a 56% yield from the Amberlite step. Both methods yielded a homogeneous metal-free apoprotein with a molecular weight of 8838.0 +/- 8.8 as analyzed by MALDI TOF mass spectrometry corresponding to N-acetylated porcine calbindin. The isolated apocalbindin was fully reconstituted with 2 molar equivalents of Ca(2+) and the protein displayed UV and fluorescence spectra characteristic of those of native calbindin D9k.

Isolation of peptides from porcine intestinal tissue that induce extracellular acidification in CHO cells: identification of the active peptide as IGF-I and characterization of a fragment of calponin H1 processed at a dibasic site.

Chinese hamster ovary (CHO) cells are widely used as hosts for receptor expression and pharmacological studies. However, several endogenous receptor populations are present on these cells. Intestinal tissue extracts were found to induce strong extracellular acidification responses (ECAR) in CHO cells, yet several pure hormonal peptides, such as VIP, secretin, CCK, GIP, and galanin were ineffective. It is not known, which are the active compounds in the extracts that can stimulate the extracellular acidification in CHO cells. These active substances may be ligands for yet unknown receptors that are present natively in this cell type. We therefore decided to identify the active compound(s) by isolation from intestinal extract and structural characterization. Using chromatographic separations in combination with microphysiometry we have purified and characterized one such bioactive ligand. Structural analysis indicated that the isolated peptide was identical to insulin-like growth factor I (IGF-I). In the intestine, IGF-I is present in low amounts and has previously been detected only with radioimmunoassays. The results indicate that CHO cells express functional receptors for IGF-I. Among the peptides extracted from the intestine IGF-I is probably the strongest stimulator of ECAR in CHO cells. Moreover, IGF-I acts synergistically with other factors present in the crude tissue extract. Additionally, a fragment of calponin H1 (residues 1-43), previously not described at the protein level, was identified in the IGF-I containing fractions. The fragment was characterized by mass spectrometry and found to be N-terminally modified by acetylation suggesting that the whole protein bears the same posttranslational modification.

Seroprevalencia de la enfermedad de Chagas en El Espìnillo y Villa Río Bermejito en la provincia de Chaco / Seroprevalence of Chagas disease in El Espinillo and Villa Río Bermejito in the province of Chaco

En el marco del proyecto "Pediatría ambulatoria itinerante para comunidades rurales y aisladas" (P.A.I.C.R.A.) diagramado por LA HIGUERA.ONG en cooperación con el Laboratorio de Villa Río Bermejito y Sistema de Salud de provincia de Chaco; entre los meses de julio a diciembre de 2007 se realizó el relevamiento serológico en el área programática correspondientes a Villa Río Bermejito y El Espinillo del Monte Impenetrable (Zona Sanitaria VI) de la provincia de Chaco. En este área viven alrededor 9200 personas pertenecientes a comunidades de pueblos originarios (tobas) y criollos, que ocupan un área geográfica de 4000 km2, en la cual las condiciones sociosanitarias son propicias para el desarrollo de esta enfermedad. Se realizaron 1296 serologías para la enfermedad de Chagas con sangre extraída por venopunción. Se utilizaron 3 técnicas de procesamiento diferentes: hemaglutinación indirecta (HAI, Chagas Polycraro S.A.I.C), enzimainmunoensayo (ELISA Chagas Test, Wienner lab) e Inmuno fluorescencia Indirecta (IFI). Se consideraron positivas las muestras que fueron reactivas por dos técnicas serológicas. Se obtuvo evidencia serológica de infección por Tripanosoma Cruzi en 529(40,8%) de 1296 personas, en el grupo de edad de 0-15 años en 191 (29%); y en los menores de 5 años de 44(22,2%). Los datos obtenidos muestran un escaso control vectorial con valores muy similares a estudios previos que se hicieron en esta misma área hace siete años. Un dato preocupante es que el 66,6% de las mujeres en edad fértil está infectada, lo que lleva a predecir un alto riesgo de chagas congénito. Este trabajo demuestra que son imprescindibles políticas sanitarias sostenidas en el tiempo, con equipos de trabajo con funciones preestablecidas destinadas por un lado al control del vector y por otro al diagnóstico, tratamiento y seguimiento en toda el área endémica, por profesionales especializados, preferentemente del área pediátrica (AU)

Within the framework of the project "Itinerary outpatient pediatrics for rural and isolated communities" (P.A.I.C.R.A.) created by LA HIGUERA.ONG in cooperation with the Laboratory of Villa Río Bermejito and the Chaco Health System, a serological survey was conducted in the study area of Villa Río Bermejito and El Espinillo del Monte Impenetrable (Sanitary zone VI) in the province of Chaco between July and December 2007. The area has around 9200 inhabitants belonging to indigenous (tobas) and immigrant-descendant communities and covers 4000 km2 where socio-sanitary conditions favor the spread of Chagas disease. By venipuncture, 1296 blood samples were drawn to be evaluated for Chagas disease. Three different techniques were used: indirect hemagglutination assay (IHA, Chagas Polycraro S.A.I.C), enzyme-linked immunosorbent assay (ELISA Chagas Test, Wienner lab), and indirect immunofluorescence assay (IFA). Samples that showed reactivity in at least two techniques were considered positive. Serological evidence of infection by Trypanosoma cruzi was found in 529 (40.8%) of 1296 persons; 191 (29%) in the age group of 0-15 years and 44 (22.2%) under 5 years of age. The data show poor vectorial control and values similar to studies conducted in the same area 7 years previously. Worrisome is the fact that 66.6% of women of child-bearing age are infected, leading to a high risk of congenital Chagas disease in the future. The present study shows that preventive policies sustained over time are necessary with teams working both on vector control and diagnosis, treatment, and follow-up of patients in endemic areas consisting of specialized health care workers, preferably in the field of pediatrics (AU)

A C-terminally elongated form of PHI from porcine intestine.

A C-terminally elongated form of peptide histidine isoleucine amide (PHI) was isolated from porcine intestine based on its effect on cAMP production in IMR-32 cells. The structure was determined by amino acid sequence analysis of tryptic fragments and by mass spectrometry. The peptide has 42 amino acid residues like those described from human, rat and mouse, but the amino acid sequence of the C-terminal extension of pig PHI is unique. Unlike the other peptides, it has a C-terminal Ala and it differs at five positions from the human form and at six positions from the rat form, while the human and the rat forms differ by only two substitutions. To avoid confusion arising from different C-terminal residues, a unifying nomenclature is proposed: PHI-27 for the hormone and PHI-42 for the elongated product.

Purification of the aldehyde oxidase homolog 1 (AOH1) protein and cloning of the AOH1 and aldehyde oxidase homolog 2 (AOH2) genes. Identification of a novel molybdo-flavoprotein gene cluster on mouse chromosome 1.

We report the cloning of the AOH1 and AOH2 genes, which encode two novel mammalian molybdo-flavoproteins. We have purified the AOH1 protein to homogeneity in its catalytically active form from mouse liver. Twenty tryptic peptides, identified or directly sequenced by mass spectrometry, confirm the primary structure of the polypeptide deduced from the AOH1 gene. The enzyme contains one molecule of FAD, one atom of molybdenum, and four atoms of iron per subunit and shows spectroscopic features similar to those of the prototypic molybdo-flavoprotein xanthine oxidoreductase. The AOH1 and AOH2 genes are 98 and 60 kilobases long, respectively, and consist of 35 coding exons. The AOH1 gene has the potential to transcribe an extra leader non-coding exon, which is located downstream of exon 26, and is transcribed in the opposite orientation relative to all the other exons. AOH1 and AOH2 map to chromosome 1 in close proximity to each other and to the aldehyde oxidase gene, forming a molybdo-flavoenzyme gene cluster. Conservation in the position of exon/intron junctions among the mouse AOH1, AOH2, aldehyde oxidase, and xanthine oxidoreductase loci indicates that these genes are derived from the duplication of an ancestral precursor.