Wilmar L. Salo

Expression of Carbamoyl-phosphate Synthetase III mRNA during the Early Stages of Development and in Muscle of Adult Rainbow Trout (Oncorhynchus mykiss)

It has been reported that the activities of the urea cycle-related enzymes ornithine carbamoyltransferase and carbamoyl-phosphate synthetase III (CPSase III) are induced during early life stages of ammonotelic rainbow trout (Oncorhynchus mykiss), suggesting that the urea cycle may play a physiological role in early development in teleost fish (Wright, P. A., Felskie, A., and Anderson, P. M. (1995) J. Exp. Biol. 198, 127-135). CPSase III cDNA prepared from embryo mRNA was sequenced, confirming the existence of the CPSase III gene in trout and its expression. The deduced amino acid sequence of the CPSase III is homologous to other CPSases. Supporting evidence for the expression of CPSase III activity in trout embryos was obtained by demonstrating expression of CPSase III mRNA as early as day 3 post-fertilization, reaching a maximum at 10-14 days, declining to a minimum at day 70, and then increasing to a relatively constant level from days 90 to 110 (relative to total RNA). Unexpectedly, in tissues of adult and fingerling trout, CPSase III mRNA was found to be present in muscle but not in other tissues, including liver. This finding was confirmed by assay of extracts, which showed CPSase III and ornithine carbamoyltransferase activity in muscle but not in other tissues. The pyrimidine nucleotide pathway-related CPSase II mRNA was expressed in all tissues.

The specificity of UDP-glucose 4-epimerase from the yeast Saccharomyces fragilis.

1. 1. The reactivity of UDP-glucose 4-epimerase (EC 5.1.3.2) from the yeast Saccharomyces fragilis toward 11 analogs of UDPG has been examined. The compounds tested involve stereochemical modifications of the hexosyl and ribosyl moieties of UDPG. 2. 2. Of the 11 compounds only UDP-β-l-arabinose, UDP-d-fucose and UDP-d-xylose are epimerized. However, evidence is presented indicating that these epimerizations are due to contaminating enzymes and are not due to UDPG 4-epimerase. 3. 3. The substances UDP-d-mannose, UDP-N-acetyl-d-galactosamine, UDP-d-allose and UDP-3-O-methyl-d-glucose were not epimerized. These observations are in agreement with the hypothesis of Budowskyet al.9 since the postulated hexose: uracil hydrogen bond, which is necessary for enzyme action in their mechanism, would be hindered in these substances. 4. 4. dUDPG is not acted upon by the yeast epimerase. It has been reported to serve as a substrate for the epimerase from calf liver. 5. 5. Other substrates which were not epimerized are UDP-β-d-glucose, UDP-d-glucuronic acid, UDP-4-O-methyl-d-glucose, ADPG, CDPG, GDPG, IDPG and dTDPG.

THE BIOARCHAEOLOGICAL VALUE OF HUMAN MUMMIES WITHOUT PROVENIENCE

En este estudio se presentan los analisis morfologicos, osteopatologicos y quimicos de trece cuerpos con momificacion natural cuyo contexto cultural era pobre o no existente. El proposito de este estudio fue verificar la hipotesis de que a pesar de que los cuerpos no presentaban un contexto arqueologico bien definido, estos podian ser muy utiles para investigar importantes problemas bioarqueologicos y biomedicos de la prehistoria local. Los resultados permitieron ubicar a estos trece cuerpos en un marco cultural y establecer la presencia de exostosis auditivas, condicion que se relaciona con actividades maritimas como el buceo en aguas marinas frias. Tambien los resultados indican la masticacion y consumo de hojas de coca, actividad que probablemente gatillaba la perdida prematura de piezas dentales. Ademas los estudios de laboratorio revelaron la presencia de la enfermedad de Chagas (tripanosomiasis americana) en seis de los casos estudiados, dos de los cuales probablemente murieron de esta infeccion durante la fase aguda de la enfermedad. Se encontraron ademas dos cuerpos con osteomielitis tuberculosa, uno mostraba multiples lesiones del tipo miliar. Varios cuerpos presentaron neumonias curadas y presencia de osteopenia y uno tenia fracturas multiples de compresion de los cuerpos vertebrales. A pesar de la falta de contexto, los analisis de laboratorio permitieron reconstruir parte del contexto cronocultural y recolectar importante informacion cientifica que puede ser integrada a las bases de datos antropologicas y biomedicas.

ASPECTS OF INGESTION TRANSMISSION OF CHAGAS DISEASE IDENTIFIED IN MUMMIES AND THEIR COPROLITES

Molecular study of Trypanosoma cruzi (T. cruzi) anciant DNA (aDNA) in the soft (nonskeletal) tissues of 283 naturally (spontaneously) mummified bodies from coastal sites located in southern Peru and northern Chile demonstrated a Chagas disease prevalence rate of about 41% over the past 9,000 years. This rate is similar to that of several endemic areas within this region prior to initiation of public health control programs. This report focuses on the presence of T. cruzi aDNA in the coprolites of some of these mummies. Review of the possible mechanisms that may explain the presence of this parasite in the coprolites indicates numerous antemortem and postmortem circumstances that conceivably could have been responsible. In given conditions, all of these may need to be considered. These considerations indicate that the presence of T. cruzi aDNA in mummy coprolites cannot categorically be considered as evidence of ingestion of the parasite

The promoter region of the carbamoyl-phosphate synthetase III gene ofSqualus acanthias

Carbamoyl-phosphate synthetase III (CPSase III) ofSqualus acanthias (spiny dogfish) is a nuclear-encoded mitochondrial enzyme that catalyzes glutamine-dependent formation of carbamoyl phosphate for urea synthesis. In this paper we report the results of cloning a 10-kb segment of genomic DNA which includes the region flanking the 5′ end of the spiny dogfish CPSase III gene. A total of 1,295 base pairs of sequence straddling the start codon was obtained. Primer extension experiments revealed that the transcription start site is the G located 114 residues upstream of the translation start codon ATG. The first exon has 240 base pairs, including the 5′ untranslated region, the coding sequence for the signal peptide (38 amino acids), and the four N-terminal amino acids of the mature enzyme. The boundary of the first exon and the first intron of the CPSase III gene is concordant with that of rat and frog (Rana catesbeiana) CPSase I, which have been suggested to have evolved from CPSase III. The putative TATA box sequence, TACAAA, is located at position −31 with an uncommonly found C at the third position. Two C/EBP binding site sequences, ATTCTGCAAG (−405 to −397) and GTGCAGTAAG (−168 to −160), were identified in the promoter region, which suggests that spiny dogfish CPSase III might be subjected to transactivation of transcription by C/EBP-related proteins, as has been reported for rat CPSase I. The preparation and binding of a recombinant RcC/EBP-1 protein (theR. catesbeiana homolog of the mammalian C/EBPα) to the two spiny dogfish C/EBP binding sequences are described. Two putative heatshock binding elements were also identified in the promoter region.

The incorporation of tritium from tritium-enriched water into UDP-N-Acetylglucosamine and UDP-N-Acetyl Mannosamine catalyzed by UDP-N-Acetylglucosamine 2-Epimerase from Escherichia coli

Uridine diphosphate N-acetylglucosamine 2-epimerase from Escherichia coli 014 K7 H- catalyzes the reversible epimerization of uridine diphosphate N-acetylglucosamine to uridine diphosphate N-acetylmannosamine. During epimerization, tritium from tritium-enriched water is incorporated into both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylmannosamine. The position of incorporation is C-2 of the N-acetylhexosamine moieties.

Differentiation of mucinous from non-mucinous pancreatic cyst fluid using dual-stained, 1 dimensional polyacrylamide gel electrophoresis

BackgroundPancreatic cysts are being increasingly identified in patients. Mucinous cysts have malignant potential whereas non-mucinous cysts do not. Distinguishing potentially malignant cysts from harmless ones by the characterization of cyst fluid contents remains a difficult problem. This study was undertaken to determine whether cyst fluid mucin glycoprotein analysis could differentiate mucinous from non-mucinous pancreatic cysts.MethodsCyst fluid from 28 patients who underwent resection of a pancreatic cyst was used for the study. In each case the type of cyst was histologically identified. One dimensional SDS polyacrylamide gel electrophoresis (1D-SDS PAGE) was performed on cyst fluid samples. For the detection of the separated proteins, we employed a novel dual staining technique. The gel was first stained with periodic acid Schiff (PAS), a mucin histochemical stain followed by a secondary protein staining with Simply Blue Safestain (Invitrogen).ResultsVisual scoring (based on the presence of mucins) gave a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 88% for prediction of mucinous histology.ConclusionsOne dimensional SDS polyacrylamide gel electrophoresis of pancreatic cyst fluid, followed by mucin (PAS) and protein (Simply Blue Safestain) staining, provides a means of concentrating and visualizing mucins, which allows the accurate differentiation of mucinous from non-mucinous histology in pancreatic cysts.