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[Subcloning and sequencing of DNA fragment related to salt tolerance in Sinorhizobium meliloti 042B].

A 4 kb ClaI DNA fragment related to salt tolerance from S. meliloti 042B was digested by HindIII down 2.4 kb fragment, and a 1.6 kb ClaII-HindIII fragment was retained on plasmid pML122. Then, the 2.4 kb DNA fragment was ligated with plasmid pBBR1-MCS2, and the recombinant plasmid was transformed to E. coli DH5 alpha, and transformant GS2 was obtained. Three-parental mating experiments were carried out with transformant GS2 as donor, salt sensitive strains GZ17 as recipient and pRK2013 as helper plasmid, then the transconjugant GG2 was selected on FY plates containing kanamycin and 0.4 mol/L NaCl. The remaining DNA fragment was self ligated with pML122 and then transformed into E. coli S17-1 and transformat GS0 was obtained. Two-parental mating experiment was carried out with transformant GS0 as donor and salt sensitive strain GZ17 as recipient, but no transconjugant was obtained on the FY plates. Then, the 2.4 kb HindIII DNA fragment was ligated into sequencing vector pGEM-7Zf(+) for sequencing. The result of sequencing and analysis showed that the 2.4 kb DNA fragment contained three ORFs. According to the result of sequencing, further subcloning was conducted and 1.9 kb HindIII-Sac II DNA fragment related to salt tolerance was obtained.

Amino Acid Sequence↗

Cloning and expressing of the three repeat fragments of Plasmodium falciparum 11.1 gene.

OBJECTIVE: To clone and express the 3R, 6R and 9R repeat fragments of Plasmodium falciparum(Pf11.1) gene. METHODS: Three repeat fragments from the genomic DNA of Plasmodium falciparum 3D7 strain cultivated were amplified by using the designed primers. The PCR products were cloned into the pT7 vector for bi-direction sequencing. The sequencing results were analysised by GENETYX-MAC. And then the amplified fragments were subcloned into pET32a(+) or pET32b(+) in order to express the recombinant proteins under the induction of IPTG in E. coli BL21. RESULTS: 3R,6R and 9R fragments with sizez of 552 bp, 630 bp and 444 bp respectively were successfully amplified by PCR. The sequence analysis showed that there were 4 more 3AA units and one more 6AA unit in Pf11.1 gene of 3D7 strain as compared with Palo Alto strain. The homologies of the nucleotide sequence between the 3R fragment and the 6R fragment of the two strains were 92.8% and 95.1%, respectively. The amplified 9R fragment contains 139AA repeat units. The three recombinant proteins were expressed in BL21 strain with molecular weights of 45, 60 and 42 kDa. CONCLUSION: We got the 3R, 6R and 9R fragments separately by PCR and expressed them in E. coli successfully. The Pf11.1 gene of 3D7 strain is highly homologous to that of the Palo Alto strain.

Animals↗

Application of restriction fragment differential display-polymerase chain reaction in study on differential expression profiles of human diseases.

OBJECTIVE: To establish the restriction fragment differential display-polymerase chain reaction (RFDD-PCR) as an efficient technique for constructing and studying the gene expression profile of human tissues. METHODS: The tissues of mamma adenocarcinoma (T), cancerometastasis lymph node (L) and normal mammary (N) from one mammary infiltrating ductal carcinoma case were collected, and the gene expression profile of each kind of tissue was constructed using RFDD-PCR technique at equal pace according to the operating manual of Qbio-gene Company. Then all fragments of the three gene expression profiles were separated and displayed by electrophoresis. With the use of gene database at the website http://www.Qbio-gene.com/display, the authors identified the names of the probable fragments by bioinformatics analysis. Through comparison of the three profiles, the numbers and types of most differentially expressed gene fragments were displayed. RESULTS: The expression profiles of the three kinds of tissue have been constructed covering 1716 fragments of mammary adenocarcinoma, 1769 of cancerometastasis lymph nodes and 1922 of normal mammary tissue. Among these 5407 fragments, 39.39% were exactly the same. While 33.9% sequences of T and L showed differences in abundance or presence, 40.9% of T and N and 39.6% fragments of L and N were observed differentially expressed. These differentially expressed gene fragments were found to relate with metastasis, differentiation, inflammation and so on. CONCLUSION: RFDD-PCR is an efficient technique for research in human diseases genomics as a mass screening for complete gene expression profile with high-flux. Through comparison among three or more profiles, the screening for candidate genes of a certain disease can be accomplished, and there is probably a chance to identify novel gene or expressed sequence tag.

Adenocarcinoma↗

[Spatial-temporal dynamics of landscape fragmentation in North Shannxi Loess Plateau].

The study of landscape fragmentation is an important means to reveal regional ecological processes. By using the remote sensing data of 1987, 1997 and 2002, and the GIS tools, some representative quantitative indices of the landscape spatial structure and landscape fragmentation in North Shannxi Loess Plateau were calculated, aimed to investigate the relationships between landscape ecological structure and landscape pattern in this area. The results showed that cropland contributed most to the extent of landscape fragmentation, but unused land lacked obvious relationship with it. To get large-area images accurately and promptly is very important for monitoring ecological process in regional scale. Moderate-Resolution Imaging Spectroradiometer (MODIS) images in 2003 were used to derive monthly landscape fragmentation information calculated with Local Index of Spatial Associations (LISA) based on Spatial Statistical Analysis (SSA). The analysis of the fragmented landscape types showed that human disturbance had a main contribution to the seasonal landscape fragmentation. Furthermore, with the increasing percentages of cropland and unused land, landscape became more fragmented, while forestland coverage had inverse effects on the fragmentation.

China↗

DNA helicase and duplex DNA fragment unwinding activities of polyoma and simian virus 40 large T antigen display similarities and differences.

We have characterized the biochemical activities of purified polyoma (Py) large T antigen (T Ag) that was capable of mediating the replication of a plasmid containing the Py origin (ori(+) DNA) in mouse cell extracts. We report here that like the T Ag encoded by simian virus 40 (SV40), Py T Ag has DNA helicase and double-stranded DNA fragment unwinding activities. Py T Ag displaced DNA fragments greater than 1,600 nucleotides which were annealed to complementary sequences in single-stranded M13 by translocating in the 3' to 5' direction. Both helicase and double-stranded DNA fragment unwinding reactions were completely dependent upon NTP hydrolysis, displaying a strong preference for ATP and dATP. At low T Ag concentrations, significantly more Py ori(+) DNA fragment was unwound compared with a fragment lacking the replication origin. However, at higher ratios of Py T Ag to DNA, equivalent to those used in replication reactions, unwinding of both ori-containing and -lacking fragments was equally efficient. This is in contrast to SV40 T Ag which exhibited a more stringent requirement for SV40 origin sequences under similar conditions. Furthermore, some of the nucleotides that supported the helicase and unwinding activities of Py T Ag were different from those for the same SV40 T Ag reactions. We have also observed that in contrast to the very poor replication of linear SV40 ori(+) DNA by SV40 T Ag in human cell extracts, linear Py ori(+) DNA was replicated efficiently in mouse cell extracts by Py T Ag. However, despite the fact that linear Py ori(+), SV40 ori(+), and ori(-) DNA fragments could be unwound with comparable efficiency by Py T Ag, only fragments containing the Py replication origin were replicated in vitro. These results suggest that the initiation of DNA synthesis at the Py origin of replication requires features in addition to unwinding of the template.

Antigens, Polyomavirus Transforming↗

Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol.

Pseudomonas exotoxin (PE) was incubated with cells and extracts analyzed for processed fragments. PE was proteolytically cleaved to produce a N-terminal 28-kDa and a C-terminal 37-kDa fragment, the latter being composed of a portion of domain II and all of domain III (the ADP-ribosylating domain). Cleavage was evident at 10 min after toxin addition and endosome preparations contained the processed fragments. Initially, the two fragments were linked by a disulfide bond. Subsequently, the 37-kDa fragment was reduced and translocated to the cytosol where it inactivated protein synthesis. Cytosol from toxin-treated cells was greatly enriched in the 37-kDa fragment. The 37-kDa fragment appears to be essential for toxicity since mutant PE molecules that do not produce this fragment, or cannot deliver it to the cytosol, fail to kill cells.

ADP Ribose Transferases↗

[Hydrophobic constant values (delta RMO) of functional groups. II. Interpretation of fragment interactions].

The delta RM0(-CH2-) values have been calculated from fragment values of various alkyl groups (methyl, ethyl, n-propyl). On the basis of delta RM0(-CH2-) values it has been established that the value of the so-called "second" methyl group from aromatic ring has slightly differed from that of the so-called third methylene group and the value of methylene functional group is constant independently from positions of substituents and from electronic interactions. The delta RM0 Me values of methyl group--which is directly attached to aromatic ring--have been regarded as the so-called first methylene group from aromatic ring since value of free energy needed for transfer of methyl group as well as the other physico-chemical data (mole volume, parachor) are nearly the same as those of methylene group according to the literature. Fragment values of methyl groups substituted at various positions have been compared to relating delta RM0(-CH2-) value which is free practically from electronic interactions and it has been ascertained that fragment values have been higher as well as lower. In accordance with literature lower delta RM0 Me values have been interpreted with hyperconjugation effect of aromatic system. On the other hand lower methylene fragment values of alkyl substituted compounds at N1 atom have been attributed to sigma conjugation. In literature higher delta RM0 Me values than relating delta RM0(-CH2-) value are known only in case of orto position to another functional group or by a methyl group on a sterically hindered aromatic ring. Results of tests have shown that values of delta RM0 Me can substantially increase with extension of conjugated aromatic system or with simultaneous substitutions of methyl, methylene groups, respectively at certain locations. The authors have assigned that increase to change of solvate cuver which surrounds the molecule. The same has been manifested by methyl fragment values of quinazoline derivates (tetrahydropyrroloquinazoline, hexahydroazepinoquinazoline) of different electronic distributions. On basis of calculations it has been proved that delta RM0 2-Me and delta RM0 6-Me fragment values of monosubstituted pyrido (1,2-a)-pyrimidine (PP) derivatives have significantly differed from delta RM0 Me values of at other positions (3,7,8,9) methyl substituted compounds. Among disubstituted derivatives only delta RM0 7-Me and delta RM0 8-Me values of 2-phenyl- and 3-carbetoxymethyl-PP derivatives have not differed from each other but methyl fragment values of other positions (6,9) have significantly differed from each other and from other fragment values too.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical Phenomena↗

Thrombin binding to the A alpha-, B beta-, and gamma-chains of fibrinogen and to their remnants contained in fragment E.

In order to study thrombin interaction with fibrinogen, thrombin binding to fragments D and E (prepared by plasmin digestion of fibrinogen) and to intact S-carboxymethylated chains of fibrinogen (A alpha, B beta, and gamma) was analyzed by autoradiography, immunoblotting, and affinity chromatography. Complex formation was observed between late fragment E and thrombin but not with fragment D. The three reduced chain remnants of fragment E all formed complexes with thrombin. Also, thrombin bound to the intact, separated A alpha, B beta, and gamma chains of fibrinogen as well as to the alpha and beta chains of fibrin. In these experiments the extended substrate-binding site, but not the catalytic-binding site, was being examined because fragment E had as its amino-terminal amino acids Val20 in the alpha chain, Lys54 in the beta chain, and Tyr1 in the gamma chain. Also, thrombin inhibited in its active center by D-phenyl-alanyl-L-prolyl-L-arginine-chloromethyl ketone bound to fragment E and to the separated chains in the same manner as unmodified thrombin. A lysine residue to thrombin was essential for its binding to fibrinogen. Thrombin attached to CNBr-activated Sepharose through its amino groups did not bind to fragment E, but when thrombin was attached through its carboxyl groups, it bound fragment E.

Amino Acid Sequence↗

Influence of A-T content on the fractionation of DNA restriction fragments by RPC-5 column chromatography.

The properties of the fractionated Hae III fragments of pRZ2 DNA (Patient, R.K., Hardies, S.C., and Wells, R.D. (1979) J. Biol. Chem. 254, 5542-5547) were studied in an effort to determine why several of the fragments bind more tightly to RPC-5 than expected on the basis of their length. The purified fragments were analyzed for their nucleotide composition by direct determination of their constituent mononucleotides and by analytical CsCl and Cs2SO4 density gradient analyses. A-T-rich fragments elute at higher salt concentrations than fragments of equivalent size which are not A-T-rich. In addition, denaturation mapping studies by electron microscopy indicate that an A-T-rich run within an otherwise G-C-rich fragment can give rise to delayed elution. At least one other factor influences the separation of DNA restriction fragments by RPC-5 chromatography. Some of the fragments in this digest which elute later than predicted from their size either contain known genetic regulatory sites or bind regulatory proteins.

Adenine↗

[Isolation and physical study of the 13S fragment of 16S RNA and its complex with ribosomal protein S4].

A fragment of E. coli 16S RNA has been obtained by its hydrolysis with pancreatic RNAase A coupled to Sepharose 4B. This fragment has a molecular weight of 170 000 and a sedimentation coefficient of 13S. It does not aggregate in solution and binds with the ribosomal protein S4. The 13S fragment and it complex with the protein S4 have been studied by different physical methods in the first place, by neutron scattering. It has been shown that this fragment is compact in solution. The radii of gyration of the fragment (50 +/- 3 A) and of the protein S4 within the complex (17 +/- 3 A) coincide, within limits of experimental error, with the radii of gyration for the free RNA fragment (47 +/- 2 A) and the free ribosomal protein S4 in solution (18 +/- 2 A). Hence, the conclusion is made that the compactness of the 13S fragment of the 16S RNA and the ribosomal protein S4 does not change at the complex formation. The compact 13S fragment of the 16S RNA is shown to be contrast matched in the H2O/D2O mixture containing 70% D2O which corresponds to its partial specific volume v equal to 0.537 cm3/g.

Chemical Phenomena↗

[Nonsurgical technique for removal of catheter fragments in the heart and the great vessels].

With the widespread use of intravenous polyethylene or silicone elastomer catheters for continuous infusion of fluids and drugs and monitoring central venous pressure, loss of catheter fragments into the central circulation has become an increasing problem. These catheter fragments have been removed from all areas of the right side circulatory system by various techniques not requiring thoracotomy. Many kinds of instruments for nonsurgical removal of these fragments have been utilized. The majority of them have been accomplished using a loop snare or a helical basket. As with loop snares and baskets, a free end of fragments is necessary for fragment entrapment. In this report the authors describe four cases with catheter fragments in the heart or the great vessels and a new nonsurgical technique using a "forceps with three or two grasping prongs for endoscope" for their removal. In the first case, we could remove the embolized catheter fragment without accessible free ends in the pulmonary artery by this method. This special forceps with two grasping prongs was applicable even for the infant and could be inserted through a No. 8 French Cordis catheter introducer. This method is extremely promising for removal of catheter fragments in the central circulatory system.

Adult↗

Metastatic melanoma cell heparanase. Characterization of heparan sulfate degradation fragments produced by B16 melanoma endoglucuronidase.

Heparan sulfate (HS), a prominent component of vascular endothelial basal lamina, is cleaved into large Mr fragments and solubilized from subendothelial basal lamina-like matrix by metastatic murine B16 melanoma cells. We have examined the degradation products of HS and other purified glycosaminoglycans produced by B16 cells. Glycosaminoglycans 3H-labeled at their reducing termini or metabolically labeled with [35S]sulfate were incubated with B16 cell extracts in the absence or presence of D-saccharic acid 1,4-lactone, a potent exo-beta-glucuronidase inhibitor, and glycosaminoglycan fragments were analyzed by high speed gel permeation chromatography. HS isolated from bovine lung, Engelbreth-Holm-Swarm sarcoma, and subendothelial matrix were degraded into fragments of characteristic Mr, in contrast to hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, keratan sulfate, and heparin which were essentially undegraded. Heparin, but not other glycosaminoglycans, inhibited HS degradation. The time dependence of HS degradation into particular Mr fragments indicated that HS was cleaved at specific intrachain sites. In order to determine specific HS cleavage points, HS prereduced with NaBH4 was incubated with a B16 cell extract and HS fragments were separated. The newly formed reducing termini of HS fragments were then reduced with NaB[3H]4, and the fragments hydrolyzed to monosaccharides by trifluoroacetic acid treatment and nitrous acid deamination. Since 3H-reduced terminal monosaccharides from HS fragments were overwhelmingly (greater than 90%) L-gulonic acid, the HS-degrading enzyme responsible is an endoglucuronidase (heparanase).

Animals↗

Fragmentation and myelin formation in hereditary xerocytosis and other hemolytic anemias.

Erythrocytes from a heterogeneous group of hemolytic anemias have been found to release acetylcholinesterase-enriched fragments and show myelin forms during ATP depletion in vitro. The highest amount of fragmentation was found in hereditary spherocytosis and xerocytosis, two inherited membrane defects. Our data suggest ATP depletion plays a role in producing fragmentation or myelin forms. The addition of external CaCl2 1 mM had no effect on the degree of fragmentation. However, propranolol hydrochloride, a cationic anesthetic that does not prevent ATP depletion, inhibited fragmentation and the appearance of myelin forms in both hereditary spherocytes and xerocytes. A more detailed study of the xerocyte fragments showed that they had the same protein composition as those from normal red cells, primarily integral membrane proteins and glycoproteins. The red cells from patients with PNH and G6PD deficiency had the shortest survival in vivo (51Cr) and produced the smallest amount of fragmentation and myelin forms in vitro, whereas xerocytosis with only mild to moderate hemolysis in vivo was associated with the highest amount of myelin forms and membrane fragments in vitro.

Acetylcholinesterase↗

[Nature of changes in the fluorescence process of P700-enriched chloroplast fragments].

Spectral and photochemical properties of P700-enriched chloroplast fragments, obtained by ether treatment of liophylized digitonin fragments, were studied. It was shown that time course of fluorescence changes of isolated fragments (in contrast to digitonin fragments) at 20 degrees does not correspond to time course of absorption changes at 700 nm. Differences in low temperature fluorescence spectra of fragments, initially distinguished by redox states of photosystem 1 reaction centers were found. However, the fragments under study were incapable of light-induced changes of fluorescence yield at--196 degrees, independently of spectral region of measured fluorescence (lambda greater than 660 nm or lambda greater than 710 nm), though these fragments reveal phototransformation of P700. Thus changes in the low temperature fluorescence spectra cannot be accounted for by redox changes of P700. The fragments, isolated by ether treatment at 20 degrees as well at--196 degrees do not reveal light-induced fluorescence changes caused by redox changes of P700. The fluorescence changes observed may be due to accessory photoprocesses of chlorophyllprotein complex.

Cell Fractionation↗

Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis.

Apoptosis is characterized by the nonrandom cleavage of DNA. After continuous treatment of MOLT-4 human T lymphoblastoid cells with the topoisomerase II inhibitor etoposide (50 microM) and the nongenotoxic agent N-methylformamide (300 mM), apoptosis was confirmed by electron microscopy. Analysis of DNA integrity by conventional gel electrophoresis failed to detect internucleosomal DNA cleavage. Resolution of DNA by field inversion gel electrophoresis showed fragments of 50 kilobases (kb). Etoposide induced the transient appearance of an additional DNA band of > 600 kb, which was temporally coincident with DNA-protein complex formation and was rapidly reversible upon drug removal. This DNA band was not observed after N-methylformamide treatment. In situ DNA end-labeling showed the incorporation of biotinylated dUTP into 50-kb DNA fragments but not etoposide-induced DNA fragments of > 600 kb. DNA end-labelling with terminal deoxynucleotidyltransferase was therefore not dependent upon intenucleosomal DNA cleavage, and fragments of approximately 50 kb were characterized by free 3'-OH termini that were not occluded by topoisomerase II protein. Although we considered that topoisomerase II potentially played an active role in the fragmentation of higher order chromatin during apoptosis, the results showed that DNA cleavage by topoisomerase II induced reversible, protein-associated fragments of > 600 kb and not irreversible cleavage to 50-kb fragments. The reversible cleavage of DNA to fragments of > 600 kb appears to be a signal for the engagement of apoptosis and is not an initial step in the sequential unwinding of chromatin.

Apoptosis↗

Role of DNA fragmentation in T cell activation-induced apoptosis in vitro and in vivo.

Apoptotic cell death, characterized by DNA fragmentation and morphologic changes, has previously been shown to occur in immature thymocytes and some T cell hybridomas after activation. Like some other forms of apoptosis, DNA fragmentation during activation-induced cell death precedes the morphologic events. For apoptosis to proceed, activation of the cells must persist at least to the time of DNA fragmentation, before which the cells can remain viable if the activation signal is removed. Aurintricarboxylic acid (ATA) blocks activation-induced apoptotic cell death in a T cell hybridoma, and kinetic studies show that this inhibition occurs at or near the time of DNA fragmentation in the cells. Taken together with the ability of ATA to inhibit DNA fragmentation in isolated nuclei exposed to Ca2+ and Mg2+, these data strongly suggest that ATA prevents apoptosis via its ability to inhibit endogenous endonuclease activity, and, conversely, that this activity is required for this form of cell death. In vivo, ATA inhibits thymocyte depletion and DNA fragmentation induced by anti-CD3 Ab. Further, specific loss of V beta 8+ thymocytes after administration of staphylococcal enterotoxin B is blocked by administration of ATA. These observations support an essential role for DNA fragmentation as an irreversible step in activation-induced apoptosis in T cell hybridomas and during T cell development. This is contrasted with heat shock-induced cell death, in which inhibition of DNA fragmentation does not prevent loss of cell viability.

Animals↗

Immunotherapy with human monoclonal antibodies. Fragment A specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin.

To investigate the feasibility of substituting human mAb (HmAb) for human polyclonal preparations in the treatment of infections, we employed anti-tetanus toxin (TT) as a model system. We established a large panel of hybridomas secreting anti-TT HmAb and compared their fine specificities and protectivity with those exhibited by tetanus immune globulin (TIG). Analysis of three different commercial TIG preparations indicated that the majority of anti-TT antibodies is directed against epitopes expressed by the A fragment, the L chain of TT. Absorption of TIG with purified A fragment completely abolished its protective capacity in mice. Absorption with C fragment, the carboxy-terminal portion of the H chain of TT, had no discernible effect, illustrating the crucial importance of anti-A fragment antibody. The vast majority of more than 100 generated TT-specific HmAb showed specificity for the A fragment. Six HmAb with significant neutralizing activity were identified and further characterized. Five of them recognized the A fragment, whereas one, ST12, bound to both the A fragment and the C fragment with equal affinity. ST12 by itself conferred long lasting protection against TT intoxication when singly administered, and the remainder mediated only a delayed death. ST12 conferred a protection of 13.2 IU/100 micrograms IgG. However, when individual HmAb were combined, synergistic effects were observed. Optimal potency (43 IU/100 micrograms IgG) was obtained with a combination of two HmAb. To obtain a 250-IU dose, only 0.7 mg of this mixture was required in contrast to 100 to 170 mg IgG for TIG.

Antibodies, Monoclonal↗

[Studies on the fragments of FDP in 3 non-DIC patients with increased FDP levels in the sera].

We previously studied fibrinolysis and fibrinogenolysis by analyzing fragments of fibrin/fibrinogen degradation products (FDP) employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. In this report, we characterized the fragments of FDP in three patients with increased serum FDP, that were caused by various diseases. In the patient suffering from tuberculous constrictive pericarditis (case 1), the most part of the FDP fragments were DD and D. In the patient suffering from infection in addition to liver cirrhosis (case 2), the most part of the FDP fragments were high molecular weight (HMW) and D. In case 1 and 2, serum FDP levels were increased in parallel with the elevations of CRP levels. Although DD and HMW fragments were remarkably increased in case 1 and 2 with our immunoblotting analysis, DD levels assayed with LPIA system were much lower than FDP levels. The reason this discrepancy was explained by the observation that affinities of the monoclonal antibody used in LPIA system with DD and HMW fragment were markedly lower than that to DD-E fragment. In the patient suffering from deep vein thrombosis probably caused by steroid therapy of nephrotic syndrome (case 3), the most part of detected FDP fragments were DD and HMW in the period when APTT was shorter than normal, whereas D was mainly observed in the period when APTT was normal. In case 3, FDP and DD levels were increased in parallel with the shortening of APTT. In these non-DIC patients, increased serum FDP levels were induced by the presence of ascites and/or pleural effusion plus infection.(ABSTRACT TRUNCATED AT 250 WORDS)

C-Reactive Protein↗