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Neutropenia is a common sequela of neonatal sepsis. Recent clinical trials have shown the beneficial effects of colony-stimulating factors (CSFs) on outcome in this group, but the exact mechanism remains unknown. Neonates and mothers who were at high-risk for infection were recruited for cord blood sampling in a university tertiary referral maternity hospital. Neonatal and adult neutrophils were evaluated for their ability to combat bacterial infection by examining their functional activity (CD11b and reactive oxygen intermediates) and their persistence at inflammatory sites (apoptosis). The mechanism for altered apoptotic responses was assessed by caspase activation assays, X chromosome-linked inhibitor of apoptosis protein expression, and cytosolic cytochrome c release. Although granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly delayed neutrophil apoptosis in normal adults, only G-CSF had a similar effect in normal neonates. Neutrophils from neonates who are at high risk for infection are unresponsive to the antiapoptotic effects of G-CSF or GM-CSF, unlike maternal neutrophils, which have delayed apoptosis in response to GM-CSF. However, CD11b expression and reactive oxygen intermediate production were significantly increased in normal neonatal neutrophils that were incubated with GM-CSF versus controls but not G-CSF or lipopolysaccharide. Decreased cytosolic cytochrome c release and caspases 3 and 9 activity are associated with the CSF-mediated delay in apoptosis in adults but not in newborns. The antiapoptotic X chromosome-linked inhibitor of apoptosis protein is up-regulated in neonates compared with adults and may mediate their differential spontaneous apoptosis. These results have important implications for the use of CSFs in neonatal sepsis, as responses differ from those seen in adults. Further delineation of neonatal neutrophil responses to CSFs may improve their therapeutic potential.

Objectives: To develop a novel beta-galactosidase leakage assay for Staphylococcus aureus and to evaluate this alongside other simple methods for detection of agents that cause membrane damage in staphylococci., Methods: Using a PCR-based approach, a derivative of S. aureus RN4220 was constructed carrying the Escherichia coli lacZ gene under the control of the strong staphylococcal promoter, cap1A. Leakage of beta-galactosidase (BG) from this strain was examined after exposure for 10 min to various membrane-damaging agents at 4 x MIC, using a fluorescence assay and the substrate 4-methylumbelliferyl-beta-d-galactoside. Other assays for membrane damage involving protoplast lysis (PL), leakage of material absorbing at 260 nm (OD) and ATP release as well as the BacLight (BL) assay were carried out using established methods., Results: All the assays, with the exception of the PL assay, detected membrane damage induced by cetyltrimethylammonium bromide, nisin, clofazimine and protegrin IB-367. However, the ability to detect membrane damage induced by these agents differed between the assay systems. The assays also varied considerably in their signal-to-noise ratio, with the ATP assay providing values for nisin approaching 100-fold that of the control., Conclusions: The PL assay is unsuitable for detection of membrane-damaging agents in S. aureus. The other assays, including the BG assay, detect membrane damage. The OD assay should be sufficient for most purposes since it is effective, rapid and cheap to perform. Studies requiring maximum sensitivity and discrimination should employ the ATP assay.

Chromosomal resistance to mupirocin in clinical isolates of Staphylococcus aureus arises from V(588)F or V(631)F mutations in isoleucyl-tRNA synthetase (IRS). Whether these are the only IRS mutations that confer mupirocin resistance or simply those that survive in the clinic is unknown. Mupirocin-resistant mutants of S. aureus 8325-4 were therefore generated to examine their ileS genotypes and the in vitro and in vivo fitness costs associated with them before and after compensatory evolution. Most spontaneous first-step mupirocin-resistant mutants carried V(588)F or V(631)F mutations in IRS, but a new mutation (G(593)V) was also identified. Second-step mutants carried combinations of previously identified IRS mutations (e.g., V(588)F/V(631)F and G(593)V/V(631)F), but additional combinations also occurred involving novel mutations (R(816)C, H(67)Q, and F(563)L). First-step mupirocin-resistant mutants were not associated with substantial fitness costs, a finding that is consistent with the occurrence of V(588)F or V(631)F mutations in the IRS of clinical strains. Second-step mutants were unfit, but fitness could be restored by subculture in the absence of mupirocin. In most cases, this was the result of compensatory mutations that also suppressed mupirocin resistance (e.g., A(196)V, E(190)K, and E(195)K), despite retention of the original mutations conferring resistance. Structural explanations for mupirocin resistance and loss of fitness were obtained by molecular modeling of mutated IRS enzymes, which provided data on mupirocin binding and interaction with the isoleucyl-AMP reactive intermediate.

Fusidic acid-resistant epidemic Staphylococcus aureus strains causing impetigo bullosa have been reported in Scandinavia. We show that these strains form part of a European epidemic clonotype that carries the fusB determinant. In contrast, resistance to fusidic acid in a collection of nonepidemic strains resulted primarily from mutations in fusA.

Increasing numbers of industrial, agricultural and natural chemicals are present in sewage effluent and are known to elicit toxic effects in laboratory exposures, but little is known of their combined sub-lethal effect in the field. In this study, a combination of esterase activity and ventilation rate assays was performed to determine the neurological and physiological function of the freshwater crustacean Asellus aquaticus (L.) at sites above and below a sewage treatment works (STW). Cholinesterase and carboxylesterase activities were significantly inhibited (n=8, P<0.05) and ventilation rates increased (n=8, P=0.0001) in A. aquaticus at STW sites compared to those from reference sites, indicating a decrease in neurological and physiological function. The ecological relevance of these findings for the population dynamics of the organisms in the field is discussed.

Objectives: We sought to investigate the anti-staphylococcal activity of indolmycin, with particular emphasis on comparing its activity with fusidic acid and mupirocin., Methods: Established procedures were used to examine the activity of indolmycin against a range of clinical isolates, including strains resistant to fusidic acid and mupirocin. Indolmycin-resistant mutants were recovered and characterized phenotypically and genotypically., Results: Indolmycin was bacteriostatic and demonstrated good activity against MSSA (methicillin-susceptible Staphylococcus aureus), MRSA (methicillin-resistant S. aureus) and VISA (vancomycin-intermediate S. aureus), including strains resistant to mupirocin or fusidic acid. Spontaneous indolmycin-resistant mutants occurred at a lower frequency than those selected by mupirocin or fusidic acid and exhibited no cross-resistance with the comparative drugs. High-level resistance (indolmycin MIC 128 mg/L) that was associated with an H43N mutation in tryptophanyl-tRNA synthetase (TrpS), the target enzyme of indolmycin, resulted in loss of bacterial fitness. However, the locus responsible for low-level indolmycin resistance (indolmycin MICs 8-32 mg/L) was not identified., Conclusions: Indolmycin is a potent anti-staphylococcal agent, which exhibits activity against mupirocin- and fusidic acid-resistant strains. Indolmycin might be a candidate for development as a topical agent in the treatment of staphylococcal infections and nasal carriage of MRSA.

The defining property of an antibacterial agent is its ability to selectively interfere with bacterial growth and/or survival. Consequently, a considerable and crucial part of the preclinical evaluation of any novel antibacterial drug involves judging and characterising its effects on bacteria in vitro. These critical stages in drug development are sometimes made to appear somewhat trivial, sandwiched as they are between the highly demanding antibacterial discovery process and the formidable task of demonstrating safety and efficacy in vivo. However, careful biological evaluation in vitro is key to quantifying and understanding the basis of the antibacterial activity, providing preliminary indications and evaluations of therapeutic potential, assessing the likelihood for the development of bacterial resistance, guiding chemical refinement and assisting subsequent stages of the appraisal of any new antibacterial drug. This review covers concepts in, and strategies for, the in vitro microbiological and molecular evaluation of antibacterial drug candidates.

Labor is a mild proinflammatory state that is associated with fetal leukocytosis. Elective cesarean section has been linked with increased neonatal morbidity, which may be partially immune mediated. We hypothesized that labor may alter neutrophil phenotype and thereby decrease neonatal complications. We characterized neutrophil function and survival in normal neonates after either uncomplicated vaginal delivery (VD) or elective cesarean section (CS) without labor. Spontaneous neutrophil apoptosis is delayed in cord blood neutrophils of neonates after normal labor (VD) compared with CS, as assessed by propidium iodide DNA incorporation using flow cytometry. This demonstrates their ability to maintain an inflammatory response. CD11b expression on neonatal neutrophils after CS is decreased, providing further evidence of altered activation or priming. Lipopolysaccharide responsiveness, characterized by CD11b and apoptosis, is similar in VD and adults, but CS-derived neutrophils are unresponsive. Baseline TLR-4 levels are elevated in CS in contrast to the other groups, although expression is not up-regulated by lipopolysaccharide co-incubation. Neonatal neutrophil survival and function are altered by labor and may increase antibacterial function and neutrophilia. This suggests that labor of any duration may be immunologically beneficial to the normal term neonate.

The aim of this study was to test the hypothesis that ongoing aspirin therapy preserves neutrophil apoptosis after cardiac surgery with cardiopulmonary bypass (CPB) by a cyclooxygenase mechanism. Twenty patients undergoing coronary revascularization with CPB were enrolled in a prospective cohort study. Patients who had continued taking 300 mg of aspirin until the day before surgery (n = 10) were compared with 10 patients not taking aspirin or who had discontinued it more than 5 days before surgery. Neutrophils were isolated from arterial blood before and 6 h after surgery and apoptosis was measured after 24 h in culture using flow cytometry. Serum was collected and assessed for IL-6, IL-8 and PGE2 by enzyme-linked immunoabsorbant assay. Patients were followed for clinical indices of sepsis for 7 days postoperatively. Spontaneous rates of neutrophil apoptosis were significantly reduced in postoperative compared with preoperative samples. There was no difference between aspirin and control preoperative neutrophil apoptosis rates (23.0% +/- 11.3% vs. 23.0% +/- 20.7%, P = 0.99). Postoperative neutrophil apoptosis was delayed in control patients (3.6% +/- 1.2% apoptosis), but this was significantly (P = 0.045) reversed in the aspirin-treated group (7.2% +/- 5.1% apoptosis). There were lower postoperative PGE2 levels in the aspirin group (136 +/- 69 pg/mL vs. 372 +/- 210 pg/mL, P = 0.04). There was no difference in clinical indices of sepsis. We conclude that the delay in postoperative neutrophil apoptosis is significantly preserved in patients taking 300 mg of aspirin on the day before surgery. This was associated with greater inhibition of PGE2, consistent with the hypothesis that aspirin exerts its effect on apoptosis after CPB via a cyclooxygenase-mediated mechanism.

Inflammatory mediators delay neutrophil apoptosis, which contributes to the persistence of inflammation. The mechanisms responsible for this delay and resistance to Fas antibody-induced apoptosis are unknown but are dependent on protein synthesis. These proteins have been shown to inhibit caspase activity central to the induction of apoptosis. The inhibitors of apoptosis proteins have been shown to inhibit caspase activity and prevent apoptosis in a number of cellular systems. We hypothesize that the regulation of neutrophil apoptosis is dependent on the expression of the IAPs. c-IAP-1, c-IAP-2, and XIAP are expressed in the neutrophil at both the mRNA and protein level, but their relative protein expression is low compared with other cell types. The in vitro aging of human neutrophils results in their induction of apoptosis, which is associated with the loss of c-IAP-1 expression. The pancaspase inhibitor (zVAD-FMK) and LPS, which delay spontaneous apoptosis, also prevented this loss of c-IAP-1. Gene chip microarrays have shown that LPS increases c-IAP-1 and c-IAP-2 mRNA expression in neutrophils. However, this does not correspond to an increase in protein. Neutrophils from septic patients with delayed apoptosis show an increase in XIAP, with no change in cIAP-1 or cIAP-2 mRNA, demonstrating that different mechanisms contribute to the delay in neutrophil apoptosis. This study demonstrates that the loss of IAP expression may facilitate the induction of neutrophil apoptosis, and preventing this loss of IAP expression may represent a more significant contribution to delayed apoptosis rather than an increase in their expression.

HL-60 cell differentiation into neutrophil like cells is associated with their induction of apoptosis. We investigated the cellular events that occur pre and post mitochondrial permeability transition to determine the role of the mitochondria in the induction of differentiation induced apoptosis. Pro-apoptotic Bax was translocated to and cleaved at the mitochondrial membrane in addition to t-Bid activation. These processes contributed to mitochondrial membrane disruption and the release of cytochrome c and Smac/DIABLO. The release of cytochrome c was caspase independent, as the caspase inhibitor Z-VAD.fmk, which inhibited apoptosis, did not block the release of cytochrome c. In contrast, the release of Smac/DIABLO was partially inhibited by caspase inhibition indicating differential release pathways for these mitochondrial pro-apoptotic factors. In addition to caspase inhibition we assessed the effects of the Bcl-2 anti-apoptotic family on differentiation induced apoptosis. BH4-Bcl-xl-TAT recombinant protein did not delay apoptosis, but did block the release of cytochrome c and Smac/DIABLO. Bcl-2 over-expression also inhibited differentiation induced apoptosis but was associated with the inhibition of the differentiation process. Differentiation mediated mitochondrial release of cytochrome c and Smac/DIABLO, may not trigger the induction of apoptosis, as BH4-Bclxl-TAT blocks the release of pro-apoptotic factors from the mitochondria, but does not prevent apoptosis.

Objectives: Infections caused by Staphylococcus aureus might be treated with agents whose primary indications are for other infections. Clofazimine, an established anti-mycobacterial drug, could be such a candidate. However, the anti-staphylococcal properties of clofazimine have not been fully described and its mode of action, possibly involving inhibition of both RNA polymerase and a membrane-located target, has not been explored in detail. We have now conducted experiments to address these issues., Methods: Using established procedures, we examined the activity of clofazimine against a range of clinical isolates of S. aureus and determined whether it was bactericidal, exhibited a post-antibiotic effect (PAE), or interacted synergically with other agents. The potential for emergence of clofazimine-resistant mutants was also examined. Mode of action studies involved macromolecular synthesis assays, cross-screening against rifampicin-resistant mutants, susceptibility of RNA polymerase to clofazimine in vitro and several methods to detect drug-induced membrane damage., Results: Clofazimine demonstrated good anti-staphylococcal activity encompassing MSSA, MRSA and GISA. It was bactericidal and resistant mutants could not be isolated. Clofazimine did not exhibit a PAE and failed to act synergically with other drugs. No evidence for specific inhibition of RNA polymerase was obtained. Clofazimine caused non-specific inhibition of DNA, RNA and protein synthesis, consistent with membrane-damaging activity that was detected in three independent assays for membrane disrupting agents., Conclusions: Clofazimine is a potent anti-staphylococcal agent. It appears to be a membrane-disrupting agent and does not inhibit RNA polymerase.

Objective: We studied the effect of labor on maternal neutrophil phenotype., Study Design: Neutrophil apoptosis with inflammatory cytokines and the expression of CD11b, CD34 and toll-like receptor 4 (TLR4) were assessed with flow cytometry in women at uncomplicated vaginal delivery (VD), and elective cesarean section (ElCS) without labor, emergency cesarean section with (EmCSL+) or without (EmCSL-) labor., Results: Spontaneous neutrophil apoptosis is delayed in maternal neutrophils after VD, EmCSL+ or EmCSL- versus ElCS. In all groups lipopolysaccharide delayed apoptosis and increased CD11b expression. Elevated TLR4 expression in ElCS was associated with lipopolysaccharide responsiveness. CD34 was diminished in VD, indicating increased cell maturity., Conclusion: Normal labor primes the neutrophil and may enhance antibacterial function by inducing a mild maternal inflammatory response syndrome. Delayed neutrophil apoptosis may promote the neutrophilia seen in women after VD. We suggest that labor of any duration may be immunologically beneficial to the parturient.

The potential maternal and developmental toxicity of dimethylpiperidone (DMPD) was assessed in rats. Groups of 25 mated female Crl:CD (SD)IGS BR rats were exposed by inhalation (whole-body exposures) for approximately six hours per day over days 7-21 of gestation (G); day 1G was the day of copulation plug detection. The exposure levels were 0, 52, 260, or 340 (vapor plus aerosol) mg/m3 DMPD. During the in-life portion, body weights, food consumption, and clinical observation data were collected. On day 22G, the dams were euthanized and examined for gross external and internal alterations. The uterine contents were described and the fetuses were weighed and examined for external, visceral, and skeletal alterations. Maternal toxicity was seen at both 260 and 340 mg/m3. At 340 mg/m3, evidence of maternal toxicity included mortality, increased clinical observations, and decreased body weight and food consumption. At 260 mg/m3, maternal toxicity was limited to increased clinical observations and decreased food consumption. Developmental toxicity was also produced at 260 and 340 mg/m3. At 340 mg/m3, evidence of developmental toxicity included decreased fetal weight, increased embryofetal lethality with concomitant reductions in litter size, and increased fetal malformations and variations. At 260 mg/m3, effects in fetuses were limited to slightly decreased fetal weight and increased fetal variations; additionally, one litter from this level consisted entirely of resorptions. There were no compound-related effects in either dams or fetuses at 52 mg/m3. It was, therefore, concluded that DMPD was not selectively toxic to the rat conceptus.

Mutators may present an enhanced risk for the emergence of antibiotic resistance in bacteria during chemotherapy. Using Escherichia coli mutators as a model, we evaluated their ability to develop resistance to antibiotics routinely used for the treatment of urinary tract infections (UTIs). Under conditions that simulate therapeutic drug concentrations in humans, low-level resistance to trimethoprim, gentamicin, and cefotaxime emerged more frequently in mutators than normal strains. Resistance to trimethoprim in both cell types arose from a single point mutation in folA (Ile94-->Leu) and cefotaxime resistance resulted from loss of outer membrane porin OmpF. The mechanisms of gentamicin resistance could not be defined, but resistance did not result from mutations in ribosomal protein L6 (rplF). Although similar mechanisms of low-level antibiotic resistance probably arise in these strains, mutators are a risk factor because the increased generation of mutants with low-level resistance enhances the opportunity for subsequent emergence of high-level resistance.

Objectives and Methods: Failure to eradicate nasal carriage of a glycopeptide-intermediate Staphylococcus aureus (strain GISA-2) with mupirocin was recently attributed to a mutation that confers low-level mupirocin resistance. To identify this mutation the ileS genes of GISA-2 and its mupirocin-susceptible progenitor GISA-1 were sequenced. For comparison, the ileS genes of 10 laboratory-derived mupirocin-resistant mutants of the GISA strain Mu50 were also examined. The fitness of GISA-2 and mupirocin-susceptible GISA-1, as well as Mu50 and its mupirocin-resistant derivatives, were compared by evaluation of growth rates and performance in mixed-culture competition assays., Results: The point mutation V588F in the isoleucyl-tRNA synthetase was identified from the ileS sequences of GISA-2 and mupirocin-resistant mutants of Mu50. The V588F mutation was not associated with a significant fitness burden., Conclusions: The low fitness cost of the V588F substitution in isoleucyl-tRNA synthetase is consistent with the frequent appearance and maintenance of this mutation in mupirocin-resistant clinical isolates, including GISA-2.

Women are conferred with greater immunologic and survival benefits compared to men. Female sex steroids contribute to this sexual dimorphism. Furthermore, during human pregnancy when female sex hormones are elevated, neutrophil apoptosis is delayed. This study examines the specific effects of estradiol and progesterone on neutrophil apoptosis and function in healthy adult men and women. We also examined the contribution of these hormones to the persistence and resolution of an inflammatory response. Spontaneous apoptosis was significantly decreased in women compared with men. Physiologic doses of estradiol and progesterone caused a further delay in spontaneous apoptosis in both men and women but did not diminish Fas antibody-induced apoptosis. The delay in apoptosis was mediated at the level of the mitochondria with decreased release of cytochrome c, which may alter caspase cleavage and activity. There were no associated alterations in neutrophil CD11b, but production of reactive oxygen intermediates (ROIs) in women was increased. Thus, female sex hormones mediate delayed neutrophil apoptosis in both sexes and enhance female intracellular production of ROIs. Modulating hormonal responses may be an effective therapeutic tool in combating inflammatory diseases.

Bacteria contain a number of error prevention and error correction systems that maintain genome stability. However, strains exhibiting elevated mutation frequencies have recently been reported amongst natural populations of pathogenic Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Neisseria meningitidis, Helicobacter pylori and Streptococcus pneumoniae. The majority of naturally occurring, strong mutators contain defects in the methyl-directed mismatch repair (MMR) system, with mutations in mutS predominating. MMR-deficient strains possess superior genetic backgrounds for the selection of some antibiotic-resistance mutations since mutation frequencies up to 1000-fold higher than normal strains have been reported, and resistance levels achieved in mutators can be greater than those arising in non-mutator hosts. MMR is a major constraint to interspecies recombination events. Removal of this barrier, as in the case of MMR defective mutators, also enhances the frequency of horizontal gene transfer, which is an important mechanism of acquired drug resistance in bacteria. Permanent global mutator status is associated with loss of fitness as mutators accumulate deleterious mutations more frequently than non-mutators. Fitness limitations of mutators may be overcome simply by the high bacterial cell densities that can be achieved during acute infection or by the adoption of transient mutator status. Mutators are a risk factor during the treatment of bacterial infections as they appear to enhance the selection of mutants expressing high- and low-level antibiotic resistance and have the capacity to refine existing plasmid-located resistance determinants.

Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the apoptotic phenotype. An important route to caspase activation proceeds via assembly of the "apoptosome" as a result of the cell stress-associated release of mitochondrial cytochrome c. Previous studies have shown that primary neutrophils are largely incapable of mitochondrial respiration, suggesting that these cells either lack functional mitochondria or possess a defective respiratory chain. This prompted us to examine whether neutrophils retain an intact cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) pathway to caspase activation and apoptosis. We show that primary human neutrophils contain barely detectable levels of cytochrome c as well as other mitochondrial proteins. Surprisingly, neutrophil cell-free extracts readily supported Apaf-1-dependent caspase activation, suggesting that these cells may assemble cytochrome c-independent apoptosomes. However, further analysis revealed that the trace amount of cytochrome c present in neutrophils is both necessary and sufficient for Apaf-1-dependent caspase activation in these cells. Thus, neutrophils have a lowered threshold requirement for cytochrome c in the Apaf-1-dependent cell death pathway. These observations suggest that neutrophils retain cytochrome c for the purpose of assembling functional apoptosomes rather than for oxidative phosphorylation.

A mixture of 1,3-dimethyl-2-piperidinone and 1,5-dimethyl-2-piperidinone (DMPD) (approximately 63-37 parts by weight) was tested for its inhalation toxicity in rats following 90-day repeated exposures. Male and female rats were exposed whole-body to either 0, 51, 230, or 310 mg/m(3) DMPD for 6 h/day, 5 days/weak for 90 days. Clinical signs, growth, clinical pathology, tissue pathology, neurobehavior, neuropathology, and semen quality were evaluated. No compound-related adverse effects were noted in clinical signs, body weights, food consumption, clinical laboratory evaluations, neurobehavioral evaluations, neuropathology, or sperm counts. Laryngeal changes consisting of minimal squamous epithelial hyperplasia and degeneration/necrosis of the cartilage were present in male and female rats exposed to 310 mg/m(3) both immediately following exposure and after the 1-month recovery period Male rats exposed to DMPD had increased relative kidney weights, increased formation of hyaline droplets and granular casts, and increased incidence of chronic progressive nephropathy. These kidney effects are consistent with increased accumulation of the urinary protein alpha(2 mu)-globulin, which has been well essential for several xenobiotics. The subsequent increased incidence of progressive nephropathy was specific to male rats with the alpha(2 mu) syndrome. Male and female rats exposed to 230 or 310 mg/m(3) had centrilobular hepatocellular hypertrophy, and male rats exposed to 310 mg/m(3) had increased relative liver weights. These liver changes were reversible following the recovery period and were considered not to represent adverse toxicological effects of treatment. Since the male rat-specific renal findings do not connote adversity for man and are net considered relevant to human hazard assessment, the no-observed-effect level in male and female rats was 230 mg/m(3), based on the microscopic changes in the larynx exposed to 310 mg/m(3).

The antibacterial properties of novel quinoline-indole (QI) agents were examined. QI agents demonstrated potent bactericidal activities against Staphylococcus aureus, killing by lytic and nonlytic mechanisms. S. aureus mutants resistant to a lytic QI agent (SEP 155342) and a nonlytic QI agent (SEP 118843) arose at frequencies of 1.4 x 10(-9) and 1.2 x 10(-8), respectively, by selection at four times the MICs. Mutants resistant to QI agent SEP 155342 were unstable, but mutants resistant to QI agent SEP 118843 displayed stable resistance. Mutants resistant to QI agent SEP 118843 were not cross resistant to other inhibitors, including QI agent SEP 155342. Addition of QI agents SEP 118843 and SEP 155342 at four times the MIC caused nonspecific inhibition of several macromolecular biosynthetic pathways in S. aureus. Within 10 min, QI agents SEP 118843 and SEP 155342 both interfered with bacterial membrane integrity, as measured by uptake of propidium iodide. Agents from the two classes of the QI agents probably kill staphylococci by separate mechanisms which, nevertheless, both involve interference with cytoplasmic membrane function. Precise structure-activity relationships for the division of QI agents into two classes could not be determined. However, lytic activity was often associated with substitution of a basic amine at position 4 of the quinoline nucleus, whereas compounds with nonlytic activity usually contained an aromatic ring with or without a methoxy substituent at position 4. Nonlytic QI agents such as SEP 118843 may possess selective activity against the prokaryotic membrane since this compound failed to lyse mouse erythrocytes when it was added at a concentration equivalent to four times the MIC for S. aureus.

Purpose: Unilateral ureteral obstruction results in decreased blood flow and tissue loss in the obstructed kidney. This condition is compensated by increased perfusion and trophic changes in the contralateral kidney. Vascular mediators' effects are central to these changes and of these mediators endothelin is the most potent vasoconstrictor known. We explored the role of endothelin and the effects of endothelin receptor blockade in unilateral ureteral obstruction., Materials and Methods: Rats were subjected to unilateral ureteral obstruction for 24 hours. Endothelin-1 mRNA expression was determined in kidney extracts from control, obstructed and contralateral (nonobstructed) kidneys. Cortical and medullary blood flow was determined in control and obstructed kidneys, and after endothelin receptor blockade with bosentan. Apoptotic rates were determined in control and obstructed kidneys after treatment with bosentan using the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end technique. RESULTS After 24 hours of unilateral ureteral obstruction endothelin-1 mRNA expression was increased in the obstructed kidney and decreased in the contralateral kidney. Obstruction was associated with a decrease in renal blood flow, which was reversed by endothelin receptor blockade. Unilateral ureteral obstruction also increased apoptosis, which was blocked by endothelin inhibition., Conclusions: Endothelin expression increases in the obstructed kidney. Inhibition of its action protects against vascular and cellular changes. Decreased endothelin expression in the contralateral kidney may facilitate trophic changes and compensatory increased blood flow.

The antimicrobial properties of cephalosporin P1, an antibiotic structurally related to fusidic acid, were examined. Cephalosporin P1 exhibited potent activity against methicillin-sensitive Staphylococcus aureus, methicillin-resistant S. aureus and vancomycin-intermediate S. aureus. Mutants of S. aureus resistant to cephalosporin P1 arose with a frequency of 1.6 x 10(-6) for selections at 4 x MIC, a frequency similar to that for fusidic acid. The mutations conferred cross-resistance to fusidic acid and mapped in fusA, the gene encoding elongation factor G. Cross-resistance between cephalosporin P1 and fusidic acid also occurred for S. aureus fusA mutants selected with fusidic acid, and in fusidic acid-resistant clinical isolates. Plasmid pUB101, which mediates resistance to fusidic acid in S. aureus, also conferred resistance to cephalosporin P1. Escherichia coli was intrinsically resistant to both fusidic acid and cephalosporin P1, but deletion of the AcrAB efflux pump resulted in susceptibility to both antibiotics. Although complete cross-resistance between fusidic acid and cephalosporin P1 was demonstrated, the nature and location of fusA mutations in S. aureus when cephalosporin P1 was the selective agent frequently differed from those selected with fusidic acid. This may reflect differences in the interaction of the two antibiotics with the translational apparatus, which results in the selection of separate mutation classes for each antibiotic. Furthermore, in three of 14 mutants selected with fusidic acid, resistance was attributed to mutations lying outside fusA. In contrast, mutations in 10 mutants selected with cephalosporin P1 were all located in fusA.

Background: Previous studies demonstrate that androgen is capable of exerting a protective effect in the androgen-sensitive human prostate cancer cell line LNCaP. Limited studies, however, have addressed the underlying mechanisms involved, in particular the effects of androgen on both pro- and anti-apoptotic gene expression., Methods: We investigated the effects of androgen on apoptotic sensitivity and the expression of the caspases and specific members of the Bcl-2 family in the LNCaP cell line. The effects of androgen on NF-kappaB activation were also investigated by using a gel mobility shift assay., Results: 5alpha-Dihydrotestosterone (5-alphaDHT) conferred resistance to radiation (5 Gy) and etoposide-induced apoptosis in the LNCaP cell line. This finding was associated with a time-dependent decrease in the expression of the caspases and pro-apoptotic Bcl-2 family members. 5-alphaDHT did not confer protection against apoptosis in the LNCaP line transfected with the IkappaB super repressor of NF-kappaB, nor in the androgen insensitive PC-3 and DU-145 cell lines., Conclusion: The ability of 5-alphaDHT to raise the apoptotic threshold in the LNCaP cell line by altering specific pro-apoptotic gene expression suggests that androgen may serve as a general survival signal against diverse pathways that ultimately signal for apoptosis. We hypothesize that NF-kappaB serves as an important mediator in androgen survival signaling., (Copyright 2002 Wiley-Liss, Inc.)

Populations defective in mismatch repair that exhibit elevated mutation frequencies to antibiotic resistance have been reported amongst pathogenic Gram-negative bacteria. Whether such mutators occur widely in clinical isolates of Gram-positive species, and in important pathogens such as Staphylococcus aureus, is unknown. Insertional inactivation of the mutS gene of S. aureus RN4220 by targeted plasmid integration produced a strain with mutation frequencies for antibiotic resistance up to 78-fold greater than those exhibited by RN4220, thereby providing proof of the concept that staphylococcal mutators could arise. Subsequently, 493 clinical S. aureus isolates were examined for the presence of mutators. However, no strain exhibited a > or =10-fold increase in mutation frequency compared with laboratory strain 8325-4. Detailed phenotypic and genotypic analysis of vancomycin-intermediate S. aureus strain Mu50 was performed, since the published genome sequence of this organism suggests that mutS is inactive as a result of a frameshift. However, elevated mutation frequencies were not observed in Mu50, and re-sequencing of a portion of mutS from this strain indicated that this gene was intact. Transient increases in mutation frequency during the stationary phase of growth occur in other bacteria, although no such increases were observed in S. aureus. We conclude that neither permanent increases in the basal mutation frequency, nor transient increases in mutation frequency under starvation, are likely to play a significant role in the development of antibiotic resistance in S. aureus.

Background: The earliest insult to the kidney following the onset of ureteral obstruction is a marked elevation in collecting system pressure. This imparts a mechanical stress that is transmitted directly from the collecting system to the kidney substance. Renal tubular injury is the principal functional and histological change encountered, with glomerular changes being less marked and occurring later. Nitric oxide (NO) has been shown to protect against renal injury in UO, but its mode of action has not been clearly defined., Methods: MDCK (canine) and HK-2 (human) renal tubular cells were grown under control conditions or subjected to mechanical strain for periods of 24 and 48 hours. Cells were studied treated with or without Fas-antibody, etoposide or diethyl maleate (DEM) alone or in combination with NG-monomethyl l-arginine (L-NMMA), sodium nitroprusside (SNP) or l-arginine. Cell proliferation and apoptosis was determined using propidium iodide DNA staining. NO production and inducible NO synthase (iNOS) expression were measured by the Griess reaction and Western blotting, respectively., Results: Cells subjected to mechanical strain displayed a decrease in the proportion of cells undergoing cell division. They also showed an increased susceptibility to apoptosis. Associated with this was a decrease in Bcl-2 expression. An increase in iNOS expression was seen in cells subjected to mechanical strain, but no increase in NO production. The cellular effects of mechanical strain were reversed by SNP and l-arginine., Conclusions: Culture of renal tubule cells in an environment of mechanical strain results in an imbalance in homeostasis and a net cell loss. This can be reversed by the administration of an NO donor or precursor.

The responses of hypermutable Escherichia coli strains to selection with antibiotics having different endogenous resistance potentials were determined. Selections with rifampicin or ciprofloxacin at 4 x MIC, i.e. conditions where they act as single target agents against RpoB and GyrA, respectively, demonstrated that some hypermutators generated resistant mutants with frequencies up to 1000-fold higher than normal strains. Furthermore, individual mutants recovered from hypermutable hosts often exhibited higher levels of resistance to the drugs than mutants arising in normal hosts. Exposure to ciprofloxacin at 16 x MIC, i.e. conditions where it has low endogenous resistance potential, failed to select resistant mutants in hypermutable or normal hosts (mutation frequency <10(-11)). Consistent with these findings, the highest estimated mutation frequency for selection at 16 x MIC in a hypermutable host would be 4.4 x 10(-15) (mutT), calculated by determining the individual mutation frequencies for first-step ciprofloxacin resistance and second-step resistance arising in hosts already harbouring single first-step mutations in gyrA at codons 83 or 87. The frequency with which second-step ciprofloxacin resistance mutations arose was suppressed in hypermutators and demonstrated at most a 10-fold increase in mutation rate compared with non-hypermutator hosts. Second-step mutants may contain mutations in mar, since a survey of 170 second-step ciprofloxacin-resistant mutants derived from both hypermutator and non-hypermutator parents demonstrated that they all possessed increased resistance to chloramphenicol, a phenotype associated with mar mutations. Exposure to 4 x MIC of D-cycloserine, cefotaxime or polymyxin B (agents with multiple targets or membrane activity) failed to select resistant mutants in normal or hypermutator hosts (mutation frequency <10(-11)); however, continuous culture in the presence of sub-lethal concentrations of D-cycloserine (0.25 x MIC) selected resistant mutants in hypermutators after c. 33 generations, compared with c. 44 generations in normal hosts. Since hypermutable bacteria occur naturally, our data emphasize that successful new drugs will need to possess low endogenous resistance potentials.

Background: The caspases are the central executioners of apoptosis. The inhibitors of apoptosis proteins (IAPs) are a family of recently described caspase inhibitors. We hypothesised that tumor resistance to apoptosis could be due in part to IAP expression., Methods: The expression of NAIP, cIAP-1, cIAP-2, XIAP, and survivin was investigated in the prostate cancer cell lines LNCaP, PC3, and DU145. RNase protection assays and Western blotting were used to assess RNA and protein expression. Apoptotic susceptibility was determined using etoposide and assessed by propidium iodide (PI) DNA incorporation using flow cytometry., Results: DU145 and PC3 cells were more resistant to apoptosis than LNCaP cells. All the IAPs were identified in the cell lines with variation in IAP expression between different cell types. Immunohistochemistry demonstrated cIAP-1 expression in PC3 cells was nuclear, while the expression of cIAP-2 and XIAP was perinuclear. Growing LNCaP cells in charcoal-stripped or androgen-supplemented medium resulted in no alteration in IAP expression., Conclusions: This study characterises the expression of IAP in three of the most commonly used prostate cancer cells. IAP may make an important contribution to apoptotic resistance in patients with prostate cancer., (Copyright 2002 Wiley-Liss, Inc.)

Human promyelocytic leukaemia cells (HL-60) differentiate into neutrophil-like cells that die spontaneously by apoptosis when treated with retinoic acid (RA). Inhibitors of apoptosis proteins (IAP) bind to and inhibit caspases 3, 7, and 9 activity and the induction of apoptosis. In this study, we demonstrate that undifferentiated HL-60 cells express IAP. During their differentiation, IAP expression is decreased at the mRNA and protein levels. In addition, we show that there is a corresponding increase in the expression and functional activity of active caspases 3 and 9. This activity was associated with the cleavage of XIAP, NAIP, and cIAP-2. Most importantly, we demonstrate that blocking caspase activity does not alter the decrease in IAP protein expression during differentiation but prevents caspase activation, IAP cleavage, and the induction of apoptosis. This result shows that the loss of IAP expression is independent of the induction of apoptosis and is solely related to the differentiation process. However, IAP cleavage is caspase-dependent. Terminal differentiation results in an altered apoptotic phenotype that is associated with the induction of HL-60 cell apoptosis.

The introduction of antibiotics for the chemotherapy of bacterial infections has been one of the most important medical achievements of the past 50 years. However, the emergence of bacterial resistance to antibiotics undermines the therapeutic utility of existing agents, creating a requirement for the discovery of new antibacterial drugs. Several drug discovery strategies have emerged, including incremental improvements to existing antibiotics by chemical manipulation and the search for novel drug targets based on genomic approaches. An alternative strategy seeks to exploit opportunities for drug discovery arising from an understanding of the mode of action of existing antibiotics. Thus biochemical pathways or processes inhibited by antibiotics already in clinical use may nevertheless contain key functions that represent unexploited targets for further drug discovery. A major benefit of employing pathways or processes that are already known to contain drug targets is that proof of principle for drug intervention is already established. This approach to drug discovery is illustrated by reviewing target sites for existing antibiotics and considering how this information might be applied for the discovery of new agents inhibiting peptidoglycan synthesis, tRNA synthesis, transcription and DNA replication

Tetramethylurea (TMU, CAS No. 632-22-4) was tested for its inhalation toxicity in rats following repeated exposures. Male rats were exposed whole-body to TMU for 6 hours/day, 5 days/week for a total of 9 exposures over 2 weeks. Concentrations of 0 (control), 2, 20, and 100 ppm were studied. Four groups of 10 rats each were used to measure clinical signs and growth, clinical pathology (including hematology, biochemistries, and urine analysis), and tissue pathology. One-half of the rats were sacrificed 1 day following the 9th exposure; the other half underwent an 18-day recovery period prior to being sacrificed (recovery group). Body weight gains in rats exposed to 100 ppm were reduced as compared to the controls; no body weight effects were seen in either 2 or 20 ppm rats and no clinical signs of toxicity were observed in rats at any of the levels throughout the study. No compound-related clinical pathology changes were seen in any of the test groups and tissue pathology effects only occurred in the nasal tissue. In rats exposed to 100 ppm, microscopic observations of degeneration, necrosis, and ulceration of olfactory mucosa was seen. These lesions were still present but seen as recovering and healing after the 18-day recovery. Under the conditions of this test, the no-observed-adverse-effect level (NOAEL) was determined to be 20 ppm based upon both body weight changes and upper respiratory tract pathologic changes in 100 ppm rats.

The developmental toxicity of tetramethylurea (TMU) was assessed in rats by inhalation exposure of the test material over days 6-20 of gestation. Groups of 25 mated female Crl:CD BR rats were exposed whole-body for 6 hours/day to concentrations of either 0, 2, 20 or 100 ppm TMU. The dams were euthanized on day 21 and the offspring were weighted, sexed, and examined for external, visceral, and skeletal alterations. Maternal toxicity was demonstrated at both 20 and 100 ppm. Maternal body weights, weight changes, and food consumption were statistically significantly reduced at these concentrations; effects were more pronounced at 100 ppm. There was evidence of developmental toxicity only at 100 ppm. The only finding was a decrease in mean fetal weight. No fetal malformations or variations occurred in fetuses derived from rats exposed to all 3 test concentrations (up to 100 ppm). The maternal no-observed-effect-level (NOEL) was 2 ppm, the fetal NOEL was 20 ppm. Thus, TMU was not considered to be uniquely toxic to the rat conceptus.

Background: Apoptotic resistance to androgen ablation represents a significant problem in the treatment of prostate cancer. Over expression of antiapoptotic proteins such as Bcl-2 and mutations in p53 contribute to this resistance. The caspase family of proteases are central executioners of the cell death pathway. They are expressed in normal prostate secretory epithelial cells. Altered expression may represent an additional component leading to cell resistance. The aim of this study was to determine by immunohistochemistry caspase 3 expression in benign prostatic hyperplasia and prostate cancers., Methods: Twenty-two patients with histologically determined prostate cancer and benign prostatic hyperplasia (BPH) were investigated. All specimens were obtained from patients undergoing surgical resection of the prostate. Immunohistochemical analysis was performed on formalin fixed paraffin embedded sections to assess caspase 3 expression., Results: Caspase 3 was expressed in 18/22 (81.1%) samples, with high expression in BPH which demonstrated staining in both basal and secretory epithelial cells. Increasing grades of prostatic cancer showed a significant loss of expression in secretory epithelial layers and little staining in epithelial cells in high-grade prostatic carcinoma., Conclusions: Altered caspase 3 expression may represent an additional mechanism of apoptotic resistance to androgen ablation. Prostate 47:183-188, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Pentane (CAS No. 109-66-0) is a chemical being used as a co-solvent in a polymer production facility with potential for inhalation exposure in humans. To assess the toxicity of pentane, groups of 10 male rats each were exposed by inhalation, 6 hr/day, 5 days/week for 2 weeks to either 0 (control), 1,000, 3,000 or 10,000 ppm. Five rats per group were killed following the 10th exposure; the remaining 5/group were killed after a 14-day post-exposure recovery period. Parameters investigated were clinical signs of toxicity, functional behavior, body weights, clinical pathology, and gross and microscopic pathology including organ weights. No unusual clinical observations were seen in the pentane-treated rats, and body weights were not altered. Test rats generally exhibited normal behavioral responses in the functional observational battery. Increases in serum calcium and phosphorus concentrations were seen in rats exposed to either 3,000 or 10,000 ppm. These were reversible during the 2-week recovery period. No other clinical pathology changes were observed and no pentane-related tissue pathology was seen in any of the groups. The no-observed-adverse-effect level was 1,000 ppm with reversible clinical pathology changes produced at 3,000 and 10,000 ppm.

Frequencies at which mutants resistant to fusidic acid and/or rifampicin arose in vitro were determined in Staphylococcus aureus strains including methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-intermediate resistant S. aureus (VISA) and hetero-VISA. The concentrations of fusidic acid (30 and 15 mg/L) and rifampicin (16 and 1 mg/L) used for selection were equal to the expected maximum and minimum serum concentrations after an oral regimen of rifampicin 900 mg od, together with fusidic acid 500 mg tds. Resistant mutants arose at a frequency of around 10(-8) for selections with rifampicin, but were undetectable (frequency <10(-11)) for selections with fusidic acid. Mutants were not recovered (frequency <10(-11)) after selections in the presence of both fusidic acid and rifampicin at 30/16 and 15/1 mg/L. Our results suggest that these antibiotics, when used in combination, could have a wider role in the management of staphylococcal infections.

Background: Ureteral obstruction (UO) is characterized by reduced blood flow and loss of tissue mass in the involved kidney(s). Vasoactive mediators interact to produce an initial hyperemia, followed by a sustained decrease in renal blood flow in the obstructed kidney. Nitric oxide (NO) has been shown to play a central role in the acute hyperemic response to UO. Its role in the reduced perfusion of prolonged UO is less studied., Methods: Ureteral obstruction was achieved by ligation of the distal left ureter and maintained for 24 hours. Blood flow was studied in untreated animals and after the administration of the NO synthase (NOS) inhibitor N-mono-methyl L-arginine and the NO donor sodium nitroprusside. Tissue was collected for localization and quantitation of NOS. Serum and renal tissue L-arginine levels were measured in control and UO settings., Results: Blood flow in the obstructed kidney diminished to approximately 50% of control values after 24 hours of UO. NOS blockade led to a further decrease in blood flow. Supplementation with exogenous nitrates restored renal blood flow to levels approaching control values. Serum and tissue L-arginine levels did not change with UO. NOS expression was seen to increase with increasing duration of obstruction, with staining most pronounced in the renal tubules., Conclusions: NO plays a vasodilatory role even in the hypoperfusion of prolonged UO. The administration of exogenous nitrates has a restorative effect on blood flow, suggesting therapeutic potential in UO.

Four chemicals (Dioxole 418, Dioxolane 418, Dioxolane 416 and Dioxolane 456) which are used as stabilizers in highresolution image were tested or both their acute and repeated inhalation toxicity in the rat using nose-only exposures. Acute studies determined the lethal concentrations following a single 4-hour exposure; repeated exposure inhalation studies determined the potency and target tissue(s) following 6-hour/day exposures, 5 days/week for 2 weeks. Each of the chemicals was at least mildly toxic acutely with approximate lethal concentrations of > 1,500 ppm for Dioxole 418, 1,300 ppm for Dioxolane 418, 1,700 ppm for Dioxolane 416, and 4,300 ppm for Dioxolane 456. No specific unusual clinical signs of response were seen in the rats exposed acutely. Repeated exposures with Dioxole 418 and Dioxolane 418 resulted in no evidence of toxicity with NOAEL's being 440 and 500 ppm respectively (the highest concentrations tested). Repeated exposures to 250 ppm Dioxolane 456 were not tolerated with mortalities observed after exposure. Severe bone marrow hypoplasia along with reductions in platelet and neutrophil counts were observed at this concentration with less severe hemopoietic changes seen also at 10 and 51 ppm. The no-effect level for Dioxolane 456 was determined to be 10 ppm in female rats and I ppm in males. The same hemopoietic effects were seen with Dioxolane 416 at exposures of 53 ppm or greater in males but not in females exposed to 53 ppm Dioxolane 416. Hepatocellular hypertrophy and depression of serum alkaline phosphatase activity were seen in male rats exposed to 500 but not 53 ppm Dioxolane 416. Testicular degeneration was also seen in rats exposed to 500 ppm Dioxolane 416. The NOAEL was 5 ppm for the chemical.

Resolving inflammation is a vital step in preventing the persistence of inflammatory disorders. Neutrophils play a major role in tissue damage associated with an inflammatory response. Their death by apoptosis is central to the final resolution of this response. Thiol depletion with diethylmaleate (DEM) or diamide represent important triggers for neutrophil apoptosis. The mechanism by which this process occurs remains unknown. The apoptotic cascade is associated with a number of cellular changes, including caspase activation and mitochondrial permeability. The aims of this study were to determine the role of mitochondrial permeability and the caspase cascade in thiol depletion-induced neutrophil apoptosis. Total cellular glutathione was reduced by DEM and diamide. This reduction was associated with neutrophil apoptosis and an increase in caspase 3 activity. The effects of DEM were blocked by the caspase 3 inhibitor, Z-DEVD-FMK. Mitochondrial permeability that occurred was also increased during this induction of apoptosis. Bongkrekic acid, a mitochondrial membrane stabilizer, inhibited DEM-induced apoptosis. The inhibitors' effects of LPS or GM-CSF on spontaneous neutrophil apoptosis was reversed by DEM, which was mediated by an increase in caspase 3 activity and independent of mitochondrial disruption. Caspase activation is an important step in glutathione depletion-induced apoptosis in resting and inflammatory neutrophils. Regulation of caspase activity may represent a possible target to trigger apoptosis and resolve inflammatory disorders.

Acute pancreatitis (AP) may lead to the development of multiple organ dysfunction syndrome (MODS), especially in severe cases. Resolution of such inflammatory responses is dependent on neutrophil apoptosis. Delays in this apoptotic response are associated with persistent inflammation and subsequent tissue damage. The aim of this study is to determine the effects of AP on neutrophil apoptosis and to investigate the underlying mechanisms involved. Neutrophils and serum were isolated from control (n=10) and from patients with AP (mild, n=35, and severe, n=5). Neutrophil apoptosis was assessed by propidium iodide DNA staining using flow cytometry. Caspase, glutathione-S-transferase (GST), and Mcl-1 protein expression were assessed by SDS-PAGE western blotting. Serum interleukin (IL)-1beta and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were measured by ELISA. Neutrophils isolated from patients with AP show a significant delay in spontaneous neutrophil apoptosis. Serum factors contributed to this delay with increases in IL-1beta and GM-CSF. Isolated neutrophils were resistant to Fas antibody-induced apoptosis. Caspases represent a central mechanism for spontaneous and Fas antibody-induced neutrophil apoptosis. Procaspase 3 expression was decreased in mild and severe cases, but this effect was independent of serum factors. Increases in GST expression may also contribute to the antiapoptotic effect. Altered caspase expression may represent an additional factor contributing to delayed neutrophil apoptosis. This may contribute to the development of AP and its related complications.

Diglyme [1,1'-oxybis(2-methoxyethane)] is an organic solvent belonging to the glycol ether class of compounds. To assess the inhalation toxicity of diglyme, groups of 20 male and 10 female rats were exposed by nose-only inhalation 6 hours/day, 5 days/week for 2 weeks to either 0 (control), 110, 370 or 1100 ppm diglyme. To compare potency, 2-methoxyethanol was also tested at 300 ppm. Rats were sacrificed either immediately following exposure, after a 14-day recovery period, or after 42 and 84 days of recovery (males only). Parameters investigated included in-life observations and body weights, clinical pathology, and histopathology with organ weights. Exposure to diglyme produced a variety of concentration-related haematological, clinical chemical and histopathological changes in both sexes. The most striking effect produced in all test groups was cellular injury involving the testes, seminal vesicles, epididymides and prostate. Although these effects were more severe at the higher concentrations tested, partial or complete recovery was seen by 84 days post-exposure. Changes in the haematopoietic system occurred in both sexes and involved the bone marrow, spleen, thymus, leucocytes and erythrocytes. The testicular effects of diglyme were somewhat less pronounced than those seen with 2-methoxyethanol. The no-observed-effect level (NOEL) for repeated inhalation exposure to diglyme in female rats is 370 ppm. For males, all concentrations tested produced effects to the reproductive system, hence a no-observed-effect level could not be demonstrated.

Abl kinases encoded by the abl oncogenes inhibit apoptosis without affecting cell proliferation. The aim of this study was to examine a wide range of normal fetal and adult human tissues and a variety of tumour types for Abl immunoreactivity. Sections from 193 paraffin blocks of normal fetal and adult tissues and 72 blocks from representative tumours were stained immunohistochemically using a polyclonal antibody to c-Abl/Bcr-Abl oncoprotein. Weak Abl immunoreactivity was observed in many adult tissues. Moderately intense or strong staining (cytoplasmic, nuclear or membranous) was consistently seen in hyaline cartilage, adipocytes, and ciliated epithelium. In fetal tissues, there was a broadly similar staining pattern, but Abl expression was also seen in muscle (all types) and occasionally in endothelial cells. The most intense staining was seen in sites of endochondral ossification and in the umbilical cord stroma. Negatively staining tissues included epidermis and squamous mucosa, lymph nodes, tonsil, spleen, hepatocytes, and adrenals. Most tumours showed focal or weak Abl immunoreactivity. The most intense staining was seen in chondrosarcoma, liposarcoma, and diffuse gastric (signet ring) adenocarcinoma. In the latter two tumour types, Abl expression was also observed in tumour microvessels. These results suggest that Abl not only functions as an apoptosis inhibitor, but also may have a role in connective tissue maturation and differentiation and in tumour growth and angiogenesis.

Aims: To assess the extent of apoptosis in ovarian serous carcinoma and to examine possible relations between apoptosis, cell proliferation, p53 overexpression, and patient survival., Methods: Apoptotic and mitotic indices were obtained by examining haematoxylin and eosin stained sections from 30 patients with ovarian serous carcinoma. Apoptosis was also evaluated semiquantitatively by in situ end labelling of fragmented DNA. Expression of p53 and determination of Ki-67 labelling indices were based on immunohistochemical staining. Clinical details were obtained from patients' clinical records. For statistical analysis, Fisher's exact test, parametric (Pearson) linear correlations, and the Kaplan-Meier method were used., Results: The mean apoptotic index was 1.3% (range 0.02-3.9%), the mean mitotic index was 0.4% (range 0.02-1.1%), and the mean Ki-67 labelling index was 16% (range 4-32%). There were significant correlations between the apoptotic and mitotic indices (p < 0.0205) and between the mitotic and Ki-67 labelling indices (p < 0.024). There was a significant correlation between a high apoptotic index and poor prognosis (p < 0.02). p53 was overexpressed in 16 cases but the extent of apoptosis and outcome were both independent of p53 status., Conclusions: These results suggest that regulation of apoptosis is an integral component of tumour cell kinetics in ovarian serous carcinoma, and that increased apoptosis is indicative of aggressive tumour growth. p53 expression did not correlate with altered apoptosis, but the possibility of an attenuated apoptotic response to subsequent DNA damage by anticancer agents is not excluded.

Certain oncogenes and tumour suppressor genes are known to modulate apoptosis. To investigate whether overexpressed bcl-2 and abnormally stabilized p53 are associated with reduced apoptosis in paraffin sections of non-small cell lung carcinoma, apoptotic, mitotic, and Ki-67 labelling indices were determined and correlated with bcl-2 and p53 immunoreactivity in 54 squamous cell carcinomas and 22 adenocarcinomas. Nineteen squamous cell carcinomas (35.2%) showed over-expression of bcl-2, but all 22 adenocarcinomas were bcl-2 negative. Thirty-seven squamous cell carcinomas (68.5%) and 13 adenocarcinomas (59.1%) showed p53 over-expression. Apoptotic tumour cells were identified among p53 positive and bcl-2 positive tumour cells. There was a significant linear correlation between apoptotic indices and mitotic indices. bcl-2 over-expression and p53 over-expression were not associated with attenuated apoptosis, or altered mitotic or Ki-67 labelling indices in either tumour type. Neither bcl-2 nor p53 was of prognostic significance. These results suggest that apoptosis in non-small cell lung carcinoma occurs independently, and is not modulated primarily by, bcl-2 or p53. It is likely that the effects on apoptosis of bcl-2 and p53 are countered by those of other oncogene products and/or additional factors that regulate apoptosis in vivo.

Aims: To compare in situ end-labelling (ISEL) of apoptosis in lung carcinoma with quantitative and semiquantitative light microscopic assessment and ultrastructural observations., Methods: ISEL of apoptosis was evaluated in 42 lung carcinomas (24 squamous cell carcinomas, 12 adenocarcinomas and six small cell carcinomas). Results were correlated semiquantitatively with the extent of apoptosis in haematoxylin and eosin stained sections, with apoptotic indices and with ultrastructural observations (nine cases)., Results: In each tumour type the extent of apoptosis identified by ISEL correlated with that observed on light and electron microscopy. Tumour cells undergoing apoptosis showed either uniform nuclear staining with a surrounding "halo" or peripheral nuclear membrane staining. The latter pattern was more prominent in small cell carcinoma and correlated ultrastructurally with early apoptosis. A variable proportion of apoptotic cells and apoptotic bodies were unlabelled. Necrotic tumour cells were weakly stained but were distinguishable from apoptotic cells., Conclusions: ISEL, if used in conjunction with standard methods for investigating apoptosis, is a useful adjunct to the investigation of apoptosis in human tumour tissue.