Unfolding and refolding properties of S pili on extraintestinal pathogenic Escherichia coli

S pili are members of the chaperone-usher-pathway-assembled pili family that are predominantly associated with neonatal meningitis (S_II) and believed to play a role in ascending urinary tract infections (S_I). We used force-measuring optical tweezers to characterize the intrinsic biomechanical properties and kinetics of S_II and S_I pili. Under steady-state conditions, a sequential unfolding of the layers in the helix-like rod occurred at somewhat different forces, 26 pN for S_II pili and 21 pN for S_I pili, and there was an apparent difference in the kinetics, 1.3 and 8.8 Hz. Tests with bacteria defective in a newly recognized sfa gene (sfaX _II) indicated that absence of the sfaX _II gene weakens the interactions of the fimbrium slightly and decreases the kinetics. Data of S_I are compared with those of previously assessed pili primary associated with urinary tract infections, the P and type 1 pili. S pili have weaker layer-to-layer bonds than both P and type 1 pili, 21, 28 and 30 pN, respectively. In addition, the S pili kinetics are ~10 times faster than the kinetics of P pili and ~550 times faster than the kinetics of type 1 pili. Our results also show that the biomechanical properties of pili expressed ectopically from a plasmid in a laboratory strain (HB101) and pili expressed from the chromosome of a clinical isolate (IHE3034) are identical. Moreover, we demonstrate that it is possible to distinguish, by analyzing force-extension data, the different types of pili expressed by an individual cell of a clinical bacterial isolate.     

Characterization of small ampicillin resistance plasmids (Rsc) originating from the mutant antibiotic resistance factor R1drd-19B2.

Summary Four plasmids Rsc10–13 ranging in size from 5.1×10⁶ to 13.4×10⁶ Dalton have been isolated from a strain carrying the copy mutant R1drd-19B2 of the antibiotic resistance factor R1. The Rsc plasmids have been cloned by transformation in Escherichia coli C. They determine high level resistance to ampicillin and occur in the cell in multiple copies. Their copy number and stability in the bacterial cell depend on the plasmid and the host strain.Physical maps of these plasmids have been constructed by cleavage with restriction endonucleases HincII, EcoRI, HindIII, BamI and SmaI. The pattern of the cleavage fragments have been compared with the parent plasmid R1drd-19B2 and with a R1 deletion mutant, R1drd-16, which has lost the ampicillin resistance. For Rsc11 and Rsc10 the data indicate, that both plasmids derive from a continuous stretch of the R1drd-19B2 DNA extending from the ampicillin transposon (TnA) to the replication site of the R1 factor. The plasmids Rsc12 and 13 have lost a DNA sequence between TnA and the replication site of R1. They may be formed by translocation of TnA to different autonomously replicating fragments of R1drd-19B2 including the replication origin or by deletion of DNA sequences from Rsc10 and Rsc11.     

Restriction map of the antibiotic resistance plasmid R1drd-19 and its derivatives pKN102 (R1drd-19B2) and R1drd-16 for the enzymes BamHI, HindIII, EcoRI and SalI.

Summary The conjugative R plasmid R1drd-19, mediating antibiotic resistance to ampicillin (Ap), chloramphenicol (Cm), kanamycin (Km), streptomycin (Sm) and sulfonamides (Su) was mapped using the restriction endonucleases BamHI, HindIII, EcoRI and SalI. BamHI generates 5 fragments (A-E) with molecular weights between 46×10⁶ dalton (representing mainly the RTF) and 0.25×10⁶ dalton, and HindIII 8 (A-H) between 42×10⁶ dalton (representing the main part of the RTF) and 0.1×10⁶ dalton. EcoRI recognises 17 sites and produces fragments (A-Q) with molecular weights between 11.7 and 0.1×10⁶ dalton. SalI yields 7 fragments (A-G) of 16.5 to 2.0×10⁶ dalton.A physical map was constructed from fragments obtained by partial digestion of R1drd-19 with one restriction enzyme, by double and triple digestion of the DNA with two or three enzymes with and without isolation of individual bands from preparative gels. In addition the restriction patterns of several mutants of R1drd-19 were compared with it.Evidence is presented which indicates that the derivatives of R1 investigated are generated by extende deletions, namely the copy mutant pKN102 which has lost the Km resistance, R1 drd-16, which has lost all resistances other than Km and the Km^s derivative of R1drd-16, which represents the pure RTF. The map of R1drd-19 is remarkably different from those of R100 and R6-5. Its molecular weight was estimated to be 62.5 Md. The circular fragment order for BamHI is: A-C-B-D-E, for HindIII: A-D-C-B-F-H-E-G, for EcoRI: A-C-K-B-F-J-O-D-H-L-G-P-Q-N-I-E-M-and for SalI A-B-C-D-G-F-E.     

Selection and timing of replication of plasmids R1drd-19 and F'lac in Escherichia coli.

The selection and timing of plasmid replication was studied in exponentially growing cultures of Escherichia coli K-12 carrying the plasmid R1drd-19 and E. coli strains B/r A and B/r F carrying the plasmid F′lac. In all cases plasmid replication was studied by analysis of covalently closed circular (CCC) DNA. The turnover time of replicating plasmid DNA into CCC-DNA was found to be less than 4 min. Density shift experiments (from ¹⁵NH₄⁺, D₂O to ¹⁴NH₄⁺, H₂O) showed that plasmids R1drd-19 and F′lac are selected randomly for replication. This means that one of the plasmid copies in a cell is selected and replicated. There is no further plasmid replication in the cell until all plasmid copies, including the newly formed ones, have the same probability of being selected for replication. The early kinetics of the appearance of light plasmid DNA after the density shift showed that the time interval between successive replications of plasmids R1drd-19 and F′lac is $\text{τ}\text{n}$, where τ is the generation time and n is the average number of plasmid replications per cell and cell cycle. In a second type of experiment, exponentially growing cells were separated into a series of size classes by low-speed centrifugation in sucrose step gradients. Replication of plasmids R1drd-19 and F′lac was equally frequent in all size classes. This result is in accordance with the results of the density shift experiment. It can therefore be concluded that replication of plasmids R1drd-19 and F′lac is evenly spread over the whole cell cycle, which means that one plasmid replication occurs every time the cell volume has increased by one initiation mass.     

ColE1 plasmid mobility: Essential and conditional functions

Summary Sequences essential for the conjugal transfer of ColE1 can be divided into a cis-acting site and a region encoding trans-acting products. Each of these was successively cloned into a non-transmissible plasmid vector. The resulting chimera was transmissible by the conjugative plasmids F'lac,pro (incFI) and R64drd11 (incI). The sequences encoding colicin E1, immunity, and incompatibility were absent from this chimera: therefore they are not essential for the conjugal transmission of the ColE1 plasmid.In contrast to ColE1, however, the same chimera was deficient in conjugal transfer initiated by R751 (incP) and R388 (incW). This suggests that ColEl sequences other than those cloned in the chimeric plasmid are necessary for its mobilization by R751 and R388. Three such regions were revealed by screening a series of ColE1 insertion mutants for transfer by R751 and R388. Two of these regions encode no other known function while the third is encoded by a region which overlaps the gene for colicin E1 itself.     

The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell-surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo-GM₁) and, to a lesser extend, ll³N-acetylneuraminosylgangliotetraosyl ceramide (GM₁) in solid-phase binding assays. Asialo-GM₁, but not GM₁, inhibited both PAK and PAK pili binding to immobilized asialo-GM₁ on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo-GM₁, suggesting the presence of a structurally similar receptor-binding domain in both pilus types. The interaction between asialo-GM₁ and pili occurs at the pilus tip as asialo-GM₁ coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C-terminal disulphide-bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal-tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134–140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128–144)^ox-OH and PAO(128–144)^ox-OH, which correspond to the C-terminal disulphide-bonded region of Pseudomonas pilin are able to bind to asialo-GM₁ and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo-GM₁ Monoclonal antibody PK3B had no effect on PAK pili binding to asialo-GM₁ Thus, the adherence of the Pseudomonas pilus to glycosphingolipid receptors is a tip-associated phenomenon Involving a tip-exposed C-terminal region of the pilin structural subunit.     

Isolation of an IS1 flanked kanamycin resistance transposon from R1drd19

Summary We have isolated and identified an IS1-flanked transposon from the plasmid R1drd19. This transposon specifies resistance to kanamycin and is 10.4 kb long. It exhibits a frequency of transposition two orders of magnitude lower than that of the smaller, IS1-flanked transposon Tn9. We have named it Tn2350.     

Partitioning of plasmid R1 in Escherichia coli,: I. Kinetics of loss of plasmid derivatives deleted of the par region

The stability of inheritance of plasmid R1drd-19 was tested. The copy number of the plasmid was determined in two different ways: As the ratio between covalently closed circular DNA and chromosomal DNA, and by quantitative determination of single-cell resistance to ampicillin. In the latter case, strains carrying the R1 ampicillin transposon Tn3 on prophage λ was used as standard. The values were transformed to copy number per cell by using the Cooper-Helmstetter model for chromosome replication as well as by determination of chromosomal DNA per cell by the diphenylamine method. The copy number was found to be five to six per cell (or about four per newborn cell). Nevertheless, plasmid R1drd-19 was found to be completely stably inherited. This stability was shown not to be due to retransfer of the plasmid by the R1 conjugation system, since transfer-negative derivatives of the plasmid were also completely stably inherited. Smaller derivatives of plasmid R1drd-19 were found to be lost at a frequency of about 1.5% per cell generation. The copy-number control was not affected in these miniplasmids, since their copy numbers were the same as that of the full size plasmid. Quantitatively, the instability of the miniplasmids was in accord with random partitioning. It is, therefore, suggested that the plasmid R1drd-19 carries genetic information for partitioning (par) of plasmid copies at cell division, and that the par mechanism is distinct from the copy number control (cop) system. Finally, the par gene maps on the resistance transfer part of the plasmid, but far away from the origin of replication and the so-called basic replicon; this is in accord with the approximate location of the repB gene (Yoshikawa, 1974, J. Bacteriol.,, 118, 1123–1131).     

Adsorption of bacteriophages specific for Pseudomonas aeruginosa R factors RP1 and R1822

Short, thick pili were found by electron microscopy on bacteria carrying the P group drug resistance plasmids RP1 and R1822. The R1822-specific phage PRR1 was seen to adsorb to the bases of the pili. Three RP1-specific phages, one filamentous (Pf3), and two with very thick capsids (PR3, PR4), were seen to attach all around the surface of $\text{P. aeruginosa}$ cells, and were thought to be somatic, since pilus phages appear to be strictly polar on this species. PR3 and PR4 also lysed a strain of $\text{E. coli}$ containing an N group plasmid, suggesting a relationship between the N and P group plasmids.     

The nucleotide sequence of a DNA fragment from the replication origin of the antibiotic resistance factor R1drd19

Summary The recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication origin of the antibiotic resistance factor R1drd-19 inserted into the vector plasmid pBR322. In a spontaneously arising mutant of this plasmid (pRK 103) a deletion of about 215 base pairs (bp) has been detected by heteroduplex analysis and mapping with restriction endonucleases. Essential parts of the replication origin must be located in the deleted sequence. The deletion mutant pRK103, in contrast to its parent plasmid pRK101 is not replicated under the control of the R1 replicon, even when the R1 factor or copy mutants of it are present within the same cell. These latter plasmids can complement a plasmid-specific protein not coded by pRK101 but essential for R1-directed replication. The nucleotide sequence of a 252 bp HpaII fragment covering about 170–200 bp of the deletion was determined. This piece of DNA is rich in G and C and contains a series of small palindromes, symmetrically arranged repeated sequences and short selfcomplementary structures which may be of significance for the initiation of the DNA replication. The possibility that the sequenced DNA fragment comprises a major part of the replication origin of R1drd-19 is discussed.     

Different photoreceptors within the same retina express unique combinations of potassium channels

Single electrode current and voltage clamp recordings in Calliphora, and whole-cell voltage clamp recordings in Drosophila were used to characterise the voltage-gated K channels in both major classes of photoreceptors, R7/8 (long visual fibres, LVFs) and R1-6 (short visual fibres, SVFs). R7/8 were identified by their unique spectral properties, ca. 3–4 fold higher input resistances and 3–4 fold lower cell capacitance. In Calliphora SVFs possess both fast and slow activating delayed rectifier potassium conductances. Drosophila SVFs possess a slowly inactivating delayed rectifier (I_Ks), a very rapidly inactivating A channel encoded by the Shaker gene (I_A), and, in a minority of cells, a third K conductance with intermediate kinetics (I_Kf). In both specs the LVFs lack the slowest component, but exhibit the faster K conductance(s) with properties indistinguishable from those in the SVFs. These findings add to established evidence demonstrating the significant role played by potassium channels in tuning the photoreceptor membrane. The results also suggest that R1-6 photoreceptors and R7/8 form inputs to visual subsystems tuned to different temporal frequencies.Abbreviations LVF long visual fibre - SVF short visual fibre - R1-6 retinular cells 1 to 6 inclusive - R7/8 retinular cell 7 and 8 - I _A rapidly inactivating A type potassium conductance; channel coded by Shaker gene - I _Kf rapidly activating, slowly inactivating delayed rectifier-like potassium conductance - I _Ks slowly activating, slowly inactivating delayed rectifier-like potassium conductance - I _KDs slowly activating delayed rectifier potassium conductance - I _KDf rapidly activating delayed rectifier potassium conductance     

Morphology of pili determined by the N incompatibility group plasmid N3 and interaction with bacteriophages PR4 and IKe

The IncN plasmid N3 was transferred to bald strains of Salmonella typhimurium LT2 and Escherichia coli K-12. In both cases, transconjugants were found to carry short pili, which were designated N pili. They were very easily detached from cells and readily broken into short pieces. N pili were found to be straight inflexible rods 9.5 nm thick and sharply pointed at the distal end. The N-specific filamentous bacteriophage IKe and the lipid-containing phage PR4 both adsorbed to the pointed tips when mixed with cell-free suspensions of N pili.     

Water transport and estimated transmembrane potential during freezing of mouse oocytes

Summary Steady-state currents at hyperpolarized membrane potentials were studied in the homologous giant neurons, LP1 and R2, ofAplysia using two-electrode voltage clamp. Nearly half of the steady-state current at voltages more hyperpolarized than –70 mV had characteristics similar to the inwardly rectifying potassium current (I _R) described previously inAplysia neurons. The pharmacological agents 4-bromophenacylbromide, indomethacin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate were found to modulateI _R.I _R was stimulated with BPB and indomethacin and inhibited with TPA. These agents alteredI _R by a mechanism independent ofcAMP, which can also modulateI _R. The effects of these modulators are consistent with their actions on arachidonic acid (AA) metabolism inAplysia nervous system, suggesting AA may constitutively inhibitI _R. When ganglia were perfused for 12 hr with medium containing BSA to absorb extracellular fatty acids,I _R was increased nearly twofold. This increase was partially inhibited by addition of AA to the perfusion medium, and completely inhibited by pretreatment of ganglia with BPB. Although no direct effect of shortterm exposure to exogenous AA was observed, long term exposure to exogenous AA and several other unsaturated fatty acids was accompanied by a decrease inI _R.     

The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence βGalNAc(1–4)βGal found in glycosphingolipids asialo-GM1 and asialo-GM2

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo-GM₁ (Lee et al., 1994 —accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo-GM₁ and asialo-GM₂ proposed by Krivan et al., 1988a) using solid-phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal-NAc(1–4)βGal-Biotin to the Immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C-terminal disulphide-looped region (residues 128–144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C-terminal residues 128–144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the βGalNAc(1–4)βGal-(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc-(1–4)βGal-BSA was inhibited by PAK pili, Ac-KCTSDQDEOFIPKGCSK-OH (AcPAK(128–144)_ox-OH) and Ac-ACKSTQDPMFTPKGCDN-OH (AcPAO(128–144)_ox-OH) peptides. (In these peptides Ac denotes N^α -acetylation of the N-terminus, -OH means a peptide with a free a-carboxyl group at the C-terminus and the‘ox’denotes the oxidation of the sulphhydryl groups of Cys–129 and Cys–142.) Both acetylated peptides were also able to inhibit the binding of βGalNAc(1–4)βGal-biotin to the corresponding BSA-Peptide(128–144)_ox-OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc-biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respective C-termlnal peptides. The data above demonstrated that the P. aeruginosa pili recognize asialo-GM₁ receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficient for binding. Furthermore, the binding to βGalNAc(1–4)βGal was mediated by residues 128–144 of the pilin subunit.     

Fast uncoiling kinetics of F1C pili expressed by uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> are revealed on a single pilus level using force-measuring optical tweezers

Uropathogenic Escherichia coli (UPEC) express various kinds of organelles, so-called pili or fimbriae, that mediate adhesion to host tissue in the urinary tract through specific receptor-adhesin interactions. The biomechanical properties of these pili have been considered important for the ability of bacteria to withstand shear forces from rinsing urine flows. Force-measuring optical tweezers have been used to characterize individual organelles of F1C type expressed by UPEC bacteria with respect to such properties. Qualitatively, the force-versus-elongation response was found to be similar to that of other types of helix-like pili expressed by UPEC, i.e., type 1, P, and S, with force-induced elongation in three regions, one of which represents the important uncoiling mechanism of the helix-like quaternary structure. Quantitatively, the steady-state uncoiling force was assessed as 26.4 ±1.4 pN, which is similar to those of other pili (which range from 21 pN for S_I to 30 pN for type 1). The corner velocity for dynamic response (1,400 nm/s) was found to be larger than those of the other pili (400–700 nm/s for S and P pili, and 6 nm/s for type 1). The kinetics were found to be faster, with a thermal opening rate of 17 Hz, a few times higher than S and P pili, and three orders of magnitude higher than type 1. These data suggest that F1C pili are, like P and S pili, evolutionarily selected to primarily withstand the conditions expressed in the upper urinary tract.     

Hydrophobic adsorptive and hemagglutinating properties ofEscherichia coli possessing colonization factor antigens (CFA/I or CFA/II), type 1 pili,or other pili

Hydrophobic and hemagglutinating activities of piliated enterotoxigenicEscherichia coli possessing colonization factor antigens (CFA)/I and putative CFA/II, strains with type 1 pili, and piliated strains of nonenterotoxigenicE. coli from urinary tract infections were compared. When passed through columns of hydrophobic Phenyl Sepharose in the presence of buffered ammonium sulfate, the strains with CFA adsorbed most strongly. Similarly, the CFA strains showed a tendency to autoagglutinate at a lower (NH₄)₂SO₄ concentration than the other strains studied. The degree of hydrophobicity of the strains tested is in the order CFA/I>CFA/II>type 1 pili>urinary tract strains. Rough variants ofE. coli strains were more hydrophobic than their smooth parents. Electron microscopy showed large numbers of pili on CFA strains, whereas type 1 piliated strains possessed fewer pili. CFA-negative clones possessed few or no, pili and did not adsorb to the gel. A highly piliated mutant strain (PAK/2PfS) ofPseudomonas aeruginosa bound to the Phenyl Sepharose while the poorly piliated wild-type strains did not. Strains, lost their adsorptive capacity after blending, sonication, heating, or trypsin treatment. It is concluded that the hydrophobicity of enteric organisms, as measured by hydrophobic interaction chromatography, is a function of the type and number of pili on the cell surface.     

Gastric emptying and Ca2+ and K+ channels of circular smooth muscle cells in diet‐induced obese prone and resistant rats

Objective:

Accelerated gastric emptying that precipitates hunger and frequent eating could be a potential factor in the development of obesity. The aim of this study was to study gastric emptying in diet‐induced obese‐prone (DIO‐P) and DIO‐resistant (DIO‐R) rats and explore possible differences in electrical properties of calcium (Ca²⁺) and potassium (K⁺) channels of antral circular smooth muscle cells (SMCs).

Design and Methods:

Whole‐cell patch‐clamp technique was used to measure Ca²⁺ and K⁺ currents in single SMCs. Gastric emptying was evaluated 90 min after the ingestion of a solid meal.

Results:

Solid gastric emptying in the DIO‐P rats was significantly faster compared with that in the DIO‐R rats. The peak amplitude of L‐type Ca²⁺ current (I_Ba,L) at 10 mV in DIO‐P rats was greater than that in DIO‐R rats without alternation of the current–voltage curve and voltage‐dependent activation and inactivation. The half‐maximal inactivation voltage of transient outward K⁺ current (I_Kto) was more depolarized (～4 mV) in DIO‐P rats compared with that in DIO‐R rats. No difference was found in the current density or recovery kinetics of I_Kto between two groups. The current density of delayed rectifier K⁺ current (I_Kdr), which was sensitive to tetraethylammonium chloride but not 4‐aminopyridine, was lower in DIO‐P rats than that in DIO‐R rats.

Conclusion:

The accelerated gastric emptying in DIO‐P rats might be attributed to a higher density of I_Ba,L, depolarizing shift of inactivation curve of I_Kto and lower density of I_Kdr observed in the antral SMCs of DIO‐P rats.     

Characteristics and function of thick and thin conjugative pili determined by transfer-derepressed plasmids of incompatibility groups I1, I2, I5, B, K and Z

Eleven transfer-derepressed plasmids from incompatibility groups I1, I5, B, K and Z were constructed using the dnaG3 mutant Escherichia coli strain BW86. All were found to determine thin flexible and thick rigid pili constitutively. Immune electron microscopy was used to relate thick and thin pilus serotypes with incompatibility grouping. Mutant plasmids that determined only thick pili constitutively transferred efficiently on an agar surface but not in a liquid, whereas plasmids with both kinds of pili transferred equally well in both environments. A mutant of the IncI2 plasmid R721 determined thin pili constitutively, and thick pili at a repressed level, as indicated by electron microscopy. Experiments with this indicated that thin pili were apparently not involved directly in conjugation but were only used to stabilize mating aggregates.     

Effects of chemical group specific reagents on sodium entry and the amiloride binding site in frog skin: Evidence for separate sites

Summary Previously we have shown that the inhibition of active transport by amiloride is noncompetitive with sodium inRana catesbeiana skin, suggesting that amiloride acts at a site separate from the sodium entry site (Benos, D.J., Mandel, L.J., Balaban, R.S. 1979,J. Gen Physiol. 73: 307). In the present study, the effects of a number of sulfhydryl, amino, and carboxyl group selective reagents were studied on short-circuit current (I _sc) as well as the efficacy of amiloride in bullfrog skin, to determine those functional ligands which may be involved with either of these processes.Addition of the sulfhydryl reagent PCMBS (1mm) to the outside bathing medium produced biphasic effects, initially reversibly increasingI _sc by an average 56% followed by a slower, irreversible decay to levels below baseline. In contrast, the addition of 0.1mm PCMB always resulted in a rapid, irreversible decrease inI _sc. When a 40,000 mol wt dextran molecule was attached to PCMB, a stable, reversible increase inI _sc was observed. These observations suggest that at least two populations of-SH groups are involved in Na translocation through the entry step. Amiloride was equally effective in inhibitingI _sc before and after treatment with PCMBS both during the stimulatory as well as the inhibitory phase. The sulfhydryl reducing agent DTT, and oxidizing agent DTNB had only minor influence onI _sc and did not alter the effectiveness of amiloride.Similarly, the amino reagents, SITS and TNBS did not affectI _sc. However, TNBS decreased the ability of amiloride to inhibit Na entry. These results suggest that an amino group may be involved in the interaction of amiloride and its site, without affecting Na entry.The carboxyl reagents EEDQ, TMO, and three separate carbodiimides were without effect onI _sc or amiloride inhibition. Methylene blue (MB), a molecule that interacts with both carboxyl and hydroxylspecific groups, inhibitedI _sc by 20% and decreased amiloride's ability to inhibitI _sc. These effects, however, are likely to occur from the cytoplasmic side as MB appears to enter into the cells.These results support the notion that amiloride and Na interact with the entry protein at different regions on the membrane.     

Toxicological and biochemical characterizations of GSTs in Liposcelis bostrychophila Badonnel (Psocop., Liposcelididae)

Abstract: The toxicological and biochemical characteristics of glutathione S‐transferases (GSTs) in the resistant and susceptible strains of Liposcelis bostrychophila were investigated. The two resistant strains were the dichlorvos‐resistant strain (DDVP‐R) and PH₃‐resistant strain (PH₃‐R), and the resistance factors were 22.36 and 4.51, respectively. Compared with their susceptible counterparts, the activities per insect and specific activities of GSTs in DDVP‐R and PH₃‐R were significantly higher. The apparent Michaelis–Menten constant values (K_m) for 1‐chloro‐2,4‐dinitrobenzene (CDNB) were obviously lower in DDVP‐R and PH₃‐R (i.e. lower K_m values, 1.5625 mm for DDVP‐R and 0.6230 mm for PH₃‐R) when compared with their susceptible counterpart (K_m = 3.5520), indicating a higher affinity to the substrate CDNB in resistant strains. In contrast, the catalytic activity of GSTs towards CDNB in the susceptible strain was significantly higher than those in resistant strains. It was noticeable that when reduced glutathione (GSH) was used as substrate, GSTs from resistant strains both indicated a significantly declined affinity. For the catalytic activity of GSTs towards GSH, only the V_max value in DDVP‐R increased significantly compared with that from the susceptible strain, suggesting an overexpression of GST in this resistant strain. The inhibition kinetics of insecticides to GSTs in vitro revealed that dichlorvos and paraoxon possessed excellent inhibition effects on GSTs. The susceptible strain showed higher sensitivity (I₅₀ = 0.9004 mm ) to dichlorvos than DDVP‐R and PH₃‐R (higher I₅₀s, 8.0955 mm for DDVP‐R and 9.3346 mm for PH₃‐R). As for paraoxon, there was a similar situation. The resistant strains both suggested a higher I₅₀ (1.8735 mm for DDVP‐R, and 0.4291 mm for PH₃‐R) compared with the susceptible strain (0.2943 mm ). These suggested that an elevated detoxification ability of GSTs developed in the resistant strains.