Interleukin-12 inhibits liver-specific drug-inducible systems in vivo.

Drug-inducible systems allow modulation of the duration and intensity of cytokine expression in liver immuno-based gene therapy protocols. However, the biological activity of the transgene may influence their function. We have analyzed the kinetics of interleukin-12 (IL-12) expression controlled by the doxycycline (Dox)- and the mifepristone (Mif)-dependent systems using two long-term expressing vectors directed to liver: a plasmid administered by hydrodynamic injection and a high-capacity adenoviral vector. Daily administration of Dox or Mif was associated with a progressive loss of inducibility and a decrease of murine IL-12 production. This inhibition occurred at the transcriptional level and was probably caused by an interferon (IFN)-gamma-mediated downmodulation of liver-specific promoters that control the expression of transactivators in these systems. Genome-wide expression microarrays studies revealed a parallel downregulation of liver-specific genes in mice overexpressing murine IL-12. However, a promoter naturally induced by IL-12 was also inhibited by this cytokine when placed in a plasmid vector. Interestingly, treatment with sodium butyrate, a class I/II histone deacetylase inhibitor, was able to rescue liver-specific promoter activity solely in the vector. We conclude that biologically active IL-12 can transiently inhibit the function of drug-inducible systems in non-integrative DNA vectors by reducing promoter activity, probably through IFN-gamma and protein deacetylation-dependent mechanisms.

An oncolytic adenovirus controlled by a modified telomerase promoter is attenuated in telomerase-negative cells, but shows reduced activity in cancer cells.

The promoter for human telomerase reverse transcriptase (hTERTp) is preferentially active in malignant cells. It was recently used to control the expression of the adenoviral E1A gene for the development of oncolytic adenoviruses. To ensure maximal repression in normal cells, the inclusion of additional E-boxes in the proximal region of the core promoter was described. We found that the transcriptional activity of this artificial sequence (T-255-4DEB) is minimal in normal cells, but it is also reduced in all the cancer cell lines tested. The cancer specificity of a new oncolytic adenovirus based in this promoter (AdTE1) was evaluated by direct comparison with wild-type adenovirus type 5 (AdWT) in vitro and in vivo. In all the parameters tested, AdTE1 was attenuated in normal cells, but the efficacy in cancer cells showed a parallel reduction, suggesting a lack of specificity. However, the cytotoxicity of AdTE1 was repressed in senescent cells compared to AdWT. Therefore, we conclude that AdTE1 is preferentially attenuated only in cells that are permanently devoid of telomerase expression such as senescent cells. Further modifications in the telomerase-based promoters should be introduced in order to combine maximal attenuation of oncolytic adenoviruses in normal tissues and enhanced activity in tumors.

Recent advances in transgenic arthropod technology.

The ability to insert foreign genes into arthropod genomes has led to a diverse set of potential applications for transgenic arthropods, many of which are designed to advance public health or improve agricultural production. New techniques for expressing foreign genes in arthropods have now been successfully used in at least 18 different genera. However, advances in field biology are lagging far behind those in the laboratory, and considerable work is needed before deployment in nature can be a reality. A mechanism to drive the gene of interest though a natural population must be developed and thoroughly evaluated before any field release, but progress in this area has been limited. Likewise, serious consideration of potential risks associated with deployment in nature has been lacking. This review gives an overview of the most promising techniques for expressing foreign genes in arthropods, considers the potential risks associated with their deployment, and highlights the areas of research that are most urgently needed for the field to advance out of the laboratory and into practice.

Life-history changes that accompany the transition from sexual to parthenogenetic reproduction in Drosophila mercatorum.

In spite of the predicted genetic and ecological costs of sex, most natural populations maintain sexual reproduction, even those capable of facultative parthenogenesis. Unfertilized eggs from natural populations of Drosophila mercatorum occasionally develop into viable adults, but obligately parthenogenetic populations are unknown in this species. To evaluate the microevolutionary forces that both favor and constrain the evolution of parthenogenesis in D. mercatorum, we have measured parthenogenetic rates across a natural, sexually reproducing population and characterized the life-history changes that accompany the transition from sexual to parthenogenetic reproduction in laboratory strains. A highly significant difference in parthenogenetic rate was found between two populations in close geographic proximity, with increased rate found with lower population density. Laboratory strains of parthenogenetic females suffered increased mortality and reduced egg viability relative to their virgin counterparts from a sexual strain. Lifetime egg production was similar across all strains, but a shift in peak egg production to an earlier age also occurred. The combination of these life-history traits resulted in a higher net reproductive value for sexual females, but because they also had a longer generation time, intrinsic rate of increase was not as dramatically different from parthenogenetic females. In environments with high early mortality, there may be no fitness disadvantage to parthenogenesis, but the predicted ecological advantage of a twofold increase in intrinsic rate of increase was not realized. These results support the theory of Stalker (1956) that parthenogenesis is favored in environments in which sexual reproduction is difficult or impossible.

Role of individual monomers of a dimeric initiator protein in the initiation and termination of plasmid rolling circle replication.

Plasmids of the pT181 family encode initiator proteins that act as dimers during plasmid rolling circle (RC) replication. These initiator proteins bind to the origin of replication through a sequence-specific interaction and generate a nick at the origin that acts as the primer for RC replication. Previous studies have demonstrated that the initiator proteins contain separate DNA binding and nicking-closing domains, both of which are required for plasmid replication. The tyrosine residue at position 191 of the initiator RepC protein of pT181 is known to be involved in nicking at the origin. We have generated heterodimers of RepC that consist of different combinations of wild type, DNA binding, and nicking mutant monomers to identify the role of each of the two monomers in RC replication. One monomer with DNA binding activity was sufficient for the targeting of the initiator to the origin, and the presence of Tyr-191 in one monomer was sufficient for the initiation of replication. On the other hand, a dimer consisting of one monomer defective in DNA binding and the other defective in origin nicking failed to initiate replication. Our results demonstrate that the monomer that promotes sequence-specific binding to the origin must also nick the DNA to initiate replication. Interestingly, whereas Tyr-191 of the initiator was required for nicking at the origin to initiate replication, it was dispensable for termination, suggesting that alternate amino acids in the initiator may promote termination but not initiation.

Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids.

Single-stranded DNA (ssDNA) promoters are the key components of the single-strand origins (ssos) of replication of rolling-circle (RC) replicating plasmids. The recognition of this origin by the host RNA polymerase and the synthesis of a short primer RNA are critical for initiation of lagging-strand synthesis. This step is thought to be a limiting factor for the establishment of RC plasmids in a broad range of bacteria, because most of the ssos described are fully active only in their natural hosts. A special type of sso, the ssoU, is unique in the sense that it can be efficiently recognized in a number of different Gram-positive hosts. We have experimentally deduced the folded structure and characterized the ssDNA promoter present within the ssoU using P1 nuclease digestion and DNase I protection assays with the Bacillus subtilis and Staphylococcus aureus RNA polymerases. We have also identified the RNA products synthesized from this ssDNA promoter and mapped the initiation points of lagging-strand synthesis in vivo from ssoU-containing plasmids. Through gel mobility shift experiments, we have found that ssDNA containing the ssoU sequence can efficiently interact with the RNA polymerase from two different Gram-positive bacteria, S. aureus and B. subtilis. We have also realigned the narrow and broad host range sso sequences of RC plasmids, and found that they contain significant homology. Our data support the notion that the strength of the RNA polymerase-ssoU interaction may be the critical factor that confers the ability on the ssoU to be fully functional in a broad range of bacteria.

Identification of a new gene in the streptococcal plasmid pLS1: the rnaI gene.

The streptococcal plasmid pMV158 has been reported to harbor five genes: three involved in initiation of rolling circle replication and its control (copG, repB, and maII), one involved in conjugative mobilization (mobM), and the fifth one specifying constitutive resistance to tetracycline (tet). The mobM gene was removed in the construction of the pMV158-derivative plasmid pLS1, which was used in this study. By in vitro transcription assays, primer extension experiments, and construction of mutations, here we demonstrate the presence of another gene (the sixth of pMV158), termed maI, which is transcribed in opposite orientation with respect to the plasmid mRNAs, to render RNA I. The 5'-end of RNA I has an 8-nt sequence which is complementary to a region of the lagging-strand origin (ssoA) comprising a 6-nt consensus sequence involved in lagging strand synthesis. This suggested that RNA I could influence, positively or negatively, initiation of lagging strand synthesis from the pLS1-ssoA. However, plasmids defective in RNA I synthesis exhibited a phenotype similar to the wild type in terms of efficiency of replication from the ssoA and copy number. When the maI gene was cloned into a compatible plasmid, the resulting recombinants did not exhibit incompatibility toward plasmids with the pLS1 replicon. Thus, RNA I does not seem to be a true copy number control element. We postulate that transcription from the maI promoter may facilitate extrusion of the hairpin of the plasmid double-strand origin, which is the target of the initiator of replication protein.

The effects of verapamil and diltiazem pretreatment on potassium release in dogs after the administration of succinylcholine.

UNLABELLED: Verapamil exacerbates the increase in serum potassium after a large-dose potassium infusion or after the IV administration of succinylcholine. We conducted a study in 12 canines conditioned for 30 days acting as their own controls. The canines had chronic tracheotomies and carotid loops performed 2 wk before the experiment. Control canines were given 1 mg/kg succinylcholine at Time 0. Blood samples were analyzed for potassium at 0, 1, 3, 5, 10, 15, 30, and 60 min. One week later, the dogs received 0.15 mg/kg of either verapamil or diltiazem, followed by a 5.6-microg x kg(-1) x min(-1) 10-min infusion of the same drug. The animals were then given a bolus dose of succinylcholine 1 mg/kg, and the blood potassium was analyzed as before. There was no significant difference in the potassium concentration before the succinylcholine injection (Time 0) between the study groups. The canines pretreated with verapamil had a significantly greater increase (24% +/- 8%) in potassium concentration than the control canines (14% +/- 6%) 15 min after succinylcholine administration. There was no difference between the potassium concentrations of the diltiazem-pretreated canines and the control group at any time point. Therefore, diltiazem pretreatment does not significantly influence potassium regulation after a succinylcholine injection, whereas verapamil pretreatment has measurable hyperkalemic effects. IMPLICATIONS: Succinylcholine is a drug that causes blood potassium to increase. Potassium influences heart rhythm. Verapamil and diltiazem are drugs used for angina heart pain. We used dogs to determine the effect of verapamil or diltiazem on the blood's potassium after an injection of succinylcholine and found that verapamil had the greatest effect.

Lagging strand replication of rolling-circle plasmids: specific recognition of the ssoA-type origins in different gram-positive bacteria.

Many bacterial plasmids replicate by a rolling-circle mechanism that involves the generation of single-stranded DNA (ssDNA) intermediates. Replication of the lagging strand of such plasmids initiates from their single strand origin (sso). Many different types of ssos have been identified. One group of ssos, termed ssoA, which have conserved sequence and structural features, function efficiently only in their natural hosts in vivo. To study the host specificity of sso sequences, we have analyzed the functions of two closely related ssoAs belonging to the staphylococcal plasmid pE194 and the streptococcal plasmid pLS1 in Staphylococcus aureus. The pLS1 ssoA functioned poorly in vivo in S. aureus as evidenced by accumulation of high levels of ssDNA but supported efficient replication in vitro in staphylococcal extracts. These results suggest that one or more host factors that are present in sufficient quantities in S. aureus cell-free extracts may be limiting in vivo. Mapping of the initiation points of lagging strand synthesis in vivo and in vitro showed that DNA synthesis initiates from specific sites within the pLS1 ssoA. These results demonstrate that specific initiation of replication can occur from the pLS1 ssoA in S. aureus although it plays a minimal role in lagging strand synthesis in vivo. Therefore, the poor functionality of the pLS1 in vivo in a nonnative host is caused by the low efficiency rather than a lack of specificity of the initiation process. We also have identified ssDNA promoters and mapped the primer RNAs synthesized by the S. aureus and Bacillus subtilis RNA polymerases from the pE194 and pLS1 ssoAs. The S. aureus RNA polymerase bound more efficiently to the native pE194 ssoA as compared with the pLS1 ssoA, suggesting that the strength of RNA polymerase-ssoA interaction may play a major role in the functionality of the ssoA sequences in Gram-positive bacteria.

A comparison of the volume and pH of gastric contents of obese and lean surgical patients.

UNLABELLED: Obese surgical patients are typically considered to be more likely than lean patients to possess high-volume and low-pH (HVLP) gastric contents after a standard preoperative fast, based on a study of a population predominately consisting of patients receiving intramuscular preoperative sedation. We revisited this issue in a study population of 256 fasted surgical patients, of which 232 received no preoperative antacid or gastric prokinetic drug. Immediately after endotracheal intubation, an 18-French sump tube was placed, and gastric contents were withdrawn. Subjects' gastric contents were defined as HVLP if they exhibited a combination of a volume > 25 mL and a pH < 2.5. Obesity was defined as a body mass index > 30. Among nonmedicated obese patients, the proportion with HVLP gastric contents was 20 of 75 (26.6%). The proportion of lean patients with HVLP gastric contents was 66 of 157 (42.0%). The difference between the HVLP proportions for these two groups was found to be significant (P < 0.05) using chi 2 analysis. Obesity seems to be associated with a significantly decreased risk of HVLP gastric contents among surgical patients with no history of gastroesophageal pathology after a normal interval of preoperative fasting. IMPLICATIONS: Previous studies have shown that obese surgical patients have a greater volume of acidic stomach contents than lean patients, despite a routine preoperative fast. We have reexamined this issue and found that among otherwise healthy, fasted, obese surgical patients, there is a lower incidence of combined high-volume, low-pH stomach contents compared with lean patients.

Lagging-strand replication from the ssoA origin of plasmid pMV158 in Streptococcus pneumoniae: in vivo and in vitro influences of mutations in two conserved ssoA regions.

The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin, sso. We have used the pMV158-derivative plasmid pLS1 (containing the ssoA type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the ssoA (the recombination site B [RS(B)] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication in Streptococcus pneumoniae. Cells containing plasmids with mutations in the RS(B) accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RS(B) region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.

Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis.

Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.

Hysterectomy: indications, alternatives and predictors.

Hysterectomy, the most common major nonobstetric operation, is performed in more than 570,000 women in the United States each year. Although the number of hysterectomies has decreased in recent years, many authorities believe that hysterectomy is often unnecessary and unjustified. There is no universally accepted set of criteria regarding the appropriate indications for hysterectomy. The main indications for hysterectomy include the following conditions: uterine leiomyomas, dysfunctional uterine bleeding, endometriosis/adenomyosis, chronic pelvic pain and genital prolapse. Current literature, however, routinely recommends conservative management of most nonmalignant gynecologic conditions, with hysterectomy reserved for refractory cases. Several nonmedical factors, such as patient race, age, geographic location, medical history and background, as well as health care provider characteristics, such as time since completion of training, gender, and affiliation with teaching hospitals, are also associated with hysterectomy rates.

Lagging-strand origins of the promiscuous plasmid pMV158: physical and functional characterization.

The streptococcal plasmid pMV158 replicates by a rolling circle mechanism, which involves the generation of single-stranded plasmid DNA intermediates. This plasmid has the unique feature of having two lagging-strand origins of replication. One of these origins, termed ssoU, is functional in Streptococcus pneumoniae and in Bacillus subtilis in an orientation-dependent manner. The other origin, ssoA, is only functional in the former host. RNA polymerase seems to be involved in the initiation of the conversion of single- to double-stranded plasmid DNA from both ssoA and ssoU. Mutational and deletion analyses have allowed us to define ssoA as being within a highly structured, non-coding 199 bp region. Within this region, two elements which are conserved in several rolling-circle replicating plasmids are located, the recombination site RSB and a 6 base consensus sequence. Both elements may play a role in the conversion of single- to double-stranded plasmid DNA.

Surface-skimming stoneflies: a possible intermediate stage in insect flight evolution.

Insect wings appear to have evolved from gills used by aquatic forms for ventilation and swimming, yet the nature of intermediate stages remains a mystery. Here a form of nonflying aerodynamic locomotion used by aquatic insects is described, called surface skimming, in which thrust is provided by wing flapping while continuous contact with the water removes the need for total aerodynamic weight support. Stoneflies surface skim with wing areas and muscle power output severely reduced, which indicates that surface skimming could have been an effective form of locomotion for ancestral aquatic insects with small protowings and low muscle power output.