Analytical Isoelectric Focusing Research Articles

Emergence of Escherichia coli isolates producing conjugative plasmid-mediated DHA-1 β-lactamase in a Korean university hospital

Seven isolates of cefoxitin-resistant Escherichia coli with an inducible phenotype were detected between November 2002 and July 2003 in a Korean hospital. Conjugations were tested by the filter mating method using azide-resistant E. coli J53 as the recipient. All isolates and their transconjugants were tested for broth microdilution minimum inhibitory concentrations, isoelectric focusing (IEF), polymerase chain reaction (PCR) for SHV, TEM, CTX-M and DHA-derived beta-lactamases, and DNA sequencing. XbaI-digested genomic DNA bands of the seven isolates were separated by pulsed-field gel electrophoresis (PFGE). IEF, PCR and sequence analysis revealed that all isolates possessed a blaTEM-1-like and a blaDHA-1 gene. Two isolates also carried the blaCTX-M-14 gene. Transfer of the resistance by conjugation experiments of all seven isolates was successful, suggesting that the blaDHA-1-containing plasmids in the E. coli isolates were self-transmissible. The isolates were recovered from patients in wards or an intensive care unit, all of which had been exposed to beta-lactams before isolation of the DHA-1 producers. Five patterns among the seven isolates were demonstrated by PFGE; sporadic infections with E. coli possessing an inducible beta-lactam resistance phenotype were found. DHA-1 encoded by conjugative plasmids conferred the resistance phenotype. The spread of the DHA-1 producers was due to both clonal spread and horizontal transfer of the resistance gene.

On-Line Optical Fiber Detection in a Preparative Free-Flow Electrofocusing Apparatus

Many analytical isoelectric focusing (IEF) instruments are equipped with on-line detection, e.g., conductivity or UV-vis absorbance. Most of their preparative counterparts have not integrated such detection systems and thus require labor-intensive off-line analysis to quantify separation results. This paper describes the incorporation of an optical fiber based, on-line detection system that allows one to follow the evolution of the protein bands in a preparative IEF apparatus. An array of four optical fibers was designed to deliver light to the annulus of a free-flow electrophoresis apparatus, to detect the transmitted light passing through the separation media and to determine the protein concentration at vertical positions along the annulus of a vortex-stabilized focusing chamber using a 1024 bit CCD line camera. The final concentration of the major myoglobin band was 21.0 mg/mL at electric field strengths as high as 333 V/cm. Spectrophotometric analysis indicated a final concentration of 18.9 mg/mL, 10% less than that reported by the optical fibers.

Apolipoprotein C-III isoforms: kinetics and relative implication in lipid metabolism

Apolipoprotein C-III (apoC-III) production rate (PR) is strongly correlated with plasma triglyceride (TG) levels. ApoC-III exists in three different isoforms, according to the sialylation degree of the protein. We investigated the kinetics and respective role of each apoC-III isoform in modulating intravascular lipid/lipoprotein metabolism. ApoC-III kinetics were measured in a sample of 18 healthy men [mean age (+/-SD) 42.1 +/- 9.5 years, body mass index 29.8 +/- 4.6 kg/m2] using a primed-constant infusion of l-(5,5,5-D3) leucine for 12 h. Mono-sialylated and di-sialylated apoC-III (apo-CIII1 and apoC-III2) exhibited similar PRs (means +/- SD, 1.22 +/- 0.49 mg/kg/day vs. 1.15 +/- 0.59 mg/kg/day, respectively) and similar fractional catabolic rates (FCRs) (0.51 +/- 0.13 pool/day vs. 0.61 +/- 0.24 pool/day, respectively). Nonsialylated apoC-III (apoC-III0) had an 80% lower PR (0.25 +/- 0.12 mg/kg/day) and a 60% lower FCR (0.21 +/- 0.07 pool/day) (P < 0.0001 for comparison with CIII1 and CIII2 isoforms). The PRs of apoC-III1 and apoC-III2 were more strongly correlated with plasma TG levels (r > 0.8, P < 0.0001) than was apoC-III0 PR (r = 0.54, P < 0.05). Finally, the PR of apoC-III2 was strongly correlated with the proportion of LDL <255 A (r = 0.72, P = 0.002). These results suggest that all apoC-III isoforms, especially the predominant CIII1 and CIII2 isoforms, contribute to hypertriglyceridemia and that apoC-III2 may play a significant role in the expression of the small, dense LDL phenotype.

Synthesis of UV-absorbing and fluorescent carrier ampholyte mixtures and their application for the determination of the operational pH values of buffering membranes used in isoelectric trapping separations

Success in isoelectric trapping separations critically depends on the knowledge of the accurate operational pH value of the buffering membranes used. Currently, due to a lack of easy, rapid, accurate methods that can be used for the post-synthesis determination of the operational pH value of a buffering membrane, only nominal pH values calculated from the amounts of the reagents used in the synthesis of the membranes and their acid–base dissociation constants are available. To rectify this problem, UV-absorbing and fluorescent carrier ampholyte mixtures were prepared by alkylating pentaethylenehexamine with a chromophore and a fluorophore, followed by Michael addition of acrylic acid and itaconic acid to the resulting oligoamine. Carrier ampholyte mixtures, with evenly distributed absorbance values across the 3 < p I < 10 range, were prepared by blending. The master blend served as the feed mixture in binary isoelectric trapping separations that used the buffering membrane to be characterized. The p I value of the most basic UV-absorbing or fluorescent carrier ampholyte collected in the anodic separation compartment, determined by full-column imaging capillary isoelectric focusing analysis, indicates the operational pH value of the separation membrane.

Lectin anchored stabilized biodegradable nanoparticles for oral immunization: 1. Development and in vitro evaluation

The investigation comprises development of a stable and targeted formulation of HBsAg for the oral immunization against Hepatitis B. PLGA nanoparticles bearing HBsAg was prepared by double emulsion method. The antigen was protected from organic/aqueous interface by using protein stabilizer, trehalose. The acidic environment generated within PLGA nanoparticles was neutralized by co-encapsulation of a basic additive, Mg(OH) 2 which provides an additional stabilization to the antigen especially against acid induced antigen inactivation. Furthermore, lectin from Arachis hypogaea (PNA) was anchored on to the surface of the HBsAg loaded nanoparticles in order to enhance their affinity towards the antigen presenting cells of the Peyer's patches. The developed system was characterized for shape, size and loading efficiency. The antigen integrity was assessed by using SDS-PAGE followed by isoelectric focusing analysis. Bovine submaxillary mucin (BSM) was used as a biological model for in vitro ligand affinity determination and activity studies. The lectin anchored nanoparticles exhibited 52.18 ± 4.73% loading while ligand density was estimated to be of 17.90 ± 1.14 μg/mg. The results suggest that HBsAg can be successfully stabilized by co-encapsulation of an appropriate protein stabilizer, i.e. trehalose and a basic additive, Mg(OH) 2. The ligand-coupled nanoparticles demonstrated approximately four folds increase in degree interaction with the BSM as compared to plain nanoparticles. Additionally, the nanoparticles maintained their intrinsic sugar specificity as associated due to lectin (PNA).

Molecular epidemiology of extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit

To investigate the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in the neonatal intensive care unit of a university hospital in Italy. Antibiotic susceptibility was evaluated by disc diffusion and Etest. ESBLs were identified by isoelectric focusing, PCR and DNA sequencing analysis. Genotyping was performed by PFGE analysis. Conjugation was performed by broth mating. Molecular typing of K. pneumoniae isolates identified three distinct PFGE patterns. Isolates of PFGE profile A were isolated during an epidemic in 1996, while isolates of PFGE profiles B and C were sequentially isolated from September 2002 to December 2004, when 233 colonizations and 19 infections by K. pneumoniae occurred. All K. pneumoniae strains of different PFGE types were identified as ESBL producers. DNA sequencing of amplified beta-lactamase genes identified a novel bla(TEM) ESBL (bla(TEM-136)) along with bla(SHV-1) in chromosomal and plasmid DNA from K. pneumoniae of PFGE type A, respectively, and bla(TEM-1) and bla(SHV-12) in plasmid DNA from K. pneumoniae of PFGE types B and C. Conjugation experiments demonstrated that resistance to third-generation cephalosporins, along with an approximately 80 kb plasmid containing bla(SHV-12) and bla(TEM-1), was transferred from K. pneumoniae epidemic strains of PFGE types B and C to a susceptible Escherichia coli host at a frequency of 4 x 10(-6) and 1 x 10(-6) cfu/recipient cell, respectively. The selection of ESBL-producing clones and the transfer of the bla(SHV-12) ESBL gene between different clones were responsible for the spread of K. pneumoniae in the neonatal intensive care unit.

Dissemination of Proteus mirabilis isolates harboring CTX-M-14 and CTX-M-3 β-lactamases at 2 hospitals in Taiwan

From February to June 2003, 111 clinical isolates of Proteus mirabilis were mainly isolated from patients with respiratory or urinary tract infections hospitalized at 3 district hospitals (A, B, C) in central Taiwan. Among them, 34 (30.6%) strains, isolated within 2 hospitals (A and B), exhibited nonsusceptibility to cefotaxime with significant reduction of MIC (≥3 log 2 dilution) by the effect of clavulanic acid, which confirmed the phenotype of extended-spectrum β-lactamases (ESBLs). These ESBL producers were coresistant to gentamicin, isepamicin, and amikacin, but remained susceptible to ceftazidime (MIC, ≤0.5 μg/mL) and meropenem (MIC, <0.5 μg/mL). By isoelectric focusing analysis, polymerase chain reaction, and nucleotide sequencing, we detected the presence of CTX-M-14 in 33 strains and CTX-M-3 in 6 strains (5 strains harboring both CTX-M-14 and CTX-M-3 enzymes). These β-lactamase genes can be successfully transferred by the conjugative plasmid. Molecular epidemiology of the 34 ESBL-producing P. mirabilis strains by pulsed-field gel electrophoresis using SfiI restriction enzyme revealed 9 different genotypes, suggesting epidemic clones with intra- and interhospital spread. In conclusion, the broadly extended clonal spreading of CTX-M–type P. mirabilis was first discovered at the district hospitals in Taiwan.

Isolation and characterisation of a protein elicitor from Sclerospora graminicola and elicitor-mediated induction of defence responses in cultured cells of Pennisetum glaucum.

Sclerospora graminicola (Sacc.) Schroet., an oomycete pathogen of Pennisetum glaucum (L.) R.Br. infects the meristematic tissues of young seedlings. The motile zoospores from the sporangia encyst, germinate and penetrate the plant tissue. Resistance to the invading pathogen is governed by the specific recognition of conserved pathogen-associated proteins or elicitors. In the present study, a zoospore protein was isolated and purified to homogeneity by a combination of size exclusion and high-performance liquid chromatography (HPLC). The crude fractionated protein was able to elicit an array of defence responses in resistant and susceptible cells of pearl millet. Treatment of cultured cells of pearl millet with partially purified elicitor protein resulted in a rapid loss of cell viability in the resistant cells and the percentage of cell death was higher in the resistant than in the susceptible cells. Cultures of resistant cells showed a sharp increase in the extra cellular pH compared with susceptible cells when treated with the crude elicitor. Increased oxidative burst was also recorded in the cells treated with the crude elicitor. The purified elicitor showed unique properties. The purified protein was acidic with a pI of 5.6 as revealed by isoelectric focusing (IEF) and matrix-assisted laser desorption ionisation (MALDI) analysis showed that the elicitor had a molecular mass of 7040 daltons. The primary structure determined by N-terminal Edman degradation and searches with BLAST did not reveal similarities to any known plant pathogenic or oomycete elicitor. Higher activities of the important defence-related enzymes phenylalanine ammonia lyase (PAL) and peroxidase in the resistant cell cultures than in the susceptible cell cultures treated with the purified elicitor were clearly evident. Studies of gene expression by northern blotting with heterologus peroxidase, PAL and oxalate oxidase probes showed that the mRNA transcripts were strongly up-regulated in resistant cell cultures within 30 min of elicitor treatment. The purified elicitor also demonstrated a very strong concentration-dependent sterol binding. The purified elicitor protein belongs to a class of low molecular weight oomycete elicitors with sterol carrier properties. The identified low molecular weight protein elicitor displays unique properties that can be exploited for synthesis of novel molecules for eco-friendly crop protection.

Genetic Variation of Glutelin Acidic Subunit Polypeptides in Bangladesh Rice Genetic Resources

Bangladesh rice genetic resources collected from six distinct regions of the country were examined to obtain the diversity in glutelin acidic subunit polypeptides. Seed glutelins from 576 Bangladesh rice cultivars representing seven ecotypes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF) electrophoresis and two-dimensional electrophoresis (2-DE: SDS-PAGE/IEF) analyses. Glutelin acidic subunit was separated into four bands, α-1, α-2, α-3 and α-4, and the variation in each of bands was detected by SDS-PAGE analysis. A higher molecular size component of α-1(α-1′) was identified in the cultivars tested. In case of α-2, α-2H with high molecular mass, α-2L with low molecular mass and α-2H/L were detected. α-3 band variation showed α-3H, α-3L, and α-3H/L, while for α-4, α-4H, α-4L bands were identified. In IEF analysis, a total of 16 bands with independent pI ranging from pI 6.30 to 7.52 were identified for the glutelin acidic subunit among the cultivars. The maximum and minimum numbers of IEF bands found were 13 and 9, respectively. The α-2L less cultivars were also lacking in pI 6.80 polypeptide in IEF. Result of 2-DE showed that pI 6.80 polypeptide was the main component of α-2L band. Transplanted Aman ecotype was the most diverse with respect to glutelin variation. Geographical distribution of glutelin variation in the Transplanted Aman ecotype differed according to the regions. The above results indicate that Bangladesh rice cultivars possess great genetic diversity in glutelin acidic subunit polypeptides. This study of indigenous rice cultivars from Bangladesh provides useful information regarding their breeding potential.

Plasmid-Mediated Carbapenem-Hydrolyzing β-Lactamase KPC in a Klebsiella pneumoniae Isolate from France

Carbapenem-hydrolyzing β-lactamases can be metallo-β-lactamases (7), expanded-spectrum oxacillinases (8), or Ambler class A enzymes (1, 2, 4, 6, 9-12). The class A KPC β-lactamases hydrolyze all β-lactams except for cephamycins and have been rarely reported in studies of enterobacterial species (Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella enterica, Enterobacter sp.) found in New England and the mid-Atlantic region of the United States; when they are reported, they are found to come from New York City hospitals in particular (2, 4, 6, 9-12). We report here a K. pneumoniae isolate from Paris that produced β-lactamase KPC-2. An 80-year-old man with a 5-year history of prostatic carcinoma and metastasis was admitted in February 2005 at the Cochin hospital in Paris for acute urine retention in the right nephrostomy. The bilateral nephrostomy was performed in December 2004 at a New York City hospital. No hospitalization or antibiotic treatment is known to have taken place during the period between the patient’s stay in New York and his emergency hospitalization in Paris. K. pneumoniae YC was isolated from urine and blood cultures and, according to disk diffusion susceptibility testing, was resistant or of intermediate susceptibility to all antibiotics except colistin and fosfomycin (3). No other K. pneumoniae isolates with similar antibiotic resistance patterns were recovered from the Cochin hospital during this same period of time. The MICs of β-lactams (3) for K. pneumoniae YC confirmed the disk diffusion results, with the MICs of expanded-spectrum cephalosporins and carbapenems being only slightly modified after the addition of clavulanic acid (Table ​(Table1).1). A crude β-lactamase extract of that isolate had significant imipenem hydrolysis activity (0.37 μmol of imipenem/min/mg of total protein) (5, 8). TABLE 1. MICs of β-lactams for K. pneumoniae YC, the transconjugant strain, and the reference strain Plasmid analysis detected two plasmids of >100 kb and ca. 75 kb in isolate YC. Using ampicillin (100 μg/ml) and sodium azide (100 μg/ml; Sigma-Aldrich, Saint-Quentin-Fallavier, France) as selective agents and sodium azide-resistant Escherichia coli J53 as the recipient strain, the ca.-75-kb plasmid was self-conjugative and conferred β-lactam resistance patterns for E. coli transconjugants similar with respect to the MICs obtained, except for those for cefoxitin, cefotaxime, ceftazidime, and aztreonam, which were lower than those for the parental strain (Table ​(Table1).1). A β-lactamase extract from a transconjugant culture subjected to analytical isoelectric focusing (8) identified two β-lactamases with pI values of 5.4 and 6.8. Using primers for the detection of Ambler class A and class B β-lactamase genes (7, 8), PCR experiments followed by sequencing identified a β-lactamase gene coding for the carbapenemase KPC-2 (pI 6.8) and the narrow-spectrum TEM-1 (pI 5.4), which were also identified from K. pneumoniae YC. Then, by the use of a series of successive PCR primers, the 2.8-kb surrounding sequences of the blaKPC-2 gene were found to be identical to those surrounding the same blaKPC-2 gene from a Salmonella isolate from Maryland (6). This report identified a carbapenem-hydrolyzing β-lactamase KPC in France that likely resulted from an intercontinental transfer of the KPC producer from the United States. Up to the time of this discovery, plasmid-mediated KPC enzymes had been a unique feature of the United States, whereas most other broad-spectrum antibiotic resistance genes have been reported in the United States after their initial discoveries elsewhere. The identification of KPC enzymes is worrisome, since they hydrolyze expanded-spectrum cephalosporins and carbapenems and thus could jeopardize therapy for serious infections caused by major nosocomial pathogens. In addition, their detection, based on careful interpretation of reduced carbapenem susceptibility in Enterobacteriaceae, remains difficult.

A Family with Hemoglobin Hirosaki

A 48-year-old man had a 30-year history of hemolytic anemia of undetermined cause. Spherocytes were not observed, osmotic fragility was normal, and red cell enzyme activities were normal. His brother and daughter also had hemolytic anemia. The brother had previously undergone splenectomy, and the anemia had been ameliorated. In the proband and daughter, no abnormal hemoglobin was apparent in the results of isoelectric focusing and DEAE anion-exchange high-performance liquid chromatography analyses. On evaluation with the isopropanol test, unstable hemoglobin was not observed in the proband but was detected in the daughter. There was also a decreased ratio of 3 globin/3 globin chain production. Analysis of the 32 gene demonstrated the presence of a mutation (alpha43 [CE1] Phe --> Leu), hemoglobin Hirosaki.

Structure and Properties of the C-terminal Domain of Insulin-like Growth Factor-binding Protein-1 Isolated from Human Amniotic Fluid

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules.

Osteoinductivity of partially purified native ostrich ( Struthio camelus) bone morphogenetic protein: Comparison with mammalian species

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily. They are capable of inducing ectopic bone formation. Until now, the main interest has been focused on mammalian osteoinductive BMPs, and there are no reports of native BMP extracts of birds. In this study, we isolated and characterized native BMPs of ostrich ( Struthio camelus) and compared them with identically isolated native bovine (cow) and reindeer BMPs with regard to BMP pattern and osteoinductive capacity. The ostrich BMP pattern differed markedly from that of cow and reindeer BMP in non-reduced SDS-PAGE, reduced SDS-PAGE and Western blot. The differences in isoelectric focusing analysis were smaller. However, the ostrich BMP extract had a peak at pH 5.1, clearly differing from the BMPs of cow and reindeer. The osteoinductive capacity and density of ectopic bone, induced by BMP extracts in a mouse thigh muscle pouch, were determined radiographically. The ostrich BMP extract displayed significantly lower osteoinductive capacity and density of induced bone than the bovine and reindeer BMP extracts. In conclusion, our results indicate that the BMP pattern of birds differs considerably from that of mammals, and that the osteoinductive capacity of BMPs and the density of induced bone are lower in birds than in mammals. They also suggest that the bone metabolism of birds is adapted to make light bones suitable for flying.

Prevalence of Ambler class A and D β-lactamases among clinical isolates of Pseudomonas aeruginosa in Korea

Recently, resistance to extended-spectrum cephalosporins due to acquired beta-lactamases has been reported in Pseudomonas aeruginosa. The aim of this study was to investigate the prevalence of Ambler class A and D beta-lactamases and their extended-spectrum derivatives and antimicrobial susceptibilities of P. aeruginosa isolated from various parts of Korea. A total of 252 consecutive, non-duplicate isolates of P. aeruginosa were studied for the presence of class A or D beta-lactamase. Antibiotic susceptibility tests and PCR amplification of genes encoding class A (bla(PSE-1), bla(PER-1), bla(VEB-1), bla(TEM), bla(SHV), bla(CTX-M) and bla(GES-1)) and class D beta-lactamases (bla(OXA-groupI), bla(OXA-groupII) and bla(OXA-groupIII)) were performed. For PCR-positive isolates, isoelectric focusing (IEF) analysis, sequencing and pulsed-field gel electrophoresis (PFGE) were performed. In 64 (25.4%) isolates, structural genes for PSE-1 (6.3%), OXA-10 (13.1%), OXA-4 (4.3%), OXA-30 (2.0%), OXA-2 (2.3%) and OXA-17 (0.4%) were found; their distribution varied between provinces. None harboured bla(PER-1), bla(VEB-1), bla(TEM), bla(SHV), bla(CTX-M) and bla(GES-1). The cross-class resistance rates to other antibiotics was significantly higher in class A and D beta-lactamase producers than in non-producers (P < 0.001 for aminoglycosides, ciprofloxacin and meropenem). OXA-type beta-lactamases are widespread, but their extended-spectrum derivatives are rare among P. aeruginosa in Korea. To our knowledge, this is the first report of OXA-17, an extended-spectrum derivative of OXA-10, outside the Middle East. In addition, combined resistance to ticarcillin and aminoglycosides was a useful indicator for P. aeruginosa producing PSE- or OXA-type beta-lactamases in this study.

Identification of a New Allelic Variant of the Acinetobacter baumannii Cephalosporinase, ADC-7 β-Lactamase: Defining a Unique Family of Class C Enzymes

Acinetobacter spp. are emerging as opportunistic hospital pathogens that demonstrate resistance to many classes of antibiotics. In a metropolitan hospital in Cleveland, a clinical isolate of Acinetobacter baumannii that tested resistant to cefepime and ceftazidime (MIC = 32 microg/ml) was identified. Herein, we sought to determine the molecular basis for the extended-spectrum-cephalosporin resistance. Using analytical isoelectric focusing, a beta-lactamase with a pI of > or = 9.2 was detected. PCR amplification with specific A. baumannii cephalosporinase primers yielded a 1,152-bp product which, when sequenced, identified a novel 383-amino-acid class C enzyme. Expressed in Escherichia coli DH10B, this beta-lactamase demonstrated greater resistance against ceftazidime and cefotaxime than cefepime (4.0 microg/ml versus 0.06 microg/ml). The kinetic characteristics of this beta-lactamase were similar to other cephalosporinases found in Acinetobacter spp. In addition, this cephalosporinase was inhibited by meropenem, imipenem, ertapenem, and sulopenem (K(i) < 40 microM). The amino acid compositions of this novel enzyme and other class C beta-lactamases thus far described for A. baumannii, Acinetobacter genomic species 3, and Oligella urethralis in Europe and South Africa suggest that this cephalosporinase defines a unique family of class C enzymes. We propose a uniform designation for this family of cephalosporinases (Acinetobacter-derived cephalosporinases [ADC]) found in Acinetobacter spp. and identify this enzyme as ADC-7 beta-lactamase. The coalescence of Acinetobacter ampC beta-lactamases into a single common ancestor and the substantial phylogenetic distance separating them from other ampC genes support the logical value of developing a system of nomenclature for these Acinetobacter cephalosporinase genes.

Improved yields for baculovirus-mediated expression of human His 6-PDK1 and His 6-PKBβ/Akt2 and characterization of phospho-specific isoforms for design of inhibitors that stabilize inactive conformations

PDK1 and PKB/Akt have a pleckstrin homology (PH) domain at the C-terminus and N-terminus, respectively, which stabilizes an unphosphorylated, autoinhibited conformation. Binding of the PH domain to a phospholipid second messenger causes relief of autoinhibition, which results in kinase phosphorylation and activation. Baculovirus-mediated expression in Sf9 insect cells of both His 6-PDK1 and His 6-PKBβ/Akt2 were optimized, which significantly improved the yields (⩾5-fold) of the affinity purified enzymes over previously reported values. Isoelectric focusing (IEF) and Western analyses indicated that the apparent V max = 192 ± 13 U/mg and K m (PDK-Tide) = 55 ± 10 μM of purified His 6-PDK1 results from a mixture of at least three different phospho-specific isoforms (p I values of 6.8, 6.5, and 6.4). A purely unphosphorylated isoform of His 6-PDK1 (p I = 6.8) was generated by treatment with lambda protein phosphatase (λPP), which decreased V max to 2.4 ± 0.4 U/mg and increased K m (PDK-Tide) to 217 ± 61 μM. Isoelectric focusing and Western analyses indicated that the apparent V max = 0.21 ± 0.03 U/mg and K m (Crosstide) = 87 ± 30 μM of purified His 6-PKBβ/Akt2 results from a mixture of the enzyme monophosphorylated either at Ser-474 (∼90%) or at Thr-309 (∼10%). A purely unphosphorylated isoform of His 6-PKBβ/Akt2 (p I = 6.4) was generated by treatment with λPP, which decreased V max ∼2-fold. The optimization of high-level production and detailed characterization of purified and λPP-treated His 6-PDK1 and His 6-PKBβ/Akt2 will facilitate detailed structural and kinetic studies aimed at understanding the mechanism of second messenger-induced activation.

Genotyping Hungarian apricot cultivars for self-(in)compatibility by isoelectric focusing and PCR analysis

Self-incompatibility (SI) in flowering plants is a widespread genetic system that promotes out-crossing. In Prunus species the SI is a gametophytic trait, which is controlled by a single multiallelic locus, termed S-locus. S-alleles codify stylar glycoproteins with ribonuclease activity (S-RNases). Our objective was to assess the S-genotype of some Hungarian apricot varieties by isoelectric focusing of stylar RNases as well as by PCR analysis using cherry consensus primers. Consensus primers amplified one or two bands of various sizes. Primers amplifying the 1st intron gained fragments the size of which ranged from 250 to 500 bp; while those amplifying the 2nd intron resulted in fragments of 800-2000 by length. Our data demonstrated that the first intron of the apricot S-RNase gene is shorter than the second one, which coincides with the structure of cherry S-RNase alleles. `Hargrand' (S1S2) and `Harcoe (S1S4) possessed one common S-RNase isoenzyme. Hungarian 'Orias' apricot cultivars showed different bands compared to the previous cultivars, but they shared completely identical patterns confirming that they possess the same S-genotype. 'Bergeron', `Harmat' and 'Korai zamatos' are characterised by an evidently distinct S-RNase pattern. The self-compatible cultivar (`Bergeron') had one allele, which suggests its correspondence to the Sc. Primers for the 2nd intron was unsuccessful in gaining fragments, which indicates that the 2nd intron in the Sc allele is too long to get any amplification. On the basis of our data, identities and differences were revealed in the S-allele constitution of some economically important Hungarian apricot cultivars at protein and DNA levels.

Cytochromes c-556 from three genetic races of Agrobacterium. Purification and comparison of their properties.

1. The soluble cytochromes c-556 from three strains of Agrobacterium tumefaciens, B6, II Chrys and Apple 185 have been purified to homogeneity. The strains are representative members of the three main genetic races of Agrobacterium. The purity of the final preparations was established by electrophoresis with an without sodium dodecyl sulphate, by analytical isoelectric focusing and ultracentrifugation, and by N-terminal analysis. 2. Properties of these cytochromes were compared wih those of cytochrome c-556 from A. tumefaciens, strain B2a, a member of the same genetic race as strain B6. The four cytochromes are monohaem proteins with molecular weights of about 12300 (determined by four different methods). The isoelectric points of those from strains B6 and B2a are identical at pH 5.5, but they differ from the cytochromes of the other genetic races: cytochrome c-556 from strain Apple 185 is more acidic (ph 5.2) and that from strain II Chrys more basic (pH 6.2). The cytochromes from strains b6 and B2a have very similar but not identical amino acid compositions; both of them differ more from Apple 185 than from II Chrys c-556. 3. Comparison of the tryptic, chymotryptic and thermolytic fingerprints of cytochrome c-556 from strains B2a and II Chrys reveals strong homology between the primary structures of these cytochromes. Therefore and because of the sequence identity of the first eight residues, the cytochromes c-556 from strains II Chrys, B6 and B2a are most likely C-terminal haem-bound, of the same type as the cytochrome c' from photosynthetic bacteria.

Vitellogenin exists as charge isomers in the Indian freshwater murrel, Channa punctatus (Bloch)

Three charge isomeric forms of vitellogenin are reported in the blood of estrogen-treated murrel, Channa punctatus on the basis of the results of native and SDS–PAGE, isoelectric focusing, and Ferguson plot analysis. Vitellogenin was induced in adult murrels by exogenous administration of estradiol-17β and labelled with 32P in vivo. Labelled vitellogenin isolated from other plasma proteins on Ultrogel AcA 34 columns resolve into three bands on native-PAGE. Ferguson plot analysis reveals free electrophoretic mobilities of the three bands as 6.0, 4.5, and 3.7, which are very similar to the isoelectric point values of 5.9, 4.6, and 3.8, respectively. The apparent molecular weight of native murrel vitellogenin is 530 kDa. It consists of a single peptide with a molecular weight of 175 kDa. All the three bands on native PAGE resolves into a single peptide on SDS–PAGE. N-terminal amino acid sequence for murrel vitellogenin peptide is MKAVVLALLL. The protein phosphorus:lipid phosphorus ratio for murrel vitellogenin is 0.9. The total lipid content is 32.8%, consisting of 45% neutral lipids and 30% phospholipids. Phosphatidyl choline is the major phospholipid whereas triglycerides are the major neutral lipids. Results of these analytical analyses indicate that murrel native vitellogenin circulates as three charge isomers; poor in phosphorus but rich in lipids content.

Escherichia coli Producing an ACC-1 Class C β-Lactamase Isolated in Barcelona, Spain

Class C β-lactamases, primarily chromosomally encoded (AmpC), were first described to occur in a plasmid in 1988. Since then, these plasmid-mediated β-lactamases have been identified throughout the world. In the Mediterranean area, most belong to a homogeneous group, CMY-2 to CMY-5 and LAT-1 to LAT-4, related to the chromosomally encoded AmpC-type β-lactamase of Citrobacter freundii (9). In Spain, the first plasmidic AmpC enzyme described was FOX-4, found in an Escherichia coli strain isolated in the Canary Islands in 2000 (2). A CMY-2 enzyme was later detected in Proteus mirabilis, Klebsiella oxytoca, and Salmonella enterica serovars Mikawasima and Montevideo in Barcelona (Catalonia) and Guipuzkoa (Basque Country) (7). To date, no other ampC genes have been described in Spain. We report the first plasmid-encoded ACC-1 β-lactamase, related to the chromosomally encoded AmpC-type β-lactamase of Hafnia alvei (5), isolated in Barcelona from an E. coli strain. The E. coli strain (U49G11) was isolated from the urine of a 55-year-old female with Hodgkin's disease treated with chemotherapy and allogeneic peripheral stem cell transplantation. During her illness, she presented with various infections, including oral candidiasis, human herpesvirus 1 infection, disseminated human herpesvirus 5 (cytomegalovirus) infection, urinary sepsis due to E. coli, and, 1 month later, a Klebsiella pneumoniae bacteremia of unknown origin. Several antimicrobial agents were administered, including meropenem, cefepime, and cefuroxime as β-lactams. Nevertheless, during the month prior to the isolation of the U49G11 strain, she did not receive antimicrobial therapy. U49G11 showed a particular β-lactam-resistant phenotype, which suggested the presence of an AmpC-type enzyme. Using the disk diffusion test according to NCCLS guidelines (6), this strain was resistant to ampicillin, cefazolin, cefuroxime, cefotaxime, ceftazidime, amoxicillin-clavulanate, kanamycin, tobramycin, tetracycline, and trimethoprim-sulfamethoxazole and was susceptible to cefoxitin, cefepime, aztreonam, imipenem, gentamicin, quinolones, chloramphenicol, and nitrofurantoine. The MICs of cefoxitin, cefotaxime, ceftazidime, and cefepime, determined by Etest (AB Biodisk, Solna, Sweden), were 8, >16, >32, and 0.75 mg/liter, respectively. When clavulanate was associated with these cephalosporins, no synergistic effect was observed. The resistance pattern was successfully transferred by conjugation (frequency of 10−6) on solid media using E. coli HB101 (Nalr) as the recipient strain (7). Transconjugants have the same resistance pattern (although MICs of cephalosporins for them are lower) as that of the donors except where trimethoprim-sulfamethoxazole is concerned. The analytical isoelectric focusing (7) performed on the donor and transconjugant strains showed a band with a pI of 7.7, which, together with the susceptibility to cefoxitin, suggested the presence of an ACC-type enzyme. A positive PCR was obtained by using the ACCMF and ACCMR primers, which amplify ACC-type enzymes (8). In order to sequence the gene, an additional PCR was performed using the ACC-1up (5′-TGC GTA AAA AAA TGC AGA A-3′) and ACC-1dn (5′-CTA CTT ATT CCC TTC CA-3′) primers. The deduced amino acid sequence of the obtained product showed 100% homology with the ACC-1 enzyme (1). ACC-1 was first described in Germany (1) and later in France during a multiresistant-K. pneumoniae outbreak in an intensive care unit following the admission of a patient transferred from Tunisia (5); it has also been shown to be present in E. coli and P. mirabilis (3). Studies at Sfax Hospital (Tunisia) reveled the ACC-1 enzyme in K. pneumoniae, P. mirabilis, and S. enterica serovars Livingstone and Mbandaka (4, 10). This is the first time that the AAC-1 enzyme has been reported in Spain, now the fourth country in the world where strains carrying this enzyme have been isolated. Three of these four countries (Spain, France, and Tunisia) are in the Mediterranean area. Such findings suggest that the ACC-1 enzyme is spreading in Europe, especially in the Mediterranean area.