A Case of Benign Hepatic Cyst with Supra-elevated Cyst Fluid Tumor Markers

Digestive Diseases and Sciences -     

Pharmacokinetics of a low molecular weight heparin (Fraxiparine) in various stages of chronic renal failure.

This study investigates the pharmacokinetics of a low molecular weight heparin (Fraxiparine) after a single bolus intravenous injection of 100 antifactor Xa IC U.kg-1 in 3 groups of patients affected by chronic renal insufficiency of various severity: group A (n = 7) was composed of hemodialyzed patients; groups; B (n = 7) had a creatinine clearance ranging from 10 to 20 ml.min-1 and group C (n = 5) from 30 to 50 ml.min-1. There was no significant difference between the pharmacokinetic parameters determined in the 3 groups of patients and no correlation between these parameters and the creatinine clearance. However, when compared to the values established in a group of 12 healthy volunteers, the half-life of disappearance of the antifactor Xa activity was significantly prolonged. Therefore it is advised to monitor antifactor Xa activity in patients affected by chronic renal insufficiency of any severity to avoid a possible accumulation phenomenon.     

Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5′-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3′,8-cyclo-7,8-dihydro-guanosine 5′-triphosphate (3′,8-cH₂GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, we performed biochemical characterization of 3′,8-cH₂GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3′,8-cH₂GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3′,8-cH₂GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3′,8-cH₂GTP (GTP 3′,8-cyclase), and that of MoaC is to catalyze the rearrangement of 3′,8-cH₂GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. Thus, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.Moco is an essential enzyme cofactor that mediates redox reactions in the active sites of enzymes. Moco-dependent enzymes play central roles in purine and sulfur catabolism in mammals, anaerobic respiration in bacteria, and nitrate assimilation in plants (1, 2). Importantly, Moco must be synthesized de novo in cells because it is chemically unstable, particularly under aerobic conditions, and cannot be taken up as a nutrient (1, 2).During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of GTP into cPMP (3). This unusual transformation involves the insertion of the guanine C-8 between C-2′ and C-3′ of ribose (Fig. 1A) (4). Although this conversion has been shown to require two proteins, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively) (4–6), their individual contributions have remained elusive and are the subject of the current study. MoaA belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, of which members catalyze unique free-radical reactions (7). By contrast, MoaC shows no significant sequence or structural similarities to any functionally characterized enzyme. Therefore, the predominant view of the field has been that MoaA catalyzes the majority or all of the rearrangement of GTP to form the pterin structure, with MoaC playing little to no catalytic role in this process (2). In line with this view, studies identifying a putative MoaA product after chemical derivatization suggested that MoaA catalyzed the transformation of GTP to pyranopterin triphosphate (Fig. 1B) (8, 9). This conventional view was challenged by a recent in vitro characterization of MoaA, where formation of 3′,8-cyclic-7,8-dihydro-GTP (3′,8-cH₂GTP; Fig. 1B) rather than pyranopterin triphosphate was proposed (10). This finding suggested the possibility of a novel mechanism in which MoaC, and not MoaA, may ultimately be responsible for pyranopterin ring formation. However, it was also proposed that 3′,8-cH₂GTP could simply be a transient intermediate of MoaA (8, 9), leaving ambiguity about the functions of MoaA and MoaC.Open in a separate windowFig. 1.In vivo and in vitro functional characterization of MoaC. (A) Moco biosynthesis. Symbols indicate the fate of each atom (4). cPMP may also be in a hydrate form in solution (14). (B) Previously proposed structures for the MoaA product. (C) In situ ¹³C NMR characterization of the MoaA product. Shown are the ¹³C NMR spectra of [U-¹³C]GTP (Top), purified [U-¹³C]3′,8-cH₂GTP (Middle), and the MoaA (0.4 mM) reaction using [U-¹³C]GTP (1 mM) as the substrate (Bottom). Numbers are the signal assignments for atoms labeled in A and B. Signals highlighted by * and # are derived from toluensulfonate and glycerol, respectively. (D) Timecourse of the formation of the biosynthetically relevant MoaA product based on the quantitation of 3′,8-cH₂GTP before (open circles) or after (filled circles) its conversion to cPMP. The assay solution contained 65 μM WT-MoaA, 0.2 mM GTP, and 1 mM SAM. (E) In vitro activity of WT and variants of MoaC determined by a coupled assay with MoaA. (F) Steady-state kinetic parameters for WT and variants of MoaC. The assays were performed in the absence of MoaA by using purified 3′,8-cH₂GTP as a substrate. a, only the upper limit (1.0 μM) was determined because the reaction rate became impractically low below this substrate concentration. (G) Moco production in E. coli ΔmoaC expressing WT or variants of MoaC based on anaerobic growth rates (black bars) or NR activity (gray). All data in D–G are average of at least three replicates, and the errors are based on SDs.Most of these previous studies have focused on the characterization of MoaA; the contribution of MoaC has been largely unexplored. Here, we report a comprehensive functional and structural study that clarifies these issues and provides strong evidence that MoaC is the enzyme responsible for pyranopterin backbone formation and that MoaA provides 3′,8-cH₂GTP as the MoaC substrate. Further structural and functional studies revealed that MoaC catalyzes the complex rearrangement of 3′,8-cH₂GTP into cPMP via a unique mechanism involving dynamic conformational changes of substrate and enzyme.     

Carbonic anhydrase I is an early specific marker of normal human erythroid differentiation

The expression of carbonic anhydrase (CA) as a marker of erythroid differentiation was investigated by immunologic and enzymatic procedures. A polyclonal anti-CA antibody was obtained by immunizing rabbits with purified CA I isozyme. This antibody is reactive with CA I but not with CA II. Within blood cells, CA I was only present in erythrocytes, whereas CA II was also detected in platelet lysates by enzymatic assay. Concerning marrow cells, identifiable erythroblasts and some blast cells expressed CA I. Most of the glycophorin A-positive marrow cells were clearly labeled by the anti-CA I antibody. However, rare CA I-positive cells were not reactive with anti-glycophorin A antibodies. We therefore investigated whether these cells were erythroid precursors or progenitors. In cell sorting experiments of marrow cells with the FA6 152 monoclonal antibody, which among hematopoietic progenitors is reactive only with CFU-E and a part of BFU- E, was performed, CA I+ cells were found mainly in the positive fraction. The percentage of CA I+ cells nonreactive with anti- glycophorin A antibodies contained in the two fractions was in the same range as the percentage of erythroid progenitors identified by their capacity to form colonies. In addition, the anti-CA I antibody labeled blood BFU-E-derived colonies as early as day 6 of culture, whereas in similar experiments with the anti-glycophorin A antibodies, they were stained three or four days later. No labeling was observed in CFU-GM- or CFU-MK-derived colonies. The phenotype of the day 6 cells expressing CA I was similar to that of erythroid progenitors (CFU-E or BFU-E): negative for glycophorin A and hemoglobin, and positive for HLA-DR antigen, the antigen identified by FA6 152, and blood group A antigen. Among the cell lines tested, only HEL cells expressed CA I, while K562 was unlabeled by the anti-CA I antibody. In contrast, HEL and K562 cells expressed CA II as detected by a biochemical technique. Synthesis of CA I, as with other erythroid markers such as glycophorin A and hemoglobin, was almost abolished after 12-O-tetradecanoyl-phorbol-13 acetate treatment of HEL cells. In conclusion, CA I appears to be an early specific marker of the erythroid differentiation, expressed by a cell with a similar phenotype as an erythroid progenitor.     

Associations between the genotypes of Staphylococcus aureus bloodstream isolates and clinical characteristics and outcomes of bacteremic patients

We investigated associations between the genotypic and phenotypic features of Staphylococcus aureus bloodstream isolates and the clinical characteristics of bacteremic patients enrolled in a phase III trial of S. aureus bacteremia and endocarditis. Isolates underwent pulsed-field gel electrophoresis, PCR for 33 putative virulence genes, and screening for heteroresistant glycopeptide intermediate S. aureus (hGISA). A total of 230 isolates (141 methicillin-susceptible S. aureus and 89 methicillin-resistant S. aureus [MRSA]) were analyzed. North American and European S. aureus isolates differed in their genotypic characteristics. Overall, 26% of the MRSA bloodstream isolates were USA 300 strains. Patients with USA 300 MRSA bacteremia were more likely to be injection drug users (61% versus 15%; P < 0.001), to have right-sided endocarditis (39% versus 9%; P = 0.002), and to be cured of right-sided endocarditis (100% versus 33%; P = 0.01) than patients with non-USA 300 MRSA bacteremia. Patients with persistent bacteremia were less likely to be infected with Panton-Valentine leukocidin gene (pvl)-constitutive MRSA (19% versus 56%; P = 0.005). Although 7 of 89 MRSA isolates (8%) exhibited the hGISA phenotype, no association with persistent bacteremia, daptomycin resistance, or bacterial genotype was observed. This study suggests that the virulence gene profiles of S. aureus bloodstream isolates from North America and Europe differ significantly. In this study of bloodstream isolates collected as part of a multinational randomized clinical trial, USA 300 and pvl-constitutive MRSA strains were associated with better clinical outcomes.     

Lambda Cold Agglutinin with Anti-A1 Specificity in a Patient with Reticulosarcoma

Abstract. Erythrocyte auto-antibodies may be associated with lymphomas. We report a case of reticulosarcoma in a patient of A₁ blood group phenotype whose direct Coombs test was positive with anti-IgM and anti-complement antiglobulins. In serum, a cold antibody of IgM type was detected, which exhibited a monoclonal pattern with Λ-light chains. Moreover, it was shown to display an unusual auto-anti-A₁ specificity.     

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein

The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.The emergence of multidrug-resistant bacteria is a mounting global health crisis. In particular, multidrug-resistant Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and is resistant to most antibiotics commonly used for patient treatment (1). Hospital intensive care units in many countries, including the United States, now report methicillin-resistant S. aureus infection rates exceeding 50% (2, 3). Antibiotic resistance in contemporary infectious S. aureus strains, such as in hospitals, is often encoded by plasmids that can be transmitted between strains via horizontal DNA transfer mechanisms. These plasmids are typically classified as small (<5 kb) multicopy plasmids, which usually encode only a single resistance gene; medium-sized (8–40 kb) multirestance plasmids that confer resistance to multiple antibiotics, disinfectants, and/or heavy metals; and large (>40 kb) conjugative multiresistance plasmids that additionally encode a conjugative DNA transfer mechanism (4–6). Importantly, sequence analyses have shown that most staphylococcal conjugative and nonconjugative multiresistance plasmids encode a highly conserved replication initiation protein, denoted RepA_N (5–15). RepA_N proteins are also encoded by plasmids from other Gram-positive bacteria as well as by some phage, underscoring their ubiquitous nature (10). These RepA proteins are essential for replication of multiresistance plasmids, and hence plasmid carriage and dissemination, yet the mechanisms by which these proteins function in replication are currently unknown.The DNA replication cycle can be divided into three stages: initiation, elongation, and termination. Replication initiation proteins (RepA) mediate the crucial first step of initiation. Bacterial chromosome replication is initiated by the chromosomal replication initiator protein, DnaA, which binds the origin and recruits the replication components known as the primosome (16). In Gram-negative bacteria the primosome includes DnaG primase, the replicative helicase (DnaB), and DnaC (17). Replication initiation in Gram-positive bacteria involves DnaG primase and helicase (DnaC) and the proteins DnaD, DnaI, and DnaB (18–22). DnaD binds first to DnaA at the origin. This is followed by binding of DnaI/DnaB and DnaG, which together recruit the replicative helicase (23, 24). Instead of DnaA, plasmids encode and use their own specific replication initiator binding protein. Structures are only available for RepA proteins (F, R6K, and pPS10 Rep) harbored in Gram-negative bacteria. These proteins contain winged helix-turn-helix (winged HTH) domains and bind iteron DNA as monomers to, in some still unclear manner, drive replicon assembly (25–27).Replication mechanisms used by plasmids harbored in Gram-positive bacteria are less well understood and are distinct from their Gram-negative counterparts. Indeed, most plasmid RepA proteins in Gram-negative and Gram-positive bacteria show no sequence homology and seem to be unrelated. The multiresistance RepA proteins are arguably among the most abundant of plasmid Rep proteins, yet how they function is not known. Data suggest that these proteins are composed of three main regions: an N-terminal domain (NTD) consisting of ∼120 aa, a long and variable linker region (∼30–50 residues), and a C-terminal domain (CTD) of ∼120 residues (28–31). The NTD and CTD are both essential for replication. The NTD exhibits the highest level of sequence conservation, which has resulted in the designation of plasmids that encode these proteins as the RepA_N replicon family (10). Although not as well conserved as the NTD, RepA CTD regions show homology between plasmids found in genus-specific clusters, suggesting that this domain may perform a host-specific role (28–32). Although the function of the RepA CTD remains enigmatic, recent studies have indicated that the NTD mediates DNA binding and interacts with iterons that reside within the plasmid origin (30). The essential roles played by RepA proteins in multiresistance plasmid retention marks them as attractive targets for the development of specific chemotherapeutics. However, the successful design of such compounds necessitates structural and mechanistic insight. Here, we describe a detailed dissection of the RepA proteins from the multiresistance plasmids pSK41 and pTZ2126. The combined data reveal the molecular underpinnings of a minimalist replication assembly mediated by multiresistance RepA proteins.     

Disappearance of Hb F and i antigen during the first year of life

In order to investigate whether a common control mechanism is involved in the diminution of i antigen expression and that of Hb F content in human erythrocytes during the postnatal period, we compared changes in 72 normal infants aged from 0 to 12 months. The proportion of hemoglobins (Hb F, Hb A, Hb A₂) and the quantitation of ?i”? antigen were determined on the total population of red blood cells. In addition, the percentage of individual cells containing Hb F or ?i”? antigen or both (F cells, ?i”? cells, and F + ?i”? cells) were evaluated by using a rhodamine-conjugated anti-Hb F and a fluorescein conjugated anti-i system on the same smear preparation, The results provided by the two most sensitive techniques (F cell counting and ?i”? agglutinability) indicated that the curves of disappearance of Hb F and ?i”? antigen along the 12 first months after birth were identical. A strong correlation (r = 0.97, P < 0.0001) existed between the percentage of F cells and ?i”? antigen expression. In addition, the progressive increase in Hb A₂ concentration was inversely correlated firstly with the proportion of Hb F and second with the expression of the ?i”? antigen. These results suggest that the switch from fetal to adult hemoglobin and the transformation of ?i”? antigen expression occurring during the first year following birth are governed by a common control mechanism.     

Regulation of i- and I-antigen expression in the K562 cell line

Expression of i- and I-antigen in K562 cultured under different conditions of culture was investigated. Under standard conditions of culture, i-antigen expression was very high (100% of i-labeled cells) in contrast to I-antigen expression of which was very low (2 to 5% of I-labeled cells). The addition of hemin to K562 cells did not modify the mean antigenic density or the proportion of i- and I-labeled cells. In contrast, sodium butyrate elicited an important increase in the proportion of cells exhibiting I-antigen associated to a decrease of i-antigenic density. The effect of butyrate was reversible and dependent upon de novo protein and messenger RNA synthesis since it was abolished in the presence of cycloheximide or actinomycin D. The stimulation of i-antigen conversion to I-antigen elicited by butyrate cannot be directly related to an induction of differentiation since evidence in this sense is lacking; in fact, butyrate did not increase the hemoglobin content of K562 cells. The passage from exponential to stationary phase of growth (cell density inhibition) was associated with an increase in I-antigen expression and a slight decrease in i-antigen density on the surface of K562 cells.