Analytical Isoelectric Focusing Research Articles

Scalable Purification of Adeno-associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes

In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations. However, the conventional purification procedure with resin-based packed-bed chromatography suffers from a number of limitations, including variations in pressure, slow pore diffusion, and large bed volumes. Here we have employed disposable high-performance anion- and cation-exchange membrane adsorbers to effectively purify recombinant viruses. As a result of isoelectric focusing analysis, the isoelectric point of empty particles was found to be significantly higher than that of packaged virions. Therefore, AAV vector purification with the membrane adsorbers was successful and allowed higher levels of gene transfer in vivo without remarkable signs of toxicity or inflammation. Electron microscopy of the AAV vector stocks obtained revealed highly purified virions with as few as 0.8% empty particles. Furthermore, the membrane adsorbers enabled recovery of AAV vectors in the transduced culture supernatant. Also, the ion-exchange enrichment of retroviral vectors bearing the amphotropic envelope was successful. This rapid and scalable viral purification protocol using disposable membrane adsorbers is particularly promising for in vivo experimentation and clinical investigations.

Production of biologically active recombinant annexin B1 with enhanced stability via a tagging system

Annexin B1 is a novel Ca(2+)-dependent phospholipid-binding protein from metacestodes of Taenia solium and has been shown to have many potential biomedical applications. Although annexin B1 has been produced successfully in Escherichia coli, the purified protein has poor stability at room temperature, which has hindered our attempts to further study its structure-function relationship. To increase the stability of the protein, the construction and purification procedures were examined and changed to hopefully increase its effectiveness. In this study, we describe a new recombinant annexin B1 expressed with a hexahistidine tag fused to its N-terminal end, which was purified to homogeneity in two steps using immobilized metal affinity followed by size exclusion chromatography. The final yield was approximately 23 mg/L of bacterial culture. Isoelectric focusing and mass spectrometry analysis showed that the protein purified by this method was quite stable at room temperature, even greater than 3 days later. A series of functional tests indicated that the recombinant protein had high anticoagulant activity, and fluorescence-labeled annexin B1 could bind to the outer membranes of apoptotic mammalian cells and efficiently detect them in the early stages of apoptosis.

Tumor liberated protein from lung cancer and perspectives for immunotherapy

This review addresses peptide regions of human tumor liberated protein (TLP) complexes with antigenic activity. The review also deals with antibodies reacting with such proteins, to be used for diagnostic and clinical purposes. We identified peptide sequences of the TLP 100 kDa protein. Lung cancer cells were found positive with an immuno-histochemical test, by peroxidase-anti-peroxidase, using a specific antiserum. This test turned out negative in the presence of the synthesized peptide (blocking test). Several tumors were used to obtain antigens by various purification procedures (cell homogenization, ultracentrifugation at 100,000g, gel filtration and chromatography, preparative and analytical isoelectric focusing, IEF). The molecular weight on SDS-PAGE of an immunogenic peak detected on IEF was determined to be a value of 214,000 Da, with monomers of 54,000 Da. In vivo TLP yields delayed hypersensitivity reactions in both autologous and homologous hosts, and does not seem to have any nonspecific mytogenic activity, but does have mytogenic-specific activity since lymphocyte blastogenesis (TLP-induced in pre-sensitized patients), in the case of intradermal inoculation, could mean its ability to sensitize a lymphocytic clone.

A Patient with a Previous Diagnosis of Hemoglobin S/C Disease with an Unusually Severe Disease Course

A 17-year-old African American male presented to the hematology clinic for treatment of sickle cell disease (SCD).1 He had received the diagnosis of hemoglobin (Hb) S/C disease at an outside hospital at the age of 6 years; the diagnosis was confirmed in house at 11 years of age. His disease course had been severe, with frequent pain crises of increasing intensity and 2 episodes of acute chest syndrome requiring hospitalization and multiple blood transfusions. The patient’s physical examination was unremarkable: blood pressure, 120/64 mmHg; pulse, 83 beats/min; temperature, 36.9 °C. Laboratory results were as follows: white blood cell count, 11.8 × 109/L [reference interval (RI), 3.9–10.3 × 109/L]; Hb, 6.39 mmol/L (RI, 8.68–10.8 mmol/L); packed cell volume, 0.28 (RI, 0.42–0.50); red blood cell count, 3.59 × 1012/L (RI, 4.5–6.0 × 1012/L); platelet count, 417 × 109/L (RI, 135–370 × 109/L); mean corpuscular volume, 78 fL (RI, 83–102 fL); mean corpuscular Hb, 28.7 pg (RI, 27–31 pg); mean corpuscular Hb count, 368 g/L (RI, 320–340 g/L); red cell distribution width, 17.3% (RI, 11.5%–14.5%); and absolute reticulocyte count, 0.115 (RI, 0.02–0.10). A peripheral blood smear showed scattered target and sickle cells, rare nucleated red cells, and mild anisopoikilocytosis. Results for the qualitative sickle cell solubility test were positive. Considering the severe disease course, Hb analysis by HPLC and isoelectric focusing (IEF) was ordered (Fig. 1⇓ ). Figure 1. IEF and HPLC analysis of Hbs in the patient’s sample. (A), The migration pattern of the standard and patient’s sample by IEF. Lane 1 shows the patient’s sample. Lane 2 shows the Hb standard containing Hb C, Hb S, Hb F, and Hb A. (B), Elution chromatographs of standard and patient Hb species by HPLC with the Bio-Rad Variant II System (β-thalassemia …

A possible mechanism underlying the ceramide deficiency in atopic dermatitis: Expression of a deacylase enzyme that cleaves the N-acyl linkage of sphingomyelin and glucosylceramide

A deficiency of ordinary ceramides in the stratum corneum is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). We have proposed that the mechanism underlying that deficiency involves a novel sphingolipid metabolizing enzyme, termed sphingomyelin (SM) glucosylceramide (GCer) deacylase, which hydrolyzes SM or GCer at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) or glucosylsphingosine (GSP) instead of ceramide, leading to the ceramide deficiency in the AD skin. The enzymic characteristics observed showed a pH dependency of catalytic activity with a peak at pH 5.0 and a molecular weight of 40,000. Analytical isoelectric focusing (IEF) chromatography demonstrated that the pI values of SM deacylase, GlcCDase, SMase and ceramidase were 4.2, 7.4, 7.0 and 5.7, respectively. Those enzymic characteristics of SM-GCer deacylase are completely distinct from ceramidase as well as the other known deacylases. Our enzymic measurements demonstrated that SM-GCer deacylase activity is enhanced more than 5-fold in involved stratum corneum, more than 3-fold in uninvolved stratum corneum and approximately 3-fold in the involved epidermis from patients with AD compared with healthy controls. Our findings suggest that the novel enzyme, SM-GCer deacylase, is expressed in situ at significant levels in the epidermis of AD patients. This results in the production of SPC and GSP, instead of ceramides, which leads in turn to the ceramide deficiency seen in the stratum corneum of those patients. It is likely that the biogenesis of SM-GCer deacylase may be critical to the pathogenesis of AD.

Purification of an infection-related acidic peroxidase from pearl millet seedlings

Higher basal level of peroxidase activity was observed in highly resistant pearl millet cultivar IP 18292. Upon inoculation with downy mildew pathogen, Sclerospora graminicola, up to 60% increase in peroxidase activity was observed in highly resistant seedlings over the period of time. Iso-electric focusing analysis revealed that, two acidic isozymes of peroxidase with the pI of 5.9 and 5.1 present only in IP 18292 pearl millet seedlings. Upon inoculation with downy mildew pathogen, accumulation of these to isozymes was increased. These results indicated the possible involvement of acidic peroxidase in pearl millet defense. To study the nature of the acidic peroxidase which increases upon inoculation was purified from seedlings of highly resistant pearl millet cultivar using DEAE–Sepharose and Sephadex G-100 columns. The purified enzyme has a molecular weight of 21.8 kDa on SDS–PAGE and has a pI of 5.1. The optimum pH for maximum peroxidase activity was found to be at pH 7.0 and was resistant to high temperature (27–60 °C). The K m for H 2O 2 and V max of the enzyme reaction were 5.26 mM and 322.58 units, respectively. Purified peroxidase enzyme was found to be CaCl 2 dependent and both MgCl 2 and ZnCl 2 showed inhibitory effect on enzyme activity. Sodium azide and EDTA inhibited the enzyme and EGTA found to be specific inhibitor of peroxidase.

Characterization of vitellin protein in the twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae)

In mites, vitellogenin synthesis, regulation and uptake by the oocytes as vitellin remain practically unknown. Although a partial sequence of the gene is now available, no previous studies have been conducted that describe the native vitellin protein in mites. The objective of this study was to characterize vitellin in the twospotted spider mite, Tetranychus urticae. The native twospotted spider mite vitellin migrated as a single major band with a molecular weight of 476 ± 14.5 kDa as compared to 590 ± 25.5 kDa for vitellin from the American dog tick, Dermacentor variabilis. However, isoelectric focusing analysis of native spider mite vitellin showed five bands with p I values slightly acidic to neutral (pH 5.8, 6.2, 6.7, 7.0 and 7.2), as is the case for insect and tick vitellins. Reducing conditions (SDS-PAGE) also revealed multiple subunits ranging from 290.9 to 3.6 kDa and was similar to that found in D. variabilis. Spider mite vitellin weakly bound lipids and carbohydrates compared to the tick. Unlike D. variabilis, the spider mite egg yolk protein does not bind heme. The significance of non-heme binding in mites is discussed.

Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA

The emergence of bla(KPC)-containing Klebsiella pneumoniae (KPC-Kp) isolates is attracting significant attention. Outbreaks in the Eastern USA have created serious treatment and infection control problems. A comparative multi-institutional analysis of these strains has not yet been performed. We analysed 42 KPC-Kp recovered during 2006-07 from five institutions located in the Eastern USA. Antimicrobial susceptibility tests, analytical isoelectric focusing (aIEF), PCR and sequencing of bla genes, PFGE and rep-PCR were performed. Results By in vitro testing, KPC-Kp isolates were highly resistant to all non-carbapenem beta-lactams (MIC(90)s >or= 128 mg/L). Among carbapenems, MIC(50/90)s were 4/64 mg/L for imipenem and meropenem, 4/32 mg/L for doripenem and 8/128 for ertapenem. Combinations of clavulanate or tazobactam with a carbapenem or cefepime did not significantly lower the MIC values. Genetic analysis revealed that the isolates possessed the following bla genes: bla(KPC-2) (59.5%), bla(KPC-3) (40.5%), bla(TEM-1) (90.5%), bla(SHV-11) (95.2%) and bla(SHV-12) (50.0%). aIEF of crude beta-lactamase extracts from these strains supported our findings, showing beta-lactamases at pIs of 5.4, 7.6 and 8.2. The mean number of beta-lactamases was 3.5 (range 3-5). PFGE demonstrated that 32 (76.2%) isolates were clonally related (type A). Type A KPC-Kp isolates (20 bla(KPC-2) and 12 bla(KPC-3)) were detected in each of the five institutions. rep-PCR showed patterns consistent with PFGE. We demonstrated the complex beta-lactamase background of KPC-Kp isolates that are emerging in multiple centres in the Eastern USA. The prevalence of a single dominant clone suggests that interstate transmission has occurred.

A Comparison of IEF and MSMS for Clinical Hemoglobinopathy Screening in 40,000 Newborns

Currently, newborn haemoglobinopathy screening is carried out using HPLC or isoelectric focusing (IEF). We have previously described a rapid and specific electrospray mass spectrometry–mass spectrometry (MSMS) technique, using multiple reaction monitoring (MRM) based peptide analysis, for simultaneous detection of the clinically significant haemoglobinpathies; haemoglobin (Hb)S, HbC, HbE, HbDPunjab and HbOArab. Here we report the results of a comparison of 40,000 newborn blood spots screened by both IEF and MSMS.For both IEF and MSMS analysis, blood spots (3.2mm) were punched into separate 96 well plates. IEF was performed using the Resolve haemoglobin test kit (PerkinElmer Life Sciences, Waltham, USA) and Isoscan imaging system. For MSMS analysis, the blood spots were digested for 30min at 37°C with a trypsin reagent, and diluted in mobile phase (acetonitrile: water, 50:50, with 0.025% formic acid. Sample, 2μl, was injected directly into the mobile phase (flow rate 80μl/min) and analysed, in positive ion mode, using a Sciex API4000 (Applied Biosystems, Warrington, UK). Specific MRM transitions for HbS, HbC, HbE, HbDPunjab, HbOArab, normal beta, alpha, gamma and delta chains were acquired; total acquisition time per sample was 60 sec. This enabled identification of sickling disorders and thalassaemia major, as well as assessment of transfusion state and potential identification of HbLepore and HbBarts.40,000 blood spot samples for routine newborn haemoglobinopathy screening were analysed in parallel. HbS was detected in 199 samples; 8 were HbS/HbF only and 3 HbSC. HbC was detected in 39 samples, HbDPunjab in 52, HbE in 48. No HbOArab or HbLepore mutations were detected by either method. There have been no discrepancies between the analytical techniques. Using MSMS, mutation positive samples can be re-run in product ion scan mode to provide peptide sequence and hence unequivocal confirmation of the haemoglobin variant. In addition, 5,000 samples were analysed on a Sciex API4000 Q trap; using the information dependent acquisition facility provided “real time” peptide sequencing thus removing the requirement for re-injection.Sample preparation is very quick and simple for both methods, but the consumable costs associated with the MSMS technique are <10% of those for IEF. The capital cost of MSMS can be offset by high throughput and/or integration with current inherited metabolic disease screening by MSMS. The specificity of the MSMS analysis implies that haemoglobinopathy detection can be limited to specified conditions, based on agreed screening policy, and can eliminate the need for costly and time consuming second line testing.This study demonstrates that newborn haemoglobinopathy screening can be carried out rapidly, easily, and cost effectively using MSMS technology. It enables rationalisation of technology platforms in newborn screening by consolidating screening for haemoglobinopathies and inherited metabolic diseases onto MSMS.

Spread of CTX-M-type ESßLs in isolates of E. coli from long-term care and rehabilitation facilities in Northern Italy

During the period March 2003 – May 2004 at the Laboratory of Clinical Microbiology “Redaelli” LTCRF in Milan, Italy, a total of 529 E. coli, obtained from inpatients of 3 different Long Term Care Rehabilitation Facilities (LTCRFs) in Northern Italy, were processed and 77 ESßLs producers (14.5%) were identified by Vitek System. The results were confirmed by double-disk synergy test with tazobactam (TZP). 61/77 isolates were characterized by higher levels of resistance to cefotaxime (CTX) than to ceftazidime (CAZ). (ß-lactamase production was investigated by analytical isoelectric focusing (IEF) coupled with a bioassay and showed multiple (ß-lactamase bands including one enzyme with pI 8.4 that, in a bioassay, was more active on CTX,ATM than on CAZ. The presence of (ß-lactamase genes was investigated by colony blot hybridization and by PCR amplification of blaTEM, blaSHV and blaCTX-M alleles. 43/61 isolates produced both TEM-1 and CTX-M-type enzymes, 14/61 expressed only CTX-M-type while in 4 cases were found blaCTX-M, blaTEM and blaSHV genes.The remainders (16/77), characterized by high levels of resistance to both CTX and CAZ, produced TEM-1 and SHV-5 enzymes (1/16) and TEM type ESßLs (15/16). Conjugation experiments, performed in liquid medium, confermed that the ESßLs determinants were transferable. Pulsed-field gel electrophoresis profiles of genomic DNA, digested with NotI, were analysed and revealed clonal heterogeneity. Our work confirms the emergence of CTX-M-type enzymes and their spread in Northern Italy also in longterm care and rehabilitation facilities that may be an important reservoir of ES?L producing E. coli.

Separation of protein charge variants with induced pH gradients using anion exchange chromatographic columns

This work concerns the chromatographic separation of protein charge variants using pH gradients generated by step changes in buffer composition with weak base anion exchange columns. A local equilibrium model is first developed to describe pH transitions occurring in the column using buffers comprising neutral, zwitterionic or positively charged species. Model predictions, based solely on the resins' titration curves and obtained with the method of characteristics, show, in excellent agreement with experiments, that induced pH gradients of varying durations and shapes can be obtained with a broad range of buffer systems including Tris, Bis-Tris propane, histidine, and their mixtures and ethanolamine. The separation of protein charge variants is then demonstrated for bovine apo-transferrin and for a monoclonal antibody. The resolution of the charge variants present in these proteins, demonstrated via isoelectric focusing analyses, is obtained for conditions amenable to scale-up for preparative purposes; that is larger particle sizes (90 mum), higher flow rates (100-600 cm/h), and higher protein loads (2-5 mg/mL). Because the approach requires only step changes in buffer composition and commonly available, unretained buffers species, practical implementation is straightforward. The focusing effect of the induced pH gradient results in relatively sharp peaks and substantial resolution even for these conditions.

Neonatal screening for haemoglobin variants using filter paper-dried blood specimens

Neonatal screening for haemoglobinopathies utilizing cord blood samples is well established, although it has a high miss rate and has the inherent problem of possible misdiagnosis from maternal contamination of the sample. The use of dried Guthrie card samples which are taken at six days of age avoids these problems and has the advantage of using an established system of sample collection. Controversy exists as to the method of choice for analysis of dried samples, this study of 2406 samples found that Iso-electric focusing (IEF) analysis of dried specimens gives excellent correlation when compared with cellulose acetate/citrate agar electrophoresis of liquid cord blood samples. The IEF results were clear and relatively simple to interpret even when the samples had been stored at room temperature for 4 weeks. The commercial enzyme linked immunosorbent assay (ELISA) screening test JOSHUA reliably determines the presence or absence of haemoglobin S in dried specimens. It could therefore be used as a relatively cheap and simple method for the confirmation of sickle cell trait in neonatal screening programmes based on dried specimens.

Increase in -lactam-resistant Proteus mirabilis strains due to CTX-M- and CMY-type as well as new VEB- and inhibitor-resistant TEM-type -lactamases

The aim of this study was to characterize the different inhibitor-resistant TEM beta-lactamases, extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC beta-lactamases implicated in beta-lactam resistance in Proteus mirabilis, which has increased over recent years. From February 2000 to December 2005, 1423 clinical isolates of P. mirabilis were collected. The AmpC phenotype was checked by means of a double-disc synergy test using cloxacillin as an inhibitor of AmpC enzymes. The production of ESBL was assessed by the double-disc synergy method and by Etest ESBL. Analytical isoelectric focusing, determination of kinetic constants, conjugation, PCR and a sequencing strategy were used to characterize the enzymes. The possible relationships between isolates were analysed by PFGE. Twenty-five of 1423 isolates were found to display intermediate or full resistance to co-amoxiclav, cefotaxime or ceftazidime. Seventeen isolates had reduced susceptibility to co-amoxiclav; of these, seven produced TEM-110, eight produced the new TEM-159, one the new TEM-160 and one TEM-1. Five isolates producing TEM-110, TEM-159 or TEM-160 enzymes shared the same PFGE profile. Three isolates produced an ESBL, CTX-M-1, CTX-M-32 and the new variant, VEB-4. Finally, five isolates with an AmpC phenotype produced CMY-2, two with the same PFGE profile. Our data emphasize the diversity of beta-lactamases found in this species.

High-level carbapenem resistance in a Citrobacter freundii clinical isolate is due to a combination of KPC-2 production and decreased porin expression

An imipenem-resistant isolate of Citrobacter freundii ZJ163 (MIC 256 microg ml(-1)) isolated from a Chinese hospital was investigated. The C. freundii ZJ163 isolate exhibited high-level resistance to carbapenems, penicillins, cephalosporins, cefoxitin, aztreonam, quinolones and aminoglycosides. Isoelectric focusing (IEF) demonstrated three beta-lactamases with pIs of 5.4 (TEM-1), 6.7 (KPC-2) and 7.9 (CTX-M-14). Two different transconjugants (types A and B) were obtained by conjugation studies. The type A transconjugant exhibited reduced susceptibility or resistance to penicillins, cephalosporins and aztreonam, but was susceptible to carbapenems, quinolones and aminoglycosides. The antimicrobial susceptibility patterns of the type B transconjugant were similar to that of type A, except for its significantly reduced carbapenem susceptibility (imipenem MIC 2 microg ml(-1)). IEF, specific PCRs and DNA sequence analysis indicated that the type A transconjugant produced CTX-M-14 beta-lactamase with a pI of 7.9, that the type B transconjugant produced KPC-2 beta-lactamase with a pI of 6.7 and that the beta-lactamase with a pI of 5.4 was TEM-1. PCR analysis and sequencing confirmed the presence of the ampC gene in the chromosomal DNA from C. freundii ZJ163, although no activity of AmpC beta-lactamase was detected by IEF. Urea/SDS-PAGE analysis of outer-membrane proteins revealed that the levels of the 41 and 38 kDa porins were decreased in C. freundii ZJ163. It was concluded that production of KPC-2 combined with decreased expression of porins contributes to high-level resistance to carbapenems in C. freundii ZJ163.

Characterization of NAD-dependent malate dehydrogenases from spinach leaves

Spinach leaves were used to extract isoforms of NAD-dependent malate dehydrogenase (NAD-MDH) (EC 1.1.1.37), either soluble or bound to microsomal, plasma, or chloroplast envelope membranes. All fractions were subjected to isoelectric focusing analysis, which showed that purified chloroplast envelopes contain an NAD-MDH isoform tightly bound to the membranes, since treatment with 0.5 or 1% Triton X-100 was not able to release the enzyme from the envelopes. In contrast, plasma membranes released an isoform with a pI of 3.5 following treatment with 0.5% Triton X-100. The most abundant soluble leaf isoform had a pI of 9, while the chloroplast stroma contained an isoform with a pI of 5.3. Kinetic analysis of oxaloacetate (OAA)-dependent NADH oxidation in different fractions gave different Km values for both substrates, the envelope- and plasma membrane-bound NAD-MDH exhibiting the highest affinities for OAA. Leaf plasma membrane-bound MDH exhibited a high capacity for both reaction directions (malate oxidation and OAA reduction), while the two chloroplast isoforms (stromal and envelope-bound) preferentially reduced OAA. Our results indicate that the chloroplast envelope contains a specifically attached NAD-MDH isoform that could provide direct coupling between chloroplast and cytosol adenylate pools.

DJ-1 Decreases Bax Expression through Repressing p53 Transcriptional Activity

DJ-1, originally identified as an oncogene product, is a protein with various functions in cellular transformation, oxidative stress response, and transcriptional regulation. Although previous studies suggest that DJ-1 is cytoprotective, the mechanism by which DJ-1 exerts its survival functions remains largely unknown. Here we show that DJ-1 exerts its cytoprotection through inhibiting p53-Bax-caspase pathway. DJ-1 interacts with p53 in vitro and in vivo. Overexpression of DJ-1 decreases the expression of Bax and inhibits caspase activation, whereas knockdown of DJ-1 increases Bax protein levels and accelerates caspase-3 activation and cell death induced by UV exposure. Our data provide evidence that the protective effects of DJ-1 on apoptosis are associated with its ability of decreasing Bax level through inhibiting p53 transcriptional activity.

Screening Using Serum Percentage of Carbohydrate-Deficient Transferrin for Congenital Disorders of Glycosylation in Children with Suspected Metabolic Disease

Diagnoses of congenital disorders of glycosylation (CDG) are based on clinical suspicion and analysis of transferrin (Tf) isoforms. Here we present our experience of CDG screening in children with a suspected metabolic disease by determination of serum percentage of carbohydrate-deficient transferrin (%CDT) in tandem with isoelectric focusing (IEF) analysis of Tf and alpha(1)-antitrypsin (alpha(1)-AT). We performed approximately 8000 serum %CDT determinations using %CDT turbidimetric immunoassay (TIA). In selected samples, IEF analysis of Tf and alpha(1)-AT was carried out on an agarose gel (pH 4-8) using an electrophoresis unit. The isoforms were detected by Western blotting and visualized by color development. We performed neuraminidase digestion of serum to detect polymorphic variants of Tf. We established a cutoff value for serum %CDT of 2.5% in our pediatric population. Sixty-five patients showed consistently high values of serum %CDT. In accordance with Tf and alpha(1)-AT IEF profiles, enzyme assays, and mutation analysis, we made the following diagnoses: 23 CDG-Ia, 1 CDG-Ib, and 1 conserved oligomeric Golgi 1 (COG-1) deficiency. In addition, we identified 13 CDG-Ix non Ia, non-Ib; 3 CDG-Ix; and 9 CDG-IIx cases, albeit requiring further characterization; 9 patients with a secondary cause of hypoglycosylation and 6 with a polymorphic Tf variant were also detected. The combined use of CDT immunoassay with IEF of Tf and alpha(1)-AT is a useful 1st-line screening tool for identifying CDG patients with an N-glycosylation defect. Additional molecular investigations must of course be carried out to determine the specific genetic disease.

First Outbreak of Multidrug-ResistantKlebsiella pneumoniaeProducing both SHV-12-Type Extended-Spectrum β-Lactamase and DHA-1-Type AmpC β-Lactamase at a Korean Hospital

PurposeCoexistence of different classes of β-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC β-lactamase and an extended-spectrum β-lactamase (ESBL) in a university hospital.Materials and MethodsOver a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify blaSHV and blaDHA, and molecular typing by pulsed-field gel electrophoresis (PFGE).ResultsAll 11 isolates were highly resistant (MIC, ≥128 µg/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, ≤2 µg/ml) to imipenem. The blaSHV-12 and blaDHA-1 genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested.ConclusionThis is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 β-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.

Plasmid-Mediated Carbapenem-Hydrolyzing β-Lactamase KPC-2 in Klebsiella pneumoniae Isolate from Greece

The emergence and dissemination of Enterobacteriaceae isolates harboring carbapenemases in various geographic regions represent a significant threat to the management of nosocomial infections. Carbapenem-hydrolyzing β-lactamases can be metallo β-lactamases, expanded-spectrum oxacillinases, or Ambler class A enzymes (8, 9). The class A KPC β-lactamases hydrolyze all β-lactams except cephamycins. They are increasingly reported in data from studies of isolates of enterobacterial species (mostly Klebsiella pneumoniae species) collected in the United States, especially in New York City hospitals (1, 2, 4, 9, 12). Outside of the United States, single KPC-producing isolates containing members of the Enterobacteriaceae or containing Pseudomonas aeruginosa strains have been reported from France, China, and Colombia, but an epidemic situation has also been reported from Israel (5-7, 10, 11). We report here a K. pneumoniae isolate from Greece that produced β-lactamase KPC-2. On 17 July 2007, a 22-year-old woman was hospitalized in the intensive care unit of the hospital of Heraklion, Greece, for severe cranial trauma with coma (Glasgow coma scale score, 7) and multiple cranial and costal fractures subsequent to a traffic accident. On 10 August, she was initially transferred to the orthopedic ward and then to the intensive care unit of the hospital of Meaux, France. The day of her admission, bacteriological screening and wound samples revealed the presence of panresistant Acinetobacter baumannii (susceptible only to colistin) and of K. pneumoniae GR, which was resistant to all antibiotics except gentamicin according to disk diffusion susceptibility testing results (3). The patient was treated locally with antiseptics and fortunately did not experience a systemic infection with K. pneumoniae requiring antibiotic treatment. No other A. baumannii or K. pneumoniae isolates with similar antibiotic resistance patterns were recovered from the hospital during this same period of time. The MICs of β-lactams tested by the Etest method (AB BIODISK, Solna, Sweden) and interpreted according to CLSI standards (3) for K. pneumoniae GR showed values for expanded-spectrum cephalosporins and carbapenems only slightly modified after the addition of clavulanic acid (Table ​(Table1).1). A crude β-lactamase extract of that isolate showed significant carbapenem hydrolyzing activity, as measured spectrophotometrically (0.2, 0.06, and 0.05 μmol of imipenem, meropenem, and ertapenem/min/mg of total protein, respectively) (6, 8). TABLE 1. MICs of β-lactams for K. pneumoniae GR, its E. coli transconjugant TcGR, and an E. coli recipient strain Plasmid analysis detected a ca. 75-kb self-conjugative plasmid that conferred a β-lactam resistance pattern for Escherichia coli transconjugants that was consistent with the presence of a slightly clavulanic acid-inhibited carbapenemase (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. PCR experiments using primers for detection of Ambler class A, class D, and class B β-lactamase genes (6, 8) followed by sequencing identified β-lactamase genes coding for carbapenemase KPC-2 (pI 6.8) and the narrow-spectrum TEM-1 (pI 5.4). Then, using a series of successive PCR primers, the 2.8-kb sequences surrounding the blaKPC-2 gene were found to be identical to those surrounding the same blaKPC-2 gene in a K. pneumoniae isolate from New York (6). Up to now, carbapenemases of the KPC type seemed to be limited to Israel in this Mediterranean region. This is the first evidence of the identification of carbapenemases of the KPC type in Greece, a country located near Israel. It remains to be determined to what extent the spread of KPC-type enzymes contributes to the high level of carbapenem resistance that until now had been thought to occur only in the presence of class B carbapenemases (VIM-type enzymes).

Ethanol dependence of α1‐antitrypsin C‐terminal Lys truncation mediated by basic carboxypeptidases

BACKGROUND: Patients with hereditary emphysema are treated with α1‐antitrypsin (α1‐proteinase inhibitor [A1PI]) concentrates. High‐resolution isoelectric focusing (IEF) analysis of A1PI shows that commercial A1PI products have different glycoisoform band patterns predominantly caused by varying degrees of C‐terminal Lys truncation at position 394 from the A1PI molecule. Basic carboxypeptidases (CPs) are a group of enzymes that specifically cleave C‐terminal basic amino acids (Arg or Lys) from peptides and proteins.STUDY DESIGN AND METHODS: In this study, whether A1PI is a substrate for basic CPs was investigated. CPN and CPU, two CPs present in plasma, and CPM, a GPI‐anchored membrane protein highly expressed in lung tissues, were included.RESULTS: Basic CPs are able to mediate the C‐terminal Lys truncation of A1PI although with a very low efficiency. However, presence of ethanol, for example, during Cohn fractionation, renders A1PI highly susceptible to cleavage by CP with the extent of Lys truncation depending on the ethanol concentration. This ethanol concentration dependence elegantly explains the varying amounts of des‐Lys A1PI present in commercial preparations purified from different Cohn fractions.CONCLUSIONS: The cause of C‐terminal truncation of A1PI present in products used for augmentation therapy has been identified, and it has been shown that A1PI becomes a substrate for CPs, specifically CPN, because of the presence of ethanol during Cohn fractionation.