Edema pulmonar unilateral / Unilateral pulmonary edema

El edema de pulmón unilateral está descrito en la literatura médica, pero es una situación muy poco frecuente y de fácil confusión en su diagnóstico. El edema agudo de pulmón unilateral puede ser ipsilateral al proceso que lo produce o contralateral al mismo. Es importante comprender el mecanismo fisiopatológico que lo produce para llegara un correcto diagnóstico y adecuado tratamiento. A este respecto presentamos un caso de edema pulmonar izquierdo en un paciente de 57 años, diagnosticado de neumonía basal derecha 4 días antes, que reacude por empeoramiento de su situación clínica. La ausencia de signos de sepsis, de fallo ventricular izquierdo y la asimetría del edema permitió sospechar de tromboembolismo pulmonar unilateral izquierdo, que se confirmó con tomografía computarizada helicoidal (AU)

Unilateral pulmonary edema has been reported in the literature, but it is a very rare condition that can easily be confused with other diagnoses. Acute unilateral pulmonary edema can be either ipsilateral or contra lateral to the causative process. An understanding of the underlying pathophysiologic mechanism is essential if we are to diagnose this condition correctly and prescribe appropriate treatment. We report a case of left-sided pulmonary edema in a 57-yearoldman who had been diagnosed 4 days earlier with right basilar pneumonia. The absence of any signs of sepsis or left ventricular failure plus the asymmetric presentation of edema led us to suspect unilateral pulmonary embolism in the leftlung. The diagnosis was confirmed by spiral computed tomography (AU)

Heterologous HIV-nef mRNA trans-splicing: a new principle how mammalian cells generate hybrid mRNA and protein molecules.

Heterologous trans-splicing is a messenger RNA (mRNA) processing mechanism, that joins RNA segments from separate transcripts to generate functional mRNA molecules. We present here for the first time experimental evidence that the proximal segment of the HIV-nef RNA segment can be trans-spliced to both viral (e.g. SV40 T-antigen) and cellular transcripts. Following either microinjection of in vitro synthesized HIV-nef and SV40 T-antigen pre-mRNA or transfection of the HIV-nef DNA into T-antigen positive cells (CV1-B3; Cos7), it was found that recipient cells synthesized HIV-nef/T-antigen hybrid mRNA and protein molecules. To generate the hybrid mRNA, the cells utilized the 5' cryptic splice sites of the HIV-nef (5'cry 66 and 5'cry 74) and the SV40 T/t-antigen 3' splice site. To demonstrate that heterologous trans-splicing also occurs between the HIV-nef RNA and cellular transcripts, a cDNA library was established from HIV-nef positive CV1-B3 cells (CV1-B3/13 cells) and screened for hybrid mRNA molecules. Reverse transcription-PCR and Northern blot analysis revealed that a significant portion of the HIV-nef transcript is involved in heterologous trans-splicing. To date, eight independent HIV-nef/cellular hybrid mRNA molecules have been identified. Five of these isolates contain segments from known cellular genes (KIAA1454, PTPkappa, Alu and transposon gene families), while three hybrid segments contain sequences of not yet known cellular genes (genes 1-3).

Modulation in vitro of H-ras oncogene expression by trans-splicing.

In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours tested. An exon known as IDX, which has been described previously and is located between exon 3 and exon 4A of the c-H-ras pre-mRNA, allows an alternative splicing process that results in the synthesis of the mRNA of a putative protein named p19. It has been suggested that this alternative pathway is less tumorigenic than that which results in the activation of p21. We have used the mammalian trans-splicing mechanism as a tool with which to modulate this particular pre-mRNA processing to produce mRNA similar to that of mature p19 RNA. The E4A exon of the activated H-ras gene was found to be a good target for external trans-splicing. We reprogrammed the rat carnitine octanoyltransferase exon 2 to specifically invade the terminal region of H-ras. Assays performed with this reprogrammed trans-exon showed that the trans-splicing product was obtained in competition with cis-splicing of the D intron of the H-ras gene, and was associated with concomitant down-modulation of D intron cis-splicing. We also found that the exon 4A of the human c-H-ras gene underwent successive trans-splicing rounds with an external exon.

Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing.

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which may lead to the synthesis of two proteins. Generation of the three COT transcripts in rat does not depend on sex, development, fat feeding, the inclusion of the peroxisome proliferator diethylhexyl phthalate in the diet or hyperinsulinemia. In addition, trans-splicing was not detected in COT of other mammals, such as human, pig, cow and mouse, or in Cos7 cells from monkey. Rat COT exon 2 contains two purine-rich sequences. Mutation of the rat COT exon 2 upstream box does not affect the trans-splicing in vitro between two truncated constructs containing exon 2 and its adjacent intron boundaries. In contrast, mutation of the downstream box from the rat sequence (GAAGAAG) to a random sequence or the sequence observed in the other mammals (AAAAAAA) decreased trans-splicing in vitro. In contrast, mutation of the AAAAAAA box of human COT exon 2 to GAAGAAG increases trans-splicing. Heterologous reactions between COT exon 2 from rat and human do not produce trans-splicing. HeLa cells transfected with minigenes of rat COT sequences produced cis- and trans-spliced bands. Mutation of the GAAGAAG box to AAAAAAA abolished trans-splicing and decreased cis-splicing in vivo. We conclude that GAAGAAG is an exonic splicing enhancer that could induce natural trans-splicing in rat COT.

Post-transcriptional regulation of rat carnitine octanoyltransferase.

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which can lead to the synthesis of two proteins. The occurrence of the three COT transcripts in rat has been found in all tissues examined and does not depend on sex, fat feeding, peroxisome proliferators or hyperinsulinaemia. Rat COT exon 2 contains a putative exonic splicing enhancer (ESE) sequence. Mutation of this ESE (GAAGAAG) to AAAAAAA decreased trans-splicing in vitro, from which it is deduced that this ESE sequence is partly responsible for the formation of the three transcripts. The protein encoded by cis-spliced mRNA of rat COT is inhibited by malonyl-CoA and etomoxir. cDNA species encoding full-length wild-type COT and one double mutant COT were expressed in Saccharomyces cerevisiae. The recombinant enzymes showed full activity towards both substrates, carnitine and decanoyl-CoA. The activity of the doubly mutated H131A/H340A enzyme was similar to that of the rat peroxisomal enzyme but was completely insensitive to malonyl-CoA and etomoxir. These results indicate that the histidine residues His-131 and His-340 are the sites responsible for the interaction of these two inhibitors, which inhibit COT by interacting with the same sites.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase promoter contains a CREB binding site that regulates cAMP action in Caco-2 cells.

cAMP increases transcription of the mitochondrial (mit.) gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase, which encodes an enzyme that has been proposed as a control site of ketogenesis. The incubation of Caco-2 cells with cAMP increased mit.HMG-CoA synthase mRNA levels 4-fold within 24 h. We have identified an active cAMP-response element (CRE) located 546 bp upstream of the mit. HMG-CoA synthase promoter that is necessary for the induction of expression by dibutyryl cAMP. Co-transfections of constructs, containing the CRE element of the mit.HMG-CoA synthase promoter fused to the gene for chloramphenicol acetyltransferase, with protein kinase A and a dominant-negative mutant of cAMP-response-element-binding protein (CREB) show that the response to cAMP is mediated by the transcription factor CREB. The CRE element confers responsiveness of protein kinase A to a heterologous promoter in transfection assays in Caco-2 cells. Gel-retardation assays revealed that the mit.HMG-CoA synthase CRE binds to recombinant CREB. The shifted band obtained with the putative mit. HMG-CoA synthase CRE sequence and nuclear proteins from Caco-2 cells competed with CRE sequences of other genes such as somatostatin and phosphoenolpyruvate carboxykinase. We conclude that the regulation of the expression of the gene for mit.HMG-CoA synthase in Caco-2 cells by cAMP is mediated by a CRE sequence in the promoter.

Processing of carnitine octanoyltransferase pre-mRNAs by cis and trans-splicing.

Trans-splicing is a mechanism by which two pre-mRNAs are processed to produce a mature transcript that contains exons from both precursors. This process has been described mostly in trypanosoma, nematodes, plant/algal chloroplasts and plant mitochondria [Bonen et al. (1993) FASEB J. 7, 40-46]. Our studies clearly demonstrate that a trans-splicing reaction occurs in the processing of the carnitine octanoyltransferase (COT) gene in rat liver. Three different mature transcripts of COT have been found in vivo, the canonical cis-spliced mRNA and two trans-spliced transcripts, in which either exon 2 or exons 2 and 3 are repeated. Splicing experiments in vitro also indicate the capacity of exon 2 to act either as a donor or as an acceptor of splicing, allowing the trans-splicing reactions to occur.

Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver.

Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. During isolation of a COT clone from a rat liver library, a cDNA in which exon 2 was repeated, was characterized. Reverse transcription-PCR amplifications of total RNAs from rat liver showed a three-band pattern. Sequencing of the fragments revealed that, in addition to the canonical exon organization, previously reported [Choi, S. J. et al. (1995) Biochim. Biophys. Acta 1264, 215-222], there were two other forms in which exon 2 or exons 2 and 3 were repeated. The possibility of this exonic repetition in the COT gene was ruled out by genomic Southern blot. To study the gene expression, we analyzed RNA transcripts by Northern blot after RNase H digestion of total RNA. Three different transcripts were observed. Splicing experiments also were carried out in vitro with different constructs that contain exon 2 plus the 5' or the 3' adjacent intron sequences. Our results indicate that accurate joining of two exons 2 occurs by a trans-splicing mechanism, confirming the potential of these structures for this process in nature. The trans-splicing can be explained by the presence of three exon-enhancer sequences in exon 2. Analysis by Western blot of the COT proteins by using specific antibodies showed that two proteins corresponding to the expected Mr are present in rat peroxisomes. This is the first time that a natural trans-splicing reaction has been demonstrated in mammalian cells.

Developmental changes in the phospho(enol)pyruvate carboxykinase gene expression in small intestine and liver of suckling rats.

The cytosolic enzyme phospho(enol)pyruvate carboxykinase (PEPCK) is markedly expressed in the intestinal mucosa of suckling rats. The expression is located in the small intestine, but there is no expression in stomach, colon, or cecum. The expression changes with age. The mRNA levels at birth are very low, increase after the first lactation, reach maximum levels between 3 and 9 days after birth, and then decrease smoothly. At weaning, when animals begin to feed on a solid chow diet, the expression falls to adult levels, which are hardly detectable. Mother's milk may influence the intestinal expression, since in rats weaned at Day 18, 3 days before normal weaning, the mRNA levels decreased dramatically. mRNA levels for PEPCK in liver present a rather different developmental pattern from that of intestine, remaining high at weaning and in adult rats. On the ninth day after birth, the mRNA levels are the same in intestine and liver.

[The contamination of enteral nutrition in critical patients]. / Contaminación de nutriciones enterales en pacientes críticos.

Some previous studies have shown that administration of contaminated enteral diets may produce nosocomial infections in critical patients. There is a series of factors in these patients which may enhance the risk of clinical complications deriving from the administration of enteral nutrition (EN) contaminated by microorganisms (alteration of the immunological state, increased stomach pH, reduced intestinal motility, reduced mucosa production, etc.). This study examines EN contamination in critical patients admitted to the ICU of the Hospital Universitario de Traumatología y Rehabilitación de las C. S. Vall d'Hebron, suffering from cranial-encephalic traumatism and/or multiple traumatism. The data made it possible to create a working design which takes account of factors which may increase the risk of EN contamination. The work was done in three phases, involving different handling procedures (Phase 1, Phase 2 and Phase 3). The results of the three studies made it possible to describe a working method in which the following points are outstanding: handwashing with antiseptic soap prior to handling the EN, avoidance of reuse of containers (if necessary) for more than 24 hours, not to exceed 8 hours' perfusion of EN previously handled, and not to wash the container prior to adding new quantities of EN.