Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins. (17/2131)

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.  (+info)

The C-terminal domain of p21 inhibits nucleotide excision repair In vitro and In vivo. (18/2131)

The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.  (+info)

Identification and complementation of frameshift mutations associated with loss of cytadherence in Mycoplasma pneumoniae. (19/2131)

Mycoplasma pneumoniae cytadherence is mediated by a specialized, polar attachment organelle. Certain spontaneously arising cytadherence mutants (designated class I) lack HMW2, fail to localize the adhesin protein P1 to the attachment organelle, and exhibit accelerated turnover of proteins HMW1, HMW3, and P65. Insertional inactivation of hmw2 by Tn4001 results in a phenotype nearly identical to that of the class I mutants, suggesting that the latter may result from a defect in hmw2. In this study, the recombinant wild-type hmw2 allele successfully complemented a class I mutant when introduced by transposon delivery. Synthesis of recombinant HMW2 at wild-type levels resulted in reacquisition of hemadsorption and normal levels of HMW1, HMW3, and P65. Low-level production of HMW2 in some transformants resulted in only an intermediate capacity to hemadsorb. Furthermore, full restoration of HMW1 and P65, but not that of HMW3, was directly proportional to the amount of recombinant HMW2 produced, reflecting the importance of proper stoichiometry for certain cytadherence-associated proteins. The recombinant class I hmw2 allele did not restore cytadherence, consistent with a defect in hmw2 in this mutant. A frameshift was discovered in different oligoadenine tracts in hmw2 from two independent class I mutants. Finally, protein P28 is thought to be the product of internal translation initiation in hmw2. A transposon excision-deletion mutant produced a truncated HMW2 but no P28, consistent with this conclusion. However, this deletion mutant was hemadsorption positive, indicating that P28 may not be required for cytadherence.  (+info)

Use of a clinical Escherichia coli isolate expressing lux genes to study the antimicrobial pharmacodynamics of moxifloxacin. (20/2131)

Escherichia coli isolate 16,906 expressing lux genes was used for real-time monitoring of moxifloxacin effects on bacterial metabolism compared with effects on cell replication. Viable counts showed concentration-dependent killing by moxifloxacin; real-time measurement of bioluminescence on the same cultures showed metabolic activity over 54 h, but with greater inhibition at 1 x MIC than with higher MIC multiples. Post-antibiotic effect was longer when determined using bioluminescence than by viable counts. The control-related effective regrowth time was consistent with both methods. Bioluminescent bacteria provide a rapid and sensitive means for measuring antimicrobial effects on bacterial metabolism.  (+info)

In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution. (21/2131)

In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.  (+info)

Control of parvovirus DNA replication by a tetracycline-regulated repressor. (22/2131)

Autonomous parvoviruses are small, single strand DNA viruses which preferentially replicate in transformed and tumor cells, causing cell death by expression of the cytotoxic nonstructural protein, NS1. Several parvoviruses of the rodent group, including LuIII, efficiently infect human transformed cell lines. The potential for systemic use of these viruses in targeting metastases might be enhanced if NS1 expression and viral replication could be controlled by an innocuous drug such as tetracycline. We therefore substituted prokaryotic tetracycline operator sequences for part of P4 of LuIII, the promoter responsible for transcription of the mRNAs for nonstructural proteins. The resulting construct unexpectedly showed constitutive expression in transiently transfected cells, as indicated by efficient excision and amplification of viral replicative form (RF) DNA. This was apparently due to self-stimulatory transcriptional transactivation by NS1. This problem was overcome by cotransfection with a plasmid expressing a chimera of the repressor of the tetracycline operon with a KRAB transrepression domain. These conditions allowed efficient control of transcription and RF amplification by the tetracycline derivative, doxycycline. These observations form a basis for developing a therapeutic agent based on a drug-controlled parvovirus.  (+info)

Improving electrotransfection efficiency by post-pulse centrifugation. (23/2131)

We have demonstrated that the viability of electrotransfected adherent CHO and suspended NK-L, K-562, L1210 and MC2 cells is improved if pelleting by centrifugation is performed immediately after pulsing. The protection effect on cell viability is cell line- and pellet thickness-dependent. For forming CHO cell pellets, centrifugation force (300-13,000 g) and duration are not crucial; about five to 10 cell layers in the pellet provide the optimal protection effect. NK-L, K-562, L1210 and MC2 cell pellets are optimally formed by centrifugation at 13,000 g in an Eppendorf desktop centrifuge. Pelleting improves the cell viability over the whole range of the NK-L, K-562, L1210 and MC2 cell concentrations studied. When this pelleting method is applied to load CHO cells with FITC-dextran (41,000 MW), not only is the success rate close to 100%, but the growth rate is similar to the control, which is far better than the conventional electroporation method. Furthermore, the transfection efficiency of the five cell lines in pellet is significantly higher than that in suspension.  (+info)

Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system. (24/2131)

Gene therapy studies require techniques that allow alteration of human genomic DNA sequences. Bacterial artificial chromosome cloning systems (BACs/PACs) bridge the gap between vectors with small inserts and yeast artificial chromosomes (YACs). We report the use of a second generation BAC vector, pEBAC, containing eukaryotic selectable markers and combining some of the best features of the BAC, PAC and HAEC systems, into which a 185 kb sequence containing the human beta-globin gene cluster was retrofitted. To permit the introduction of mutations corresponding to those causing human pathology, we have adapted an inducible homologous recombination system for use in E. coli DH10B cells, the host strain for BACs and PACs. Using this system, we have introduced PCR fragments carrying a selectable marker and a reporter gene downstream of the IVS-110 splicing mutation into a specific site within the beta-globin gene sequence. The use of this inducible system minimises the risk of unwanted rearrangements by recombination between repetitive elements and allows the introduction of relevant modifications or reporters at any specific sequence within BACs/PACs in E. coli DH10B cells.  (+info)