Bands pI Research Articles

First-Principles Investigation of Phosphorus-Doped Graphitic Carbon Nitride as Anchoring Material for the Lithium-Sulfur Battery.

The utilization of lithium-sulfur battery is hindered by various challenges, including the "shuttle effect", limited sulfur utilization, and the sluggish conversion kinetics of lithium polysulfides (LiPSs). In the present work, a theoretical design for the viability of graphitic carbon nitride (g-C3N4) and phosphorus-doping graphitic carbon nitride substrates (P-g-C3N4) as promising host materials in a Li-S battery was conducted utilizing first-principles calculations. The PDOS shows that when the P atom is introduced, the 2p of the N atom is affected by the 2p orbital of the P atom, which increases the energy band of phosphorus-doping substrates. The energy bands of PC and Pi are 0.12 eV and 0.20 eV, respectively. When the lithium polysulfides are adsorbed on four substrates, the overall adsorption energy of PC is 48-77% higher than that of graphitic carbon nitride, in which the charge transfer of long-chain lithium polysulfides increase by more than 1.5-fold. It is found that there are powerful Li-N bonds between lithium polysulfides and P-g-C3N4 substrates. Compared with the graphitic carbon nitride monolayer, the anchoring effect of the LiPSs@P-g-C3N4 substrate is enhanced, which is beneficial for inhibiting the shuttle of high-order lithium polysulfides. Furthermore, the catalytic performance of the P-g-C3N4 substrate is assessed in terms of the S8 reduction pathway and the decomposition of Li2S; the decomposition energy barrier of the P-g-C3N4 substrate decrease by 10% to 18%. The calculated results show that P-g-C3N4 can promote the reduction of S8 molecules and Li-S bond cleavage within Li2S, thus improving the utilization of sulfur-active substances and the ability of rapid reaction kinetics. Therefore, the P-g-C3N4 substrates are a promising high-performance lithium-sulfur battery anchoring material.

Surface-surface contacted direct Z-scheme TiO2-BiVO4-PI heterostructure for enhanced photoelectrocatalytic degradation of bisphenols under solar driven

Bisphenols (BPs) are a series of widely used endocrine disruptors, which potentially harm the environment and human health. In this work, a novel Z-scheme TiO2-BiVO4-PI heterostructure was synthesized, characterized, and used for the simulated sunlight-driven photoelectrocatalytic degradation of BPs. Due to the existence of surface-surface contacted direct Z-scheme between BiVO4 and PI, holes were concentrated on the valence band of BiVO4 and electrons were concentrated on the conduction band of PI, resulting in a stronger redox activity. All six BPs exhibited appreciable degradation following the order of bisphenol A (BPA, 93.5%) > bisphenol B (BPB, 92.7%) > bisphenol AP (BPAP, 85.6%) > bisphenol F (BPF, 75.9%) > bisphenol AF (BPAF, 69.8%) > bisphenol S (BPS, 39.2%), within 120 min under the optimal condition. In the process of degradation, superoxide radicals (·O2−) and hydroxyl radicals (·OH) played dominant roles, and the intermediates of BPs degradation were mainly formed via the substituent shedding or C–C bond breaking of phenol ring, hydroxylation, and ring opening of phenol ring. The ECOSAR program was used to analyze the changes in the toxicity of the intermediates, and it was proved that the toxicity showed a decrease trend during the degradation process. This study provides a Z-scheme mechanism for TiO2-BiVO4-PI, which can degrade BPs and reduce their toxicity effectively.

Phase morphology and tribological properties of PI/UHMWPE blend composites

Polyimide/ultrahigh molecular weight polyethylene (PI/UHMWPE) blends with different mass ratio were produced. A ratio-image method was adopted by plotting the ratio of Raman peak intensity of a typical band of PI with that of UHMWPE as a function of position, Raman Mapping images with a relatively high accuracy and enhanced visualization were obtained. Two-phase distribution morphologies of the blends were studied using Raman Mapping. Effects of blend phase morphology and phase distribution on the tribological properties of blend composites were investigated. The results indicated that both the phase structures and their distribution are obviously different for the PI/UHMWPE blends with different mass ratios, which significantly influence the tribological properties of the blends. PI phase disperses in an island form in the UHMWPE matrix phase when the mass ratio of PI/UHMWPE is relatively small (10/90). The scattered PI dispersed phase effectively reduces the friction coefficient of the blend and improves the wear resistance. For the PI/UHMWPE blend composite with the mass ratio of 30/70, layered PI phase forms and distributes in the UHMWPE matrix phase, improving bearing capacity and wear resistance of the blends. Large continuous area structure and phase separation occurs in the blend when the mass ratio of PI/UHMWPE increases to 50/50, resulting in a significant reduction in the wear resistance of the blend composite.

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.

Vibrationally Mediated Photodissociation of Ethylene Cation by Reflectron Multimass Velocity Map Imaging

A new imaging technique, reflectron multimass velocity map ion imaging, is used to study the vibrationally mediated photodissociation dynamics in the ethylene cation. The cation ground electronic state is prepared in specific vibrational levels by two-photon resonant, three-photon ionization via vibronic bands of (pi, nf) Rydberg states in the vicinity of the ionization potential of ethylene, then photodissociated through the (B 2A(g)) excited state. We simultaneously record spatially resolved images of parent C2H4+ ions as well as photofragment C2H3+ and C2H2+ ions originating in dissociation from the vibronic excitations in two distinct bands, 7f 4(0)2 and 8f 0(0)0, at roughly the same total energy. By analyzing the images, we directly obtain the total translation energy distributions for the two dissociation channels and the branching between them. The results show that there exist differences for competitive dissociation pathways between H and H2 elimination from C2H4+ depending on the vibronic preparation used, i.e., on the vibrational excitation in the ground state of the cation prior to photodissociation. Our findings are discussed in terms of the possible influence of the torsional excitation on competition between direct dissociation, isomerization, and radiationless transitions through conical intersections among the numerous electronic states that participate in the dissociation.

Emergence of Multidrug-Resistant Salmonella enterica Serovar Typhi in Korea

A chloramphenicol-resistant strain of Salmonella enterica serovar Typhi was first noted in Korea in 1992, when a resistant isolate was detected in a returned traveler. Continued isolation of multidrug-resistant (MDR) strains thereafter in other settings prompted a retrospective analysis of laboratory records and phenotypic and genotypic analyses of 12 chloramphenicol-resistant isolates. Among these, one isolate was resistant only to chloramphenicol, and the other isolates were also resistant to ampicillin and co-trimoxazole. MDR was transferred by conjugation from 9 of the 11 isolates. PCR showed that all isolates had an incompatible group HI1 plasmid, and oriT was detected in 10 isolates, which included strains with an unsuccessful transfer of resistance. All of the ampicillin-resistant isolates had a beta-lactamase band of pI 5.4 and bla(TEM) alleles. A PCR amplicon from an isolate showed that the sequences were identical to those of bla(TEM-1), suggesting that all isolates had a TEM-1 beta-lactamase. All isolates had class 1 integrons: 10 isolates had integrons of ca. 1.2 kb with dhfr7 gene cassettes, and 1 isolate had an integron of ca. 2.3 kb with aacA4 and bla(OXA-1)-like gene cassettes. The pulsed-field gel electrophoresis patterns of 7 of 11 MDR isolates were identical and indistinguishable from those reported for isolates in India and Indonesia. In conclusion, some of the MDR strains in Korea are related to those in other Asian countries. Susceptibility testing became necessary for selection of antimicrobial agents for the optimal treatment of patients with the emergence of MDR Salmonella serovar Typhi in Korea.

Biochemical characterization of chromosomal cephalosporinases from isolates belonging to the Acinetobactet baumannii complex

Members of the Acinetobacter baumannii complex have emerged as some of the most important opportunistic pathogens within the hospital environment, being able to colonize and produce infections in most immunocompromised patients, especially in intensive care units (ICUs). Such infections are difficult to treat due to their multiple resistance to the antibiotics currently available for the treatment of nosocomial infections [1]. Various mechanisms of resistance to b-lactams have been identified in this genus, including b-lactamase production, alteration of penicillin-binding proteins, and reduced levels of penetration across the outer membrane. b-Lactamase production is one of the main mechanisms of resistance to b-lactams in Acinetobacter spp. [2,3]. Such enzymes can be either plasmid-encoded penicillinases (TEM and CARB type) or chromosomal cephalosporinases. The exact nature and character of these latter enzymes are not fully clarified for Acinetobacter spp. They belong to the group 1 b-lactamases of the classification of Bush et al [4], but several authors have found heterogeneity among the Acinetobacter cephalosporinases [5,6]. In the present work, the cephalosporinases produced by 125 strains belonging to the A. baumannii complex were characterized by determination of their pI values, b-lactamase inhibition profiles and substrate profiles. Clinical isolates (N1⁄4 125) belonging to the A. baumannii complex, obtained in the Hospital de la Princesa (Madrid, Spain) from January 1995 to December 1997, were studied. The isolates were identified by the API 20NE system (BioMerieux, Lyon, France) and growth at 44 8C [7]. Minimal inhibitory concentrations (MICs) and susceptibility percentages of 15 b-lactam agents were determined by an agar dilution method, as recommended by the National Committee for Clinical Laboratory Standards [8]. A. baumannii ATCC 19606, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains. b-Lactamase activity was detected in crude enzymatic extracts by the hydrolysis of nitrocefin (0.5 mg/mL). The pI values were determined by isoelectric focusing (IEF) (Multhiphor II Electrophoresis System; Amersham Pharmacia Biotech, Uppsala, Sweden), on wide-range ampholine (pH range 3.5–9.5) polyacrylamide gels; in some cases a narrow-range ampholine (pH 5.5–8.5) was used. The percentage of inhibition of the following inhibitors was studied at fixed concentrations for 43 enzymatic extracts: clavulanic acid (10 mM), sulbactam (20 mM), cloxacillin (4 mg/mL), aztreonam (1.148 mM), NaCl (140 mM) and EDTA (0.1 mM). This assay was performed as described by Papanicolaou et al [9] with several modifications. After incubating the extracts with nitrocefin solution, the changes in optical density at a wavelength of 492 nm were measured at 2, 5 and 10 min, and subsequently at 10-min intervals for 40 min. Duplicate determinations were performed for each extract. Sixteen crude enzymatic extracts were selected to study the substrate profile of the b-lactamases. Two methods were used. First, hydrolysis of the following b-lactams by the crude extracts was studied with a microbiological assay as described by Paton et al [10]: ampicillin, cefazolin, oxacillin, carbenicillin, cefuroxime, cefotaxime, ceftazidime, imipenem and meropenem. OXA-2 and TEM-1 were used as control enzymatic extracts. Staphylococcus aureus ATCC 25923 and E. coli ATCC 25922 were used as hydrolysis control strains depending on the antibiotic tested. Second, assay UV spectrophotometry was used to study the hydrolysis rate of cephaloridine, benzylpenicillin, cefotaxime, ceftazidime, imipenem, meropenem, carbenicillin and oxacillin. Duplicate determinations were performed for each enzymatic extract, with concentrations of either 100 mM or 1 mM. Hydrolysis rates were expressed as a percentage of hydrolysis with respect to cephaloridine. A high percentage of susceptibility was found to imipenem, meropenem and ampicillin–sulbactam (100%, 100% and 84%, respectively); however, low susceptibility was shown to most penicillins (piperacillin, 8%; ticarcillin, 18.4%), and cephalosporins (cefotaxime, 4.8%; ceftazidime, 11.2%; cefepime, 12.8%). Isoelectric focusing showed a unique band of pI> 8

Spectroscopy and photophysics of 1-phenylisatin and oxindole

The absorption and photoluminescence characteristics of 1-phenylisatin (PI) and oxindole (OI) were studied in different solvents at 300 K. In the basic solution a new absorption band system found for PI has been attributed to a structural change leading to formation of new species. Dual fluorescence for PI and OI in different solvents was explained as normal 1B 2u → 1A 1g transition and the other well-structured band as an anomalous band. This large Stokes shifted anomalous band of PI in water and alcohol was explained as fluorescence emission from a state formed due to solute–solvent relaxation producing a sort of critical inversion. Studies in different solvents including a β-cyclodextrin solution revealed that in suitable active medium PI has all the properties to be potential laser dye.

Isoelectric focusing of native urinary uromodulin (Tamm-Horsfall protein) shows no physicochemical differences between stone formers and non-stone formers.

Recent studies have suggested the occurrence of an abnormal form of uromodulin in stone formers which may be related to a reduced sialic acid content of the protein in these patients. Previous attempts to demonstrate these differences have required extensive sample processing prior to analysis, which may in itself alter the nature and behaviour of uromodulin. By employing a sensitive detection system of enhanced chemiluminescence on Western blots following isoelectric focusing we have been able to study the physicochemical properties of uromodulin in whole unprocessed urine from 50 idiopathic calcium stone formers and 15 non-stone formers. Uromodulin, desialated in vitro with either acid or neuraminidase was also analysed using the same system. All urine samples analysed from stone formers and non-stone formers showed a single band of pI 3.5 after isoelectric focusing. Desialated uromodulin showed a series of bands ranging from pI 4.0 to 5.1 reflecting different amounts of sialic acid removed. We conclude there are no charge-related differences in native uromodulin between stone formers and non-stone formers, in particular none relating to the sialic acid content of the protein.

Gibberellic acid stimulation of isoperoxidase from pedicel of grape

Soluble peroxidase activity from pedicels of cv. Thompson seedless grape was highly stimulated by post-bloom gibberellic acid (GA 3) applications to vines. This stimulation was dependent on the dose employed and increased with berry ripening. In control vines, i.e. non GA 3 treated, the level of peroxidase activity remained constant during berry ripening. Isoelectrofocusing (IEF) analysis of soluble peroxidases from pedicel extracts performed in a 3–10 pH range, showed the presence of a main band that corresponded to an acidic isoperoxidase of (pI 3.5) and the presence of a minor band of (pI 3.9). It is a well-known fact that GA 3 applications to cv. Thompson seedless grape beside producing berry enlargement cause berry-drop which has been associated with pedicel thickening. Here we found a positive correlation between pedicel thickness and peroxidase activity within the rates of GA 3 used. The involvement of the acidic isoperoxidases in pedicel thickening and its relation to berry-drop are discussed.

Isolation and immunobiochemical characterization of a major allergen (65 kDa) from Fusarium equiseti.

Fusarium equiseti is one of the most important species in the class Deuteromycetes (Fungi Imperfecti). For proper diagnosis and immunotherapy, isolation and characterization of allergens of F. equiseti are necessary. In the present study, culture filtrate (CF) extract of F. equiseti was resolved into 35-37 bands on isoelectric focusing pI (3-9) and SDS-PAGE (mol. wt. 10-100 kDa). Most of them were glycoproteins, as identified by PAS staining. F. equiseti CF revealed 15 allergenic proteins on immunoblot with an allergic serum pool. It was fractionated into nine fractions (I-IX) on a Superose-12 column by FPLC. Fraction IV (65 kDa) and fraction VI (25 kDa) were found to be highly allergenic by IgE ELISA. A 65-kDa protein was observed as a major allergen because it was recognized by most of the patient sera on immunoblot. After elution from SDS-PAGE gel, it gave two bands of pI 7.4 and 6.0. Inhibition in IgE-binding components of F. equiseti CF with CF extracts of F. solani and F. moniliforme by immunoprint inhibition assay indicated the allergenicity shared between the extracts of Fusarium species. Data suggested that the 65-kDa is the major allergen in the Fusarium species and can be used for the treatment of allergic patients.

Limited proteolysis and site-directed mutagenesis reveal the origin of microheterogeneity in Rhodotorula gracilis D-amino acid oxidase.

When analysed by isoelectric focusing, D-amino acid oxidase from the yeast Rhodotorula gracilis normally consists of three molecular isoforms (pI 7.8, 7.4 and 7.2, respectively) all with the same N-terminal sequence. However, only a single band of pI 7.8 is detected with the recombinant wild-type protein expressed in E. coli. To determine whether the molecular basis of this heterogeneity is due to proteolysed forms of the protein, we treated R. gracilis D-amino acid oxidase with various proteases. Limited proteolysis by chymotrypsin and thermolysin produced truncated and nicked monomeric holoenzymes containing two polypeptides of approximately 34 kDa (Met1-Leu312) and one of approximately 5 kDa (Ala319-Arg364 with chymotrypsin or Ala319-Ala362 with thermolysin). On the other hand, treatment with endoproteinase Glu-C gave a dimeric holoenzyme lacking the C-terminal SKL tripeptide. This cleavage of Glu365-Ser366 peptide bond caused the disappearance of the three isoelectric bands and a single homogeneous band (pI 7.2) appeared. To study this protein form, we used site-directed mutagenesis to produce a mutant form of R. gracilis D-amino acid oxidase lacking the SKL C-terminal tripeptide (which is the targeting sequence PTS1 for peroxisomal proteins). As expected, the SKL-deleted mutant gave a single band (pI 7.2) in isoelectric focusing. The three-band pattern of native yeast enzyme was generated by in vitro experiments using an equimolar mixture of the wild-type (pI 7.8) and the SKL-deleted recombinant (pI 7.2) DAAOs. The microheterogeneity of yeast DAAO thus stems from the association of two polypeptide chains differing in the C-terminal tripeptide, giving three different holoenzyme dimers.

Purification, properties and characterization of a high‐molecular‐mass b‐galactosidase isoenzyme from Thermusaquaticus YT‐I

An unusual high‐molecular‐mass b‐galactosidase isoenzyme from Thermus aquaticus YT‐1 was purified to homogeneity by chromatography using gel filtration (Ultrogel AcA 34), anion‐exchange (Mono Q) and gel filtration (Superose‐12). The molecular mass of the purified enzyme was > 700 kDa as estimated by gel filtration, and its subunit molecular mass was 59 ± 1 kDa as determined by SDS/PAGE. A single protein band of pI 4.9 was obtained by isoelectric‐focusing gel electrophoresis. The optimum tempera‐ture and pH for enzyme activity were 80 ° C and pH 5.5 respectively. The enzyme was stable over a wide pH range (pH 3‐12), and the thermostability of the enzyme was enhanced by CaCl2. The enzyme was significantly activated by alkali and alkaline‐earth‐metal salts. Whereas reducing agents enhanced b‐galactosidase activity, thiol‐binding agents drastically decreased the enzyme activity. The enzyme was strongly inhibited by the end products of the lactose hydrolysis reaction. The b‐galactosidase was specific for b‐D anomeric linkages, and the identity of the aglycone moiety also influenced enzyme activity dramatically. Oligosaccharide (OS) formation rates were directly related to lactose concentration. At a concentration of 19.4% (w/v) lactose, OS accounted for approx. 20′ of the total carbohydrates, and only two types of OS were observed. Moreover, once formed, OS were not rehydrolysed to monosaccharides, even during long residence times. OS synthesis was also observed with cellobiose and lactulose. Fructose, xylose, lyxose, rhamnose and arabinose were active as galactosyl acceptors, in contrast with glucose, ribose, fucose, tagatose and N‐acetyl‐glucosamine.

Immunobiochemical characterization of Brassica campestris pollen allergen.

Brassica campestris (BC), Eng. Mustard, is an important source of pollen allergen, responsible for type I hypersensitivity disorders. In the present study, BC pollen extract was characterized by TLIEF, SDS-PAGE and immunoprinting. The extract separated into 50 silver stained bands of pI 3-9 on isoelectric focusing whereas it resolved into 14 Coomassie blue stained protein bands of 14-100 kD on SDS-PAGE. Immunoblot analysis with individual patient sera detected four allergenic proteins of 90, 67, 60 and 14 kD. BC separated into 8 peaks (Bras 1-8) on DEAE Sephadex A-50 column. Bras 2 was found to be most potent by IgE specific ELISA, hence further fractionated on Sephadex G-200. A protein of 90 kD (Bras 2a) isolated by gel filtration was found to be most allergenic protein by ELISA inhibition. The findings shall be applicable in standardization of future batches of BC pollen extract to be used for allergy diagnosis and immunotherapy.

The effects of βbT‐lactams on the isoelectric focusing of βbT‐lactamases

A range of concentrations of ceftazidime (4–64 mg I‐1) was shown to cause no induction of the TEM‐1 and TEM‐5 β‐lactamases produced by Escherichia coli Nb. Increasing the concentration of ceftazidime in cultures of E. coli Nb caused a concomitant increase in the intensity of a satellite band of pI 5.2. The same increase in this satellite band was observed when ceftazidime was added to cell‐free β‐lactamase peparations from E. coli Nb and the separate addition of 11 different β‐lactams to TEM‐1 showed that each compound produced its own unique pattern of satellite bands. In addition, the mixing of ceftazidime with TEM‐1 and 13 other TEM‐derived β‐lactamases caused a similar satellite band to be observed but ceftazidime did not have the same effect on PSE or SHV β‐lactamases. Consequently, the addition of ceftazidime to a β‐lactamase preparation prior to isoelectric focusing (IEF) may help to verify if a particular β‐lactamase is TEM‐derived. Purification of the satellite bands by electrodialysis and their subsequent re‐focusing demonstrated that the ceftazidime‐induced satellite bands can revert to a protein which has a pI similar to the parent band, illustrating the possible reversibility and dynamic nature of β‐lactamase satellite bands on IEF. These results enable a better interpretation to be made of β‐lactamase satellite bands observed on IEF.

Purification of Cu, Zn-Superoxide Dismutase Isoenzymes from Fish Liver: Appearance of New Isoforms as a Consequence of Pollution

Liver cell-free extracts of fish (Mugil sp.) from polluted environments show new Cu,Zn-SOD isoenzymes when analyzed by polyacrylamide gel electrophoresis or isoelectrofocusing followed by in situ staining for SOD activity. The most active isoenzymes, with pI 6.1 and 5.1, were present both in control and problem samples while the isoenzymes of intermediate pI value showed significant differences. Fish from control areas showed three intermediate isoenzymes with pI 5.7, 5.5 and 5.4 (the last one quite faint) while polluted animals showed three bands of pI 5.9, 5.45 and 5.35, this last very intense. To further characterize their utility as biomarkers, Cu,Zn-SOD isoenzymes from polluted fish livers were purified to homogeneity. Five superoxide dismutase peaks were purified, named thereafter I (pI 6.1) to V (pI 5.1) respectively. Isoenzymes I and V displayed the highest specific activity. Upon incubation with moderate H2O2 concentrations, pure isoenzyme I yielded more acidic bands with pI 5.5, 5.45 and 5.35, this last being predominant. The pure isoenzyme V generated only a new band of pI 5.0. Concomitant with oxidation, the activity of peaks I and V was lost in a H2O2 concentration-dependent manner. The pattern of the new acidic bands generated upon the oxidixing treatment of isoenzyme I closely resembles that observed in crude extracts from polluted animals.

Characterization of penicillinase-producing strains ofNeisseria gonorrhoeae

Twenty-three penicillinase-producing strains of Neisseria gonorrhoeae were characterized. All strains showed hybridization with a TEM-1 probe and production of a TEM-1 type beta-lactamase (pI 5.4) on isoelectric focusing. Eight strains also showed a beta-lactamase band of pI 5.5. The penicillin MICs for the strains producing only TEM-1 were in the range 4-16 micrograms/ml; MICs of the strains with the additional pI 5.5 band were > or = 128 micrograms/ml for seven strains and 4 micrograms/ml for an arginine-hypoxanthine dependent strain. The association of the high MICs with the presence of the pI 5.5 band was statistically significant. The pI 5.5 band could represent a new beta-lactamase type or a TEM-1 mutant.

Purification and properties of a functional 47-kilodalton cytosolic factor required for NADPH-oxidase activation in bovine neutrophils

A cytosolic factor of 47 kDa required for activation of the NADPH oxidase, and referred to as p47, has been purified in its functional form from the cytosol of resting bovine neutrophils. The purification was monitored by the determination of the activating potency of p47 in a cell-free system of oxidase activation. The recovery was around 10% and the purification factor greater than 1000. P47 was phosphorylated in vitro by protein kinase A and protein kinase C. [ 32P] labeled p47 was resolved by isoelectric focusing into two major labeled bands of pI 7.0 and 8.5. Polyclonal antibodies were used to demonstrate that p47 is localized specifically in the cytosol of resting neutrophils, and that, upon activation of neutrophils, p47 is translocated from the cytosol to the membrane.

Glycogen phosphorylase isoenzymes from hepatoma 3924A and from a non-tumorigenic liver cell line. Comparison with the liver and brain enzymes.

Glycogen phosphorylase isoenzymes were isolated from normal rat liver, rat brain, the glycogen-poor Morris hepatoma (MH) 3924A, and the glycogen-rich non-tumorigenic liver cell line C1I. Electrophoretic and immunological characterization of the enzymes showed that tumour and C1I cells expressed a phosphorylase isoform similar to the brain type; the liver type was not detectable. All enzymes were obtained as dimers; the Mr of the subunits was 96,000 (liver), 93,000 (brain and MH 3924A) and 92,000 (C1I). Isoelectric focusing revealed a main band of pI 6.34 for liver phosphorylase a, pI 5.67 for the enzymes from MH 3924A and brain, and pI 5.68 for C1I phosphorylase. Partial kinetic characterization of the AMP-independent forms of the isoenzymes yielded Km values for glucose 1-phosphate of 3.5 +/- 0.5 mM (liver), 3.9 mM (brain), 1.9 +/- 0.3 mM (MH 3924A) and 2.5 +/- 0.5 mM (C1I); Km values for glycogen were 0.4 mM (liver) and 0.3 mM (MH 3924A and C1I), calculated as glucose equivalents. The AMP-independent phosphorylase was inhibited by glucose 6-phosphate (Glc6P) with Ki values of 0.32 +/- 0.03 mM (C1I), 0.50 +/- 0.04 mM (MH 3924A) and approximately 5 mM (brain). The inhibition could be abolished by 1 mM-AMP, indicating that AMP and Glc6P may partially compete for the same site on the protein. Liver phosphorylase a was not inhibited by up to 25 mM-Glc6P. In contrast with liver and brain isoenzymes, phosphorylase from the cell lines was not affected by NaF and Na2SO4. The data show that both the hepatocellular carcinoma and the non-malignant immortalized liver cells express a phosphorylase isoform different from the liver type. Furthermore, there is some evidence that the enzyme from MH 3924A and C1I cells is distinct from brain phosphorylase a, in spite of electrophoretic and immunological resemblance, and that this isoenzyme is subject to altered metabolic regulation.

Rapid detection and counting of single bacteria in a wide field using a photon-counting TV camera

Using Escherichia coli as a model bacterium, we tested a photon-counting method for enumeration of bacteria. This method is based on the principle that microscopic sized luminous particles in a wide field can be directly detected and counted using a photon-counting TV camera without the use of a microscope. E. coli cells were labeled with peroxidase and luminescence induced by adding a luminol-based reaction mixture. The number of luminous spots in the TV images was in good agreement with the number of bacterial colonies grown from labeled cells. The results show that our method provides a rapid and easy microbial counting system for such purposes as clinical diagnosis, microbial analysis in food, and environmental assessment.