Dose-associated pulmonary complication rates after fresh frozen plasma administration for warfarin reversal.

UNLABELLED: ESSENTIALS: Fresh frozen plasma (FFP) may be associated with a dose-based risk of pulmonary complications. Patients received FFP for warfarin reversal at a large academic hospital over a 3-year period. Almost 20% developed pulmonary complications, and the risk was highest after > 3 units of FFP. The risk of pulmonary complications remained significant in multivariable analysis. BACKGROUND: Fresh frozen plasma (FFP) is often administered to reverse warfarin anticoagulation. Administration has been associated with pulmonary complications, but it is unclear whether this risk is dose-related. Aims We sought to characterize the incidence and dose relationship of pulmonary complications, including transfusion-associated circulatory overload (TACO) and transfusion-related acute lung injury (TRALI), after FFP administration for warfarin reversal. METHODS: We performed a structured retrospective review of patients who received FFP for warfarin reversal in the emergency department (ED) of an academic tertiary-care hospital over a 3-year period. Logistic regression was used to explore the relationship between FFP dose and risk of pulmonary events. RESULTS: Two hundred and fifty-one patients met the inclusion criteria. Overall, 49 patients (20%) developed pulmonary complications, including 30 (12%) with TACO, two (1%) with TRALI, and 17 (7%) with pulmonary edema not meeting the criteria for TACO. Pulmonary complications were significantly more frequent in those who received > 3 units of FFP (34.0% versus 15.6%, 95% confidence interval for risk difference 7.9%-8.9%). After stratification by subtype of complication, only the risk of TACO was statistically significant (28.3% versus 7.6%, 95% confidence interval for risk difference 8.2%-16.6%). In multivariable analysis controlling for age, sex, initial systolic blood pressure, and intravenous fluids given in the ED, > 3 units of FFP remained a significant risk factor for pulmonary complications (odds ratio 2.49, 95% confidence interval 1.21-5.13). CONCLUSIONS: Almost 20% of patients who received FFP for warfarin reversal developed pulmonary complications, primarily TACO, and this risk increased with > 3 units of FFP. Clinicians should be aware of and prepared to manage these complications.

AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide.

In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is approximately 65% alpha-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H(2)O(2), and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H(2)O(2), phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H(2)O(2) detoxification.

A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities.

A Pseudomonas aeruginosa oxyR mutant was dramatically sensitive to H(2)O(2), despite possessing wild-type catalase activity. Oxygen-dependent oxyR phenotypes also included an inability to survive aerobic serial dilution in Luria broth and to resist aminoglycosides. Plating the oxyR mutant after serial dilution in its own spent culture supernatant, which contained the major catalase KatA, or under anaerobic conditions allowed for survival. KatA was resistant to sodium dodecyl sulfate, proteinase K, pepsin, trypsin, chymotrypsin and the neutrophil protease cathepsin G. When provided in trans and expressed constitutively, the OxyR-regulated genes katB, ahpB, and ahpCF could not restore both the serial dilution defect and H(2)O(2) resistance; only oxyR itself could do so. The aerobic dilution defect could be complemented, in part, by only ahpB and ahpCF, suggesting that the latter gene products could possess a catalase-like activity. Aerobic Luria broth was found to generate approximately 1.2 microM H(2)O(2) min(-1) via autoxidation, a level sufficient to kill serially diluted oxyR and oxyR katA bacteria and explain the molecular mechanism behind the aerobic serial dilution defect. Taken together, our results indicate that inactivation of OxyR renders P. aeruginosa exquisitely sensitive to both H(2)O(2) and aminoglycosides, which are clinically and environmentally important antimicrobials.

Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa.

We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa harboring the rpoA, rplQ, katA, and bfrA genes. These loci are predicted to encode, respectively, (i) the alpha subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome b1 or b557). Our goal was to determine the contributions of KatA and BfrA to the resistance of P. aeruginosa to hydrogen peroxide (H2O2). When provided on a multicopy plasmid, the P. aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli. The katA gene was found to contain two translational start codons encoding a heteromultimer of approximately 160 to 170 kDa and having an apparent Km for H2O2 of 44.7 mM. Isogenic katA and bfrA mutants were hypersusceptible to H2O2, while a katA bfrA double mutant demonstrated the greatest sensitivity. The katA and katA bfrA mutants possessed no detectable catalase activity. Interestingly, a bfrA mutant expressed only approximately 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation. Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the bfrA mutant. RNase protection assays revealed that katA and bfrA are on different transcripts, the levels of which are increased by both iron and H2O2. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the bfrA, katA, and katA bfrA mutants relative to wild-type bacteria. Our results suggest that P. aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.

Regulation of the macrophage vacuolar ATPase and phagosome-lysosome fusion by Histoplasma capsulatum.

Histoplasma capsulatum (Hc) maintains a phagosomal pH of about 6.5. This strategy allows Hc to obtain iron from transferrin, and minimize the activity of macrophage (Mo) lysosomal hydrolases. To determine the mechanism of pH regulation, we evaluated the function of the vacuolar ATPase (V-ATPase) in RAW264.7 Mo infected with Hc yeast or the nonpathogenic yeast Saccharomyces cerevisae (Sc). Incubation of Hc-infected Mo with bafilomycin, an inhibitor of the V-ATPase, did not affect the intracellular growth of Hc, nor did it affect the intraphagosomal pH. In contrast, upon addition of bafilomycin, phagosomes containing Sc rapidly changed their pH from 5 to 7. Hc-containing phagosomes had 5-fold less V-ATPase than Sc-containing phagosomes as quantified by immunoelectron microscopy. Furthermore, Hc-containing phagosomes inhibited phagolysosomal fusion as quantified by the presence of acid phosphatase, accumulation of LAMP2, and fusion with rhodamine B-isothiocyanate-labeled dextran-loaded lysosomes. Finally, in Hc-containing phagosomes, uptake of ferritin was equivalent to phagosomes containing Sc, indicating that Hc-containing phagosomes have full access to the early "bulk flow" endocytic pathway. Thus, Hc yeasts inhibit phagolysosomal fusion, inhibit accumulation of the V-ATPase in the phagosome, and actively acidify the phagosomal pH to 6.5 as part of their strategy to survive in Mo phagosomes.

Cloning and characterization of the Pseudomonas aeruginosa zwf gene encoding glucose-6-phosphate dehydrogenase, an enzyme important in resistance to methyl viologen (paraquat).

In this study, we cloned the Pseudomonas aeruginosa zwf gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that catalyzes the NAD+- or NADP+-dependent conversion of glucose-6-phosphate to 6-phosphogluconate. The predicted zwf gene product is 490 residues, which could form a tetramer with a molecular mass of approximately 220 kDa. G6PDH activity and zwf transcription were maximal in early logarithmic phase when inducing substrates such as glycerol, glucose, or gluconate were abundant. In contrast, both G6PDH activity and zwf transcription plummeted dramatically when bacteria approached stationary phase, when inducing substrate was limiting, or when the organisms were grown in a citrate-, succinate-, or acetate-containing basal salts medium. G6PDH was purified to homogeneity, and its molecular mass was estimated to be approximately 220 kDa by size exclusion chromatography. Estimated Km values of purified G6PDH acting on glucose-6-phosphate, NADP+, and NAD+ were 530, 57, and 333 microM, respectively. The specific activities with NAD+ and NADP+ were calculated to be 176 and 69 micromol/min/mg. An isogenic zwf mutant was unable to grow on minimal medium supplemented with mannitol. The mutant also demonstrated increased sensitivity to the redox-active superoxide-generating agent methyl viologen (paraquat). Since one by-product of G6PDH activity is NADPH, the latter data suggest that this cofactor is essential for the activity of enzymes critical in defense against paraquat toxicity.

Fumarase C activity is elevated in response to iron deprivation and in mucoid, alginate-producing Pseudomonas aeruginosa: cloning and characterization of fumC and purification of native fumC.

We report the discovery of fumC, encoding a fumarase, upstream of the sodA gene, encoding manganese superoxide dismutase, in Pseudomonas aeruginosa. The fumC open reading frame, which terminates 485 bp upstream of sodA, contains 1,374 bp that encode 458 amino acids. A second 444-bp open reading frame located between fumC and sodA, called orfX, showed no homology with any genes or proteins in database searches. A fumarase activity stain revealed that P. aeruginosa possesses at least two and possibly three fumarases. Total fumarase activity was at least approximately 1.6-fold greater in mucoid, alginate-producing bacteria than in nonmucoid bacteria and decreased 84 to 95% during the first 5 h of aerobic growth, followed by a rapid rise to maximum activity in stationary phase. Bacteria exposed to the iron chelator 2,2'-dipyridyl, but not ferric chloride, demonstrated an increase in fumarase activity. Mucoid bacteria produced approximately twofold-higher levels of the siderophores pyoverdin and pyochelin than nonmucoid bacteria. Northern blot analysis revealed a transcript that included fumC, orfX, and sodA, the amount of which was increased in response to iron deprivation. A P. aeruginosa fumC mutant produced only approximately 40% the alginate of wild-type bacteria. Interestingly, a sodA mutant possessed an alginate-stable phenotype, a trait that is typically unstable in vitro. These data suggest that mucoid bacteria either are in an iron-starved state relative to nonmucoid bacteria or simply require more iron for the process of alginate biosynthesis. In addition, the iron-regulated, tricarboxylic acid cycle enzyme fumarase C is essential for optimal alginate production by P. aeruginosa.

An operon containing fumC and sodA encoding fumarase C and manganese superoxide dismutase is controlled by the ferric uptake regulator in Pseudomonas aeruginosa: fur mutants produce elevated alginate levels.

The activities of fumarase- and manganese-cofactored superoxide dismutase (SOD), encoded by the fumC and sodA genes in Pseudomonas aeruginosa, are elevated in mucoid, alginate-producing bacteria and in response to iron deprivation (D. J. Hassett, M. L. Howell, P. A. Sokol, M. L. Vasil, and G. E. Dean, J. Bacteriol. 179:1442-1451, 1997). In this study, a 393-bp open reading frame, fagA (Fur-associated gene), was identified immediately upstream of fumC, in an operon with orfX and sodA. Two iron boxes or Fur (ferric uptake regulatory protein) binding sites were discovered just upstream of fagA. Purified P. aeruginosa Fur caused a gel mobility shift of a PCR product containing these iron box regions. DNA footprinting analysis revealed a 37-bp region that included the Fur binding sites and was protected by Fur. Primer extension analysis and RNase protection assays revealed that the operon is composed of at least three major iron-regulated transcripts. Four mucoid fur mutants produced 1.7- to 2.6-fold-greater fumarase activity and 1.7- to 2.3-greater amounts of alginate than wild-type organisms. A strain devoid of the alternative sigma factor AlgT(U) produced elevated levels of one major transcript and fumarase C and manganase-cofactored SOD activity, suggesting that AlgT(U) may either play a role in regulating this transcript or function in some facet of iron metabolism. These data suggest that the P. aeruginosa fagA, fumC, orfX, and sodA genes reside together on a small operon that is regulated by Fur and is transcribed in response to iron limitation in mucoid, alginate-producing bacteria.

Sexual dimorphism in the spinal cord is absent in mice lacking the ciliary neurotrophic factor receptor.

Ciliary neurotrophic factor (CNTF) has potent survival-promoting effects on motoneurons in vitro and in vivo. We examined knockout mice with null mutations of the gene for either CNTF itself or the alpha-subunit of the CNTF receptor (CNTFRalpha) to assess whether CNTF and/or its receptors are involved in the development of a sexually dimorphic neuromuscular system. Male rodents have many more motoneurons in the spinal nucleus of the bulbocavernosus (SNB) than do females. This sex difference is caused by hormone-regulated death of SNB motoneurons and their target muscles. Sexual dimorphism of SNB motoneuron number developed completely normally in CNTF knockout (CNTF -/-) mice. In contrast, a sex difference in the SNB was absent in CNTFRalpha -/- animals: male mice lacking a functional CNTF alpha-receptor had fewer than half as many SNB motoneurons than did wild-type males and no more than did their female counterparts. Size of the bulbocavernosus and levator ani muscles, the main targets of SNB motoneurons, was not affected in either CNTF or CNTFRalpha knockout males. These observations suggest that signaling through the CNTF receptor is involved in sexually dimorphic development of SNB motoneuron number and that target muscle survival per se is not sufficient to ensure motoneuron survival in this system. In addition, our observations are consistent with the suggestion that CNTF itself is not the only endogenous ligand for the CNTF receptor. A second, as yet unknown, ligand may be important for neural development, including sexually dimorphic motoneuron development.

Sequence-dependent effects of spermine on the thermodynamics of the B-DNA to Z-DNA transition.

Spermine has been shown to bind to and stabilize a number of altered DNA conformations, including left-handed Z-DNA. Here, we have quantitatively studied the effects of spermine on the negative supercoil-induced transition from B- to Z-DNA. We have determined the intrinsic association constants for and the effective number of ligands that bind to both B- and Z-DNA. The intrinsic affinity of spermine for Z-DNA is approximately 10 times higher for d(CA/TG) (KZP = 1.2 x 10(8) M-1) than for d(CG) dinucleotides (KZP = 1.5 x 10(7) M-1), and both are greater than that for B-DNA (KBP = 1.4 x 10(5) M-1). This accounts for the stabilization of Z-DNA by spermine. The number of spermine accommodated by Z-DNA (nZ) is sequence-dependent [nZ = 0.6 spermine per 18 d(CA/TG) dinucleotides and 2.3 for 12 d(CG) dinucleotides]. The value of nZ of < 1 was interpreted as evidence for negative cooperativity in spermine binding to d(CA/TG) dinucleotides. Thus, although d(CA/TG) sequences saturate at lower spermine concentrations, the ligand has an overall greater effect on the stability of d(CG) dinucleotides as Z-DNA. B-DNA accommodates more spermines per base pair than either sequence as Z-DNA. At higher concentrations (> 10 microM), spermine destabilizes Z-DNA. Using these parameters in a model for competitive spermine binding to B-DNA and Z-DNA, we can make predictions for how potential Z-DNA sequences found in the human genome are affected by cellular levels of superhelical density and spermine.

Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities.

Pseudomonas aeruginosa is considered a strict aerobe that possesses several enzymes important in the disposal of toxic oxygen reduction products including iron- and manganese-cofactored superoxide dismutase and catalase. At present, the nature of the regulation of these enzymes in P. aeruginosa Is not understood. To address these issues, we used two mutants called A4 and C6 which express altered Fur (named for ferric uptake regulation) proteins and constitutively produce the siderophores pyochelin and pyoverdin. Both mutants required a significant lag phase prior to log-phase aerobic growth, but this lag was not as apparent when the organisms were grown under microaerobic conditions. The addition of iron salts to mutant A4 and, to a greater extent, C6 cultures allowed for an increased growth rate under both conditions relative to that of bacteria without added iron. Increased manganese superoxide dismutase (Mn-SOD) and decreased catalase activities were also apparent in the mutants, although the second catalase, KatB, was detected in cell extracts of each fur mutant. Iron deprivation by the addition of the iron chelator 2,2'-dipyridyl to wild-type bacteria produced an increase in Mn-SOD activity and a decrease in total catalase activity, similar to the fur mutant phenotype. Purified wild-type Fur bound more avidly than mutant Fur to a PCR product containing two palindromic 19-bp "iron box" regions controlling expression of an operon containing the sodA gene that encodes Mn-SOD. All mutants were defective in both ferripyochelin- and ferripyoverdin-mediated iron uptake. Two mutants of strain PAO1, defective in pyoverdin but not pyochelin biosynthesis, produced increased Mn-SOD activity. Sensitivity to both the redox-cycling agent paraquat and hydrogen peroxide was greater in each mutant than in the wild-type strain. In summary, the results indicate that mutations in the P. aeruginosa fur locus affect aerobic growth and SOD and catalase activities in P. aeruginosa. We postulate that reduced siderophore-mediated iron uptake, especially that by pyoverdin, may be one possible mechanism contributing to such effect.

The effect of microabrasion on restorative materials and tooth surface.

The effect of microabrasion on human enamel has been well documented; however, no information is available on its effect on dentin or restorative materials. The purpose of this study was to evaluate the effect of the microabrasion technique on the surface roughness of restorative materials and enamel and dentin surfaces. Flat disks of amalgam, composite resin, porcelain, and glass ionomer were evaluated. Labial enamel of three maxillary incisors and three molars that were flattened buccally to expose dentin were also tested. The Prema microabrasion compound was applied to each sample with a 10:1 gear-reduction, slow-speed handpiece for 5 seconds, then rinsed for 10 seconds. Roughness was determined with a profilometer. This procedure was repeated 20 times for each sample. A polyvinylsiloxane impression of the surface was taken after 0, 5, 10, 15, and 20 applications and examined under a scanning electron microscope. Enamel surface roughness did not improve as previously reported, suggesting that changes in optical characteristics may not be as important as removal of enamel in obtaining esthetic results. Dentin and glass ionomer exhibited an increase in roughness, such that their presence contraindicates the technique. Amalgam was essentially polished. Porcelain was most resistant to the effects of microabrasion. Judicious use of the technique, especially when restorative materials are present, is advised.

Resorption-cycle-dependent polarization of mRNAs for different subunits of V-ATPase in bone-resorbing osteoclasts.

Protein sorting in eukaryotic cells is mainly done by specific targeting of polypeptides. The present evidence from oocytes, neurons, and some other polarized cells suggests that protein sorting can be further facilitated by concentrating mRNAs to their corresponding subcellular areas. However, very little is known about the mechanism(s) involved in mRNA targeting, or how widespread and dynamic such mRNA sorting might be. In this study, we have used an in vitro cell culture system, where large multinucleated osteoclasts undergo continuous structural and functional changes from polarized (resorbing) to a nonpolarized (resting) stage. We demonstrate here, using a nonradioactive in situ hybridization technique and confocal microscopy, that mRNAs for several vacuolar H(+)-ATPase subunits change their localization and polarity in osteoclasts according to the resorption cycle, whereas mRNA for cytoplasmic carbonic anhydrase II is found diffusely located throughout the osteoclast during the whole resorption cycle. Antisense RNA against the 16-kDa or 60-kDa V-ATPase subunit inhibits polarization of the osteoclasts, as determined by cytoskeleton staining. Antisense RNA against carbonic anhydrase II, however, has no such effect.

Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide.

Pseudomonas aeruginosa is an obligate aerobe that is virtually ubiquitous in the environment. During aerobic respiration, the metabolism of dioxygen can lead to the production of reactive oxygen intermediates, one of which includes hydrogen peroxide. To counteract the potentially toxic effects of this compound, P. aeruginosa possesses two heme-containing catalases which detoxify hydrogen peroxide. In this study, we have cloned katB, encoding one catalase gene of P. aeruginosa. The gene was cloned on a 5.4-kb EcoRI fragment and is composed of 1,539 bp, encoding 513 amino acids. The amino acid sequence of the P. aeruginosa katB was approximately 65% identical to that of a catalase from a related species, Pseudomonas syringae. The katB gene was mapped to the 71- to 75-min region of the P. aeruginosa chromosome, the identical region which harbors both sodA and sodB genes encoding both manganese and iron superoxide dismutases. When cloned into a catalase-deficient mutant of Escherichia coli (UM255), the recombinant P. aeruginosa KatB was expressed (229 U/mg) and afforded this strain resistance to hydrogen peroxide nearly equivalent to that of the wild-type E. coli strain (HB101). The KatB protein was purified to homogeneity and determined to be a tetramer of approximately 228 kDa, which was in good agreement with the predicted protein size derived from the translated katB gene. Interestingly, KatB was not produced during the normal P. aeruginosa growth cycle, and catalase activity was greater in nonmucoid than in mucoid, alginate-producing organisms. When exposed to hydrogen peroxide and, to a greater extent, paraquat, total catalase activity was elevated 7- to 16-fold, respectively. In addition, an increase in KatB activity caused a marked increase in resistance to hydrogen peroxide. KatB was localized to the cytoplasm, while KatA, the "housekeeping" enzyme, was detected in both cytoplasmic and periplasmic extracts. A P. aeruginosa katB mutant demonstrated 50% greater sensitivity to hydrogen peroxide than wild-type bacteria, suggesting that KatB is essential for optimal resistance of P. aeroginosa to exogenous hydrogen peroxide.

Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy.

During the course of gene therapy experiments in rodents, using intramuscular injections of plasmid DNA derived from Escherichia coli, we noted dose-related toxicity. This observation prompted a search for possible contaminants of DNA samples. We used the highly specific and sensitive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioactivity in DNA samples, and found plasmid DNA derived from standard E. coli bacterial strains, using traditional DNA isolation protocols, to be heavily contaminated with endotoxin, or lipopolysaccharide (LPA). Standard DNA isolation procedures resulted in the copurification of up to 500 micrograms/ml of LPS. LPS is a potent inducer of cytokines and other inflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has important implications for in vitro transfection studies and microinjection of DNA into embryos. A simple and efficient protocol to reduce LPS contamination of plasmid DNA was developed. The conversion of intact bacteria to spheroplasts prior to the isolation of plasmid DNA, incubation with lysozyme, treatment with the detergent n-octyl-beta-D-thioglucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This represents a 10,000-fold reduction in LPS contamination, compared to conventional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plasmid DNA.

Effects of base substituents on the hydration of B- and Z-DNA: correlations to the B- to Z-DNA transition.

We present a study of how substituent groups of naturally occurring and modified nucleotide bases affect the degree of hydration of right-handed B-DNA and left-handed Z-DNA. A comparison of poly(dG-dC) and poly(dG-dm5C) titrations with the lipotropic salts of the Hofmeister series infers that the methyl stabilization of cytosines as Z-DNA is primarily a hydrophobic effect. The hydration free energies of various alternating pyrimidine-purine sequences in the two DNA conformations were calculated as solvent free energies from solvent accessible surfaces. Our analysis focused on the N2 amino group of purine bases that sits in the minor groove of the double helix. Removing this amino group from guanine to form inosine (I) destabilizes Z-DNA, while adding this group to adenines to form 2-aminoadenine (A') stabilizes Z-DNA. These predictions were tested by comparing the salt concentrations required to crystallize hexanucleotide sequences that incorporate d(CG), d(CI), d(TA) and d(TA') base pairs as Z-DNA. Combining the current results with our previous analysis of major groove substituents, we derived a thermodynamic cycle that relates the systematic addition, deletion, or substitution of each base substituent to the B- to Z-DNA transition free energy.

Vaccinia virus ribonucleotide reductase. Correlation between deoxyribonucleotide supply and demand.

Ribonucleotide reductase has been suggested as a rate-limiting enzyme in DNA synthesis, partly because activities of the enzyme in cell-free preparations are low relative to rates needed to sustain DNA replication at observed rates. Vaccinia virus, with a large duplex DNA genome, encodes both subunits of a specific ribonucleoside diphosphate reductase. In this report, we describe quantitative analysis of ribonucleotide reductase protein levels and DNA accumulation in vaccinia virus-infected cell extracts, to correlate the supply of deoxyribonucleotides with the demand for these precursors in viral DNA synthesis. To do this, we generated polyclonal antisera to TrpE fusion proteins constructed from the carboxyl termini of both subunits of viral ribonucleotide reductase. We used S1 nuclease and immunoprecipitation analysis to determine the transcriptional and translational kinetics of vaccinia virus ribonucleotide reductase expression. Enzyme activity and ribonucleotide reductase protein stability were also assayed during the time course of viral infection. Enzyme-linked immunoassays were used to quantitate protein levels, and filter hybridizations were used to measure the accumulation of viral DNA. We show that ribonucleotide reductase activity in vaccinia virus-infected cells is severalfold higher than needed to provide deoxyribonucleotides at rates commensurate with DNA synthesis. Thus, while the enzyme is important as catalyst for the first committed reaction in DNA replication, it is not rate-limiting for this process.

Effect of grooves on resistance form of Class 2 amalgams with wide occlusal preparations.

This study evaluated in vitro the necessity of retention grooves in approximal boxes in class 2 preparations with faciolingually wide occlusal extensions (occlusal isthmus width 1.8 mm). Thirty-six class 2 mesio-occlusal cavities were prepared in sound human maxillary premolar teeth and divided into three groups of 12. In one group, the preparations had no retention grooves. In another group, retention grooves 0.3-0.5 mm deep were prepared at the axioproximal line angles and extended from the gingival floor occlusally to just gingival to the occlusal dentinoenamel junction. In the third group, a 1 mm-long groove was prepared extending from the axiopulpal line angle occlusally to just gingival to the occlusal dentinoenamel junction. The marginal ridges of the amalgam were loaded at an angle of 13.5 degrees from vertical in an Instron Testing Machine until the restorations failed. Results indicated no significant difference in load to cause failure in any of the three groups. Restorations without approximal retention grooves possessed as much resistance to failure as those with grooves.

Effect of grooves on resistance form of conservative Class 2 amalgams.

This study evaluated several means of providing retention for the approximal box in very conservative class 2 preparations (occlusal isthmus width 0.7 mm). Sixty class 2 mesio-occlusal cavities were prepared in sound human maxillary premolar teeth. Four types of retention grooves, 0.3-0.5 mm deep, were prepared at the axiofacial and axiolingual line angles and/or occlusal to those line angles. Specimens were loaded at an angle of 13.5 degrees from vertical in an Instron Universal Testing Machine until the restoration failed. Results indicate that grooves located occlusal to the axiopulpal line angle provided more resistance than conventional grooves (gingival to the axiopulpal line angle) or no grooves. The use of a short retention groove or retention point located occlusal to the axiopulpal line angle, but not extending to the occlusal cavosurface margin, provided greater retention while removing minimal tooth structure.

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.