Obstructive jaundice of a parasitic etiology.

We present the case of a patient with colic pain in the epigastrium and right hypochondrium, which was accompanied by choluria and acholia and slightly elevated levels of bilirubin and eosinophilia. Abdominal echography and magnetic resonance imaging identified a biliary obstruction and endoscopic retrograde cholangiopancreatography was used to extract 3 adult worms identified as Fasciola hepatica. This case highlights the need to consider the suspicion of biliary obstruction caused by Fasciola hepatica in the presence of obstructive jaundice, with or without eosinophilia.

Identity, virulence genes, and clonal relatedness of Aeromonas isolates from patients with diarrhea and drinking water.

Among 800 stool specimens from patients with diarrhea submitted by Primary Care Centers for routine analysis to the Hospital of León (NW Spain) Microbiology and Parasitology Service, 32 (4%) were tested positive for Aeromonas spp. Mixed infections with other enteric pathogens occurred in 12 patients. A. caviae was isolated from 23 clinical specimens. There were also patients infected with A. media, A. hydrophila, A. bestiarum, and A. veronii biovar veronii. All but three isolates carried one or more of the virulence genes. The incidence of the alt, hlyA, aerA, ast, and laf genes was 71.9, 28.1, 25.0, 18.8, and 9.4%, respectively. The alt(+)/ast(+) combination was detected in four isolates and the aerA(+)/hlyA(+) combination was detected in the two A. hydrophila isolates. None of the strains harbored the TTSS, stx1, or stx2 genes and nine bore plasmids. Thirty clinical isolates and a collection of 12 A. caviae and A. media strains obtained from León municipal drinking water over the study period were typed by pulsed-field gel electrophoresis (PFGE). PFGE patterns revealed genetic relatedness and persistence over time among water isolates and some clinical isolates. Interestingly, one A. caviae (aerA(-)/hlyA(-)/alt(+)/ast(-)/laf(+)) human isolate and two A. caviae (aerA(-)/hlyA(-)/alt(+)/ast(-)/laf(+)) drinking water isolates had indistinguishable PFGE patterns, suggesting waterborne infection.

Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae.

Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene. By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45. The disrupted genes in the transformants were characterized by Southern blots. The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected. The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium. The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture. A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable. These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.

Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.

Pulmonary tuberculosis due to Mycobacterium bovis in León.

In recent years, human infection due to Mycobacterium bovis has been considered almost eradicated in most industrialised countries. During the period 1998-2002, nine patients were diagnosed with pulmonary tuberculosis (TB) due to M. bovis in the Hospital Monte San Isidro, León, Spain. Their average age was 66 years. The response to therapy was good in seven cases, while two died, one from renal TB and the other from cancer of the pancreatic head.

Phosphorylation of the yeast ribosomal stalk. Functional effects and enzymes involved in the process.

The ribosomal stalk is directly involved in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes the acidic components correspond to the 12-kDa P1 and P2 proteins, and the RNA binding component is the P0 protein. All these proteins are found phosphorylated in eukaryotic organisms, and previous in vitro data suggested this modification was involved in the activity of this structure. Results from mutational studies have shown that phosphorylation takes place at a serine residue close to the carboxy end of the P proteins. Modification of this serine residue does not affect the formation of the stalk and the activity of the ribosome in standard conditions but induces an osmoregulation-related phenotype at 37 degrees C. The phosphorylatable serine is part of a consensus casein kinase II phosphorylation site. However, although CKII seems to be responsible for part of the stalk phosphorylation in vivo, it is probably not the only enzyme in the cell able to perform this modification. Five protein kinases, RAPI, RAPII and RAPIII, in addition to the previously reported CKII and PK60 kinases, are able to phosphorylate the stalk proteins. A comparison of the five enzymes shows differences among them that suggest some specificity regarding the phosphorylation of the four yeast acidic proteins. It has been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data suggest that although phosphorylation is not involved in the interaction of the acidic P proteins with the ribosome, it can affect the ribosome activity and might participate in a possible ribosome regulatory mechanism.

Peritoneal tuberculosis due to Mycobacterium caprae.

The incidence of tuberculosis in humans due to Mycobacterium caprae is very low and is almost confined to Europe. We report a case of a previously healthy 41-year-old Moroccan with a 6 month history of abdominal pain, weight loss, fatigue and diarrhea. A diagnosis of peritoneal tuberculosis due to M. caprae was made.

Heterologous expression of the highly conserved acidic ribosomal phosphoproteins from Dictyostelium (changed from Dictyosteliumm) discoideum in Saccharomyces cerevisiae.

The genes encoding the acidic ribosomal phosphoproteins DdP1 and DdP2 from Dictyostelium discoideum have been cloned into yeast plasmid vectors under the control of the inducible GAL1 promoter. These constructions have been used to transform S. cerevisiae strains D45 and D67 lacking the equivalent ribosomal components. The D. discoideum genes are properly transcribed when cells are grown in the presence of the inducer galactose and the mRNAs incorporated into polysomes. However, the heterologous ribosomal proteins are not able to rescue the growth deficiency in S. cerevisiae caused by the absence of their own ribosomal proteins. When the heterologous proteins are analyzed using specific antibodies, only protein DdP1 is found in the ribosomes of the transformed S. cerevisiae D67 strain. No other heterologous protein is found in any other transformed strain, suggesting that the heterologous acidic ribosomal components are rapidly degraded when they are not bound to the ribosomes. The results indicate that D. discoideum DdP1 protein is able to interact with the yeast ribosome, though the interaction is functionally inefficient. Protein DdP2, in spite of having a higher sequence similarity to its yeast counterparts, is completely inactive in S. cerevisiae. Since the P proteins from both organisms have extensive amino acid sequence similarity ranging from 60% to 70%, these results warns about establishing a direct relationship between the extent of amino acid sequence similarity and the capacity of heterologous proteins to be functional in host species. Moreover, our data suggest that evolution affected the interaction of the acidic proteins with the ribosome rather than the structural features responsible for their primary functions.

The acidic ribosomal proteins as regulators of the eukaryotic ribosomal activity.

The acidic proteins, A-proteins, from the large ribosomal subunit of Saccharomyces cerevisiae grown under different conditions have been quantitatively estimated by ELISA tests using rabbit sera specific for these polypeptides. It has been found that the amount of A-protein present in the ribosome is not constant and depends on the metabolic state of the cell. Ribosomes from exponentially growing cultures have about 40% more of these proteins than those from stationary phase. Similarly, the particles forming part of the polysomes are enriched in A-proteins as compared with the free 80 S ribosomes. The cytoplasmic pool of A-protein is considerably high, containing as a whole as much protein as the total ribosome population. These results are compatible with an exchanging process of the acidic proteins during protein synthesis that can regulate the activity of the ribosome. On the other hand, cells inhibited with different metabolic inhibitors produce a very low yield of ribosomes that contain, however, a surprisingly high amount of acidic proteins while the cytoplasmic pool is considerably reduced, suggesting that under stress conditions the ribosome and the A-protein may aggregate, forming complex structures that are not recovered by the standard preparation methods.

Deletion of 24 open reading frames from chromosome XI from Saccharomyces cerevisiae and phenotypic analysis of the deletants.

As a part of the EUROFAN program, 24 open reading frames from Saccharomyces cerevisiae (YKR010c to YKR013w, YKR015c to YKR025w, YKR081c to YKR083c, YKR087c to YKR091w and YKR096w) were disrupted in two genetic backgrounds, FY1679 and W303. Systematic deletions and phenotypic analysis were performed following a hierarchical strategy, the so-called 'mass murder'. Of the 24 genes thus deleted, four are essential, whereas the deletion of 17 did not reveal any significant difference between the parental and mutant strains. Deletions of the remaining three show some growth phenotype; ykr024c mutants grow slowly under any conditions, ykr019c mutants grow slower in a rich medium and ykr082w mutants are temperature sensitive, being unable to germinate at 30 degrees C and above.

Structure-activity relationships of sparsomycin and its analogues. Inhibition of peptide bond formation in cell-free systems and of L1210 and bacterial cell growth.

The biological activity of 14 analogues of sparsomycin (1) was studied in cell-free systems of Escherichia coli, Saccharomyces cerevisiae, and Sulfolobus solfataricus by measuring the inhibition of protein synthesis. The inhibition of L1210 colony formation in soft agar and bacterial cell growth in solid as well as in liquid medium was also examined. Each analogue possesses not more than two structural modifications of the sparsomycin molecule. This enabled us to determine unambiguously several structural and stereochemical features that are required for an optimal biological activity in these assays. Sparsomycin, having the SCRS chirality, is the most potent of the four possible stereoisomers. The results obtained with compounds 5-7 indicate that the presence of an oxygen atom on the S (alpha) atom is essential. Substitution of the bivalent sulfur atom by a CH2 group (10) or of the SCH3 moiety by a Cl atom (12) affects the activity of the molecule partially. Compound 12 is surprisingly active against intact cells. Substitution of the C(6)-CH3 group by a H(14) reduces the activity of the molecule. Isomerization of the trans double bond into the cis double bond yields cis-sparsomycin (15), which is inactive. The hydrophobic derivatives 8, 9, and 11 are considerably more active than sparsomycin; thus the ribosomal binding site for sparsomycin may have a hydrophobic character.

Ectopic pregnancy early diagnosis limitations.

A series of 219 surgically and pathologic proven ectopic gestations are reviewed to emphasize the ectopic pregnancy early diagnosis limitations. A childbearing age, low parity woman is typical of having an ectopic pregnancy. Risk factors in their past history were absent in 52% of patients. Fertility investigations, IUD, PID, and abdominal surgery are often found in their past. Six per cent of patients had a previous ectopic pregnancy. Sixty-one per cent of patients were admitted with a definite ruptured ectopic pregnancy and 37% were admitted to rule out this condition. At surgery 58% had ruptured ectopic pregnancy with intraabdominal hemorrhage. Only 12% were unruptured. The obstetric outcome after surgery was available in 74 patients. Out of these, 40.5% had term pregnancies with live children, repeat ectopic pregnancy occurred in 8.2%, spontaneous first trimester abortion in 4.1%, and subsequent infertility in 16%. Postoperative pelvic adhesions were more frequently seen, at laparoscopy, when the patients were diagnosed at the stage of ruptured ectopic pregnancy with intraabdominal hemorrhage. A diagnostic protocol based on the screening of the patients at risk, correct evaluation of symptom and signs, and liberal use of beta-hCG pregnancy tests, culdocentesis, ultrasound and laparoscopy, is finally proposed.

Structure and function of the stalk, a putative regulatory element of the yeast ribosome. Role of stalk protein phosphorylation.

The ribosomal stalk is involved directly in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes, the acidic components correspond to the 12 kDa P1 and P2 proteins, and the RNA binding component is protein P0. All these proteins are found to be phosphorylated in eukaryotic organisms. Previous in vitro data suggested this modification was involved in the activity of this structure. To confirm this possibility a mutational study has shown that phosphorylation takes place at a serine residue close to the carboxyl end of proteins P1, P2 and P0. This serine is part of a consensus casein kinase II phosphorylation site. However, by using a yeast strain carrying a temperature sensitive mutant, it has been shown that CKII is probably not the only enzyme responsible for this modification. Three new protein kinases, RAPI, RAPII and RAPIII, have been purified and compared with CKII and PK60, a previously reported enzyme that phosphorylates the stalk proteins. Differences among the five enzymes have been studied. It has also been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data available suggest that phosphorylation, although it is not involved in the interaction of the acidic proteins with the ribosome, affects ribosome activity and might participate in some ribosome regulatory mechanism.

[Behavior of vascularization during hepatic regeneration]. / Comportamiento de la vascularización durante la regeneración hepática.

In this work, out of 185 rats 120 underwent hepatectomy of 70%. Studies carried out by microradiography and Spalteholz transparence method prove that during the first days after hepatectomy the ratio hepatic parenchyma/vascular texture is increased; this fact comes to be maximal four days after, becoming stabilized toward ten days after. Studies done by Ibas I imaging analyzer in hepatic sections of rats which underwent hepatectomy as compared to those which underwent laparatomy only, show that ratio LCV-LCV, as well as areas and area form of hepatic sanguineous vessels are changing, in the first ones, in a rhythmical way including two phases of greater activity. These variations are evolved, in a different way, in the central zone than in the peripherical one of liver. We try to relate these facts to vascular and humoural theories which explain hepatic regeneration.

[Effect of alloxan-induced diabetes on hepatic regeneration]. / Influencia de la diabetes inducida por aloxana sobre la regeneración hepática.

Hepatic regeneration in alloxan-induced diabetic rats that underwent a 70% hepatectomy was compared to that of a series of non-treated rats. In diabetic rats, hepatic regeneration is lower and the liver reaches only 70% of the original weight ten days after hepatectomy. Analytical studies in these rats show a diminution of insulin and glucagon. Hepatic microcirculation as shown by measurements of CLV-CLV and CLV area undergoes changes which evolve at a different rate in the central and peripheral zones of the liver. These findings as well as the diminished hepatic regeneration may be attributed to the diminution of the insulin and glucagon values. Insulin and glucagon may be responsible for hepatic regeneration.

Pneumonia due to Nocardia cyriacigeorgica in a patient with Crohn's disease treated with infliximab.

Infliximab, an anti-tumor necrosis factor α (TNF-α) antibody, is useful in the treatment of rheumatoid arthritis, Crohn's disease etc. It has been related to increases in the rate of several infections. We present the case of a 53-year-old woman diagnosed with community-acquired pneumonia due to Nocardia cyriacigeorgica who was taking infliximab, azathioprine and prednisone for Crohn's disease.

The activity-controlling phosphorylation site is not the same in the four acidic ribosomal proteins from Saccharomyces cerevisiae.

By using site-directed mutagenesis and chemical analysis of phosphopeptides, a unique phosphorylation site has been shown at serine 73 in the amino acid sequence of the Saccharomyces cerevisiae acidic ribosomal protein YP1 beta (L44'). The mutation in this position prevents in vitro phosphorylation by protein kinases that modify the wild-type polypeptide. The unphosphorylatable mutated protein is unable to bind to the ribosomes and to rescue the growth deficiency of yeast strains in which the corresponding original gene is inactivated by gene disruption. Sequencing of tryptic phosphopeptides has shown that acidic proteins YP1 alpha and YP2 alpha (L44) are also phosphorylated at positions near the carboxyl end. These results contrast with the data indicating that in the highly homologous protein YP2 beta, phosphorylation takes place at serine 19, close to the amino terminus. The results show that phosphorylation is definitely required for the biological activity of these ribosomal proteins. However, the differences in the phosphorylation sites suggest that the effect of this modification is not the same in all of them, confirming the heterologous role of these peculiar ribosomal components. In fact, the different context of the modification sites in the four polypeptides suggests the existence of more than one protein kinase specific for this set of proteins.