Host-specific differences in the contribution of an ESBL IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4.

Objectives: To assess stability and contribution of a large ESBL-encoding IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts. Methods: Specific-pathogen-free 3-4-day-old New Zealand White rabbits and conventionally reared 6-week-old weaned lambs were orally infected with WT E. coli O104:H4 or the ESBL-plasmid-cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time. Results: Carriage of the ESBL plasmid had differing impacts on E. coli O104:H4 colonization of the two experimental hosts. The plasmid-cured strain was recovered at significantly higher levels than WT during late-stage colonization of rabbits, but at lower levels than WT in sheep. Regardless of the animal host, the ESBL plasmid was stably maintained in virtually all in vivo passaged bacteria that were examined. Conclusions: These findings suggest that carriage of ESBL plasmids has distinct effects on the host bacterium depending upon the animal species it encounters and demonstrates that, as for E. coli O157:H7, ruminants could represent a potential transmission reservoir.

Prostate cancer and consistency of reporting sexual histories in men over age 50.

We conducted an in-person interview to examine the reliability of reported sexual histories among men over age 50 y with and without prostate cancer. Marriage and cohabitation were used as memory cues to recall sexual activity. High correlations on test-retest for questions evaluating sexual histories suggest reliable answers for most factors, and specifically for age at first sexual activity, and lifetime number of sexual partners. Low correlations were seen for ill-defined and socially undesirable items. These data suggest that men consistently report most measures of sexual activity when using marriage and cohabitation as memory cues to recall sexual histories.

Edge-based identification of DP-features on free-form solids.

Numerous applications in mechanical CAD/CAM need robust algorithms for the identification of protrusion and depression features (DP-features) on geometric models with free-form (B-Spline) surfaces. This paper reports a partitioning algorithm that first identifies the boundary edges of DP-features and then creates a surface patch to cover the depressions or isolate the protrusions. The novelty of the method lies in the use of tangent continuity between edge segments to identify DP-feature boundaries that cross multiple faces and geometries.

Persistent HPV infection in postmenopausal age women.

OBJECTIVE: Persistence of human papillomavirus (HPV) is associated with an increased risk of developing cervical SIL and cancer in young women. Because this association in older, postmenopausal age women has received little attention, we evaluated persistence of HPV among women in this age group. METHODS: Women (n=105) ages 45-64 were examined annually for 7 years to evaluate HPV in cervical cytologic specimens. PCR, dot blot hybridization and DNA sequencing were used to detect HPV types. RESULTS: The cumulative prevalence of HPV was 34%, and 24% had HPV high-risk oncogenic types which are associated with genital cancers. The most common oncogenic types were HPV-16 (72%) and HPV-31 (16%). The persistence rate of HPV infection was 16%. No specific risk factors were associated with repeat viral positivity. CONCLUSION: Postmenopausal women are infected with persistent oncogenic HPV at a substantial rate, supporting the need for continued screening in postmenopausal women to detect preneoplastic genital lesions.

HPV prevalence and concordance in the cervix and oral cavity of pregnant women.

OBJECTIVES: This investigation examined human papillomavirus (HPV) in pregnant women in order to characterize viral prevalence, types and concordance between infection in the cervix and in the oral cavity. METHODS: A total of 577 pregnant women seeking routine obstetric care were evaluated for HPV infection in their cervix during gestation and immediately before delivery, and in the oral cavity during gestation. Male partners present during the gestational clinic visit also provided a specimen from their oral cavity. HPV assessment was performed by PCR, dot blot hybridization and DNA sequencing. A sexual and health questionnaire was completed by the pregnant women. RESULTS: HPV prevalence in women was 29% in the cervix and 2.4% in the oral cavity. Among those with both gestational and delivery specimens, 35% were infected at least once and 20% had infection at both intervals. At delivery, 68% of infected women had an oncogenic HPV type in the cervix. There was no type-specific HPV concordance between the two cervical specimens, nor cervical and oral results in women, nor with cervical and oral findings between partners. CONCLUSION: The lack of association in HPV positivity and types between the cervix and oral cavity in these women suggests that self-inoculation is uncommon. This source of infection does not appear to be from oral contact with a current male partner, since there also was no concordance between partners. These results suggest either other modes of HPV transmission or differences in susceptibility to HPV infection or its clearance in the oral cavity and genital mucosa.

Is use of hormone replacement therapy associated with increased detection of human papillomavirus and potential risk of HPV-related genital cancers?

Oral contraceptives (OC) are a risk factor for female genital cancers and in vivo studies have shown that progestins stimulate human papillomavirus (HPV) gene expression. A similar role for hormone replacement therapy (HRT) has received little evaluation. Cervical/vaginal specimens were obtained to detect HPV from postmenopausal women (n = 429) seeking annual gynaecologic care. HPV was detected in 14% of women and 4.4% had high-risk, oncogenic types. HPV prevalence was similar across current, past and never HRT users. After adjustment for HPV-related risk factors, current and past user status showed no increased viral detection compared with never users. HRT duration also did not elevate risk among current users. However, longer duration (adj. OR 1.5/year, 95% CI 1.0-2.3) and longer latency (adj. OR 1.2/year, 95% CI 0.9-1.7) among past users of oestrogen/progestin regimens were associated with greater risk. Overall use of HRTs was not associated with HPV detection or disease. However, past users of combination HRTs had significantly greater risk of HPV detection with longer HRT duration and latency, similar to OC-HPV findings. The recommendation that postmenopausal women continue HRTs long term may lead to an increased development of HPV-related diseases, of particular concern among those who discontinue HRTs and subsequent gynaecologic care for early cancer detection.

Toxicological response of a bioluminescent microbial assay to Zn, Pb and Cu in an artificial soil solution: relationship with total metal concentrations and free ion activities.

The relationship between toxicological response and both total concentrations and free ion activities of Pb, Cu and Zn in an artificial soil solution has been investigated using lux-marked Escherichia coli HB101 (pUCD607) as a bioassay. SO4(2-) (as K2SO4) was added as an inorganic complexing agent up to 0.01 M representing the range of ionic strengths found in soil solutions and giving a wide range of free metal ion activities. EC50 values expressed in terms of concentration, varied significantly with K2SO4 molarity for all metals. However, when EC50 values were expressed in terms of free ion activity they were not significantly different for Pb and Zn, supporting the free ion activity model. Conversely, EC50 values expressed as free Cu activity did vary significantly with K2SO4 molarity, possibly due to a greater degree of adsorption of Cu onto inactive sites on the cell surfaces than for Zn and Pb. Linear regression analysis of bioluminescence on free ion activity revealed significant correlations for each metal above the toxicity threshold. In conclusion, lux-marked E. coli is suitable for investigating the toxicity of metal ions and complexes in non saline systems although cell surface adsorption effects could be important for some metals, e.g. Cu.

Construction of a modified mini-Tn5 luxCDABE transposon for the development of bacterial biosensors for ecotoxicity testing.

A mini-Tn5 transposon was modified to introduce a promoterless luxCDABE cassette from Vibrio fischeri into environmentally relevant bacterial strains in order to develop bioluminescence-based biosensors for toxicity testing. The mini-Tn5 luxCDABE transposon was chromosomally integrated downstream from an active promoter into two Pseudomonas strains (Pseudomonas fluorescens 8866 and Pseudomonas putida F1). Characterisation of the bioluminescent transconjugants demonstrated that the transposon integration was stable and had no effect on growth rate. Both P. fluorescens 8866 Tn5 luxCDABE and P. putida F1 Tn5 luxCDABE were used to assess the toxicity of standard solutions (Cu, Zn and 3,5-DCP) as well as Cu- and 3,5-DCP-spiked groundwater samples. They were successfully used for bioluminescence-based bioassays and the potential value of using different bacterial biosensors for ecotoxicity testing was shown.

Growth arrest and spontaneous differentiation are initiated through an autocrine loop in clonally derived Schwann cells by alpha1-procollagen I C-propeptide.

Schwann cells cloned from rat sciatic nerve survive and display self-induced growth suppression, or undergo spontaneous apoptosis, on long-term serum-free subconfluent culture. Strain SCL4.1/F7 sustained the capacity to growth arrest for up to 40 generations. A soluble activity transmitted between neighbouring cells of this strain suppresses DNA synthesis within three cell cycles. Autocrine Schwann cell growth-inhibitory factor (SGIF) operates during the G1 phase of the cell cycle, overcomes the mitogenic action of Schwann cell/serum-associated (platelet-derived growth factor-BB) and axon-associated (axolemma-enriched fraction) stimuli in serum-free conditions, and suppresses DNA synthesis in sciatic nerve Schwann cell cultures in a stage-specific manner. A 35-kDa protein with N-terminal sequence and approximate molecular mass of the C-propeptide of rat alpha1-procollagen I makes a major contribution to SGIF. Growth suppression in the SCL4.1/F7 strain is mediated by the ras/extracellular signal-regulated kinase pathway, is accompanied by down-regulation of erbB2/erbB3 and of tetraethylammonium-sensitive K+ currents, and is followed by transition of cells within 5-10 days from O4+, p75 nerve growth factor receptor (p75NGF-R)+, glial fibrillary acidic protein (GFAP)+ to O4+, p75NGF-R-, GFAP-, periaxin+ phenotypes. Oct-6/SCIP mRNA is present in both proliferating and growth-arrested SCL4.1/F7 cells. These results demonstrate an autocrine/ paracrine loop for the growth arrest of clonally derived Schwann cells in the absence of axons linked in part to the metabolism of collagen. Schwann cells thus appear to self-regulate growth in a negative as well as a positive direction through characterized molecular mechanisms and signal pathways.

Effect of specific ion channel blockers on cultured Schwann cell proliferation.

Mitogenesis in a variety of tissues is known to be inhibited by K+ channel blockers such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Using radiolabeled thymidine as a proliferation index we have examined what role, if any, specific K+ channels have in cultured Schwann cells that have been induced to proliferate by pre-exposure to mitogens. TEA and 4-AP are "broad-spectrum" in that they block a variety of different types of K+ channel. In contrast, we found that alpha-dendrotoxin (alpha-DTX), a specific blocker of the type 1 fast delayed rectifier current (the largest component of Schwann cell K+ current) does not affect proliferation, suggesting that type 1 current may not be involved in mitogenesis. This suggestion is supported by our finding that the values of the KD for the mitogenic effect (722 nM, 4-AP; 13 mM, TEA) are much larger than the corresponding electrophysiological values for type 1 channels (0.1 mM, 4-AP; 0.2 mM, TEA). Charybdotoxin (200 nM) and iberiotoxin (100 nM), inhibitors of Ca2+-activated K+ channels, cesium (5 mM), an inhibitor of inward rectifier channels, and furosemide (100 pM), which blocks Na+/K+/Cl- cotransport, all had no effect on proliferation. Interestingly, 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS), which blocks voltage-gated Cl- channels, reduced proliferation. In summary, broad-spectrum K+ channel blockers inhibit Schwann cell proliferation, but inhibitors specific for type 1, Ca2+-activated, and inward rectifier K+ channels do not. Whether the inhibition is mediated by type 2 K- channels, by an as yet unidentified Schwann cell K+ channel, or by another mechanism remains unclear.

Characteristics of type I and type II K+ channels in rabbit cultured Schwann cells.

1. Voltage-dependent K+ currents were studied in rabbit Schwann cells cultured from neonatal sciatic nerve and from the lumbar or sacral spinal roots of 10-day-old animals. 2. Whole-cell K+ currents, evoked in response to depolarizing voltage-clamp steps, were categorized as type I or type II on the basis of their apparent threshold and activation kinetics. In the presence of a quasi-physiological [K+] gradient, the magnitude of the fully activated type I current varied linearly with membrane potential, whereas type II current always gave rise to a curved and outwardly rectifying current-membrane potential (I-E) relation. 3. Type II whole-cell currents, obtained with long duration voltage-clamp steps (> or = 1 s), have an apparent threshold for activation close to -40 mV. Type II current inactivated slowly, and apparently to completion. The current is more than 90% inactivated over 5 s at 0 mV (time consant of inactivation, tau h, approximately 2 s, 20-22 degrees C). Type I current, which activates at close to -60 mV, inactivated at about half this rate at the same potential, assuming that inactivation also proceeds to completion. 4. Type I whole-cell currents were reversibly blocked by superfused beta-bungarotoxin (beta-BuTX; apparent KD = 46 nM). beta-BuTX did not appear to reduce type II whole-cell currents at concentrations up to 500 nM. 5. In outside-out patches, the type I channel had an almost linear I-E relation over the potential range -60 to +60 mV with a quasi-physiological [K+] gradient. A best linear fit gave a single-channel conductance of 12 pS under these conditions. In symmetrical 170 mM K+, type I channels had a single-channel conductance of 30 pS over the same potential range. 6. More slowly activating type II single-channel currents were also recorded in inside-out patches. With symmetrical 170 mM K+, the major conductance level was close to 9.0 pS. With a quasi-physiological [K+] gradient, type II single channels exhibit outward rectification that is reasonably well described by the Goldman-Hodgkin-Katz current equation. 7. In the presence of 2 nM externally superfused alpha-dendrotoxin (alpha-DTX), or 50 nM superfused beta-BuTX, unitary currents were recorded (outside-out patches, -60 or -50 mV) that were smaller than control type I currents. Virtually all transitions in the presence of 50 nM beta-BuTX were at one-third of the control current level. The currents did not conform to the characteristics of type II. 8. The electrophysiological and pharmacological characteristics of the type I channel strongly suggest that it is a member of the mammalian K+ channel subfamily of Shaker homologues, most similar to the homomultimeric Kv1.1 translation product. The type II channel may be a member of the mammalian Shab subfamily. 9. Possible roles for Na+ channels and type I K+ channels in the Schwann cell are discussed.

Cloning of a sodium channel alpha subunit from rabbit Schwann cells.

Overlapping cDNA clones spanning the entire coding region of a Na-channel alpha subunit were isolated from cultured Schwann cells from rabbits. The coding region predicts a polypeptide (Nas) of 1984 amino acids exhibiting several features characteristic of Na-channel alpha subunits isolated from other tissues. Sequence comparisons showed that the Nas alpha subunit resembles most the family of Na channels isolated from brain (approximately 80% amino acid identity) and is least similar (approximately 55% amino acid identity) to the atypical Na channel expressed in human heart and the partial rat cDNA, NaG. As for the brain II and III isoforms, two variants of Nas exist that appear to arise by alternative splicing. The results of reverse transcriptase-polymerase chain reaction experiments suggest that expression of Nas transcripts is restricted to cells in the peripheral and central nervous systems. Expression was detected in cultured Schwann cells, sciatic nerve, brain, and spinal cord but not in skeletal or cardiac muscle, liver, kidney, or lung.

The action of capsaicin on type I delayed rectifier K+ currents in rabbit Schwann cells.

Capsaicin, a lipophilic alkaloid, blocked type I K+ currents in rabbit cultured Schwann cells when applied by superfusion. The concentration-response relation at equilibrium was well described by a rectangular hyperbola, with a KD of 8.7 microM. The kinetics of block resembled an 'inactivation', the rate of blockade increasing with increasing concentrations of capsaicin (1-100 microM). Unlike internal tetraethylammonium (TEA) ions (5-10 mM), which preferentially reduced outward current in symmetrical high [K+], capsaicin reduced both inward and outward type I current by the same proportion. The block achieved by capsaicin during a voltage-clamp step that activated the current was relieved by subsequent hyperpolarization, and the rate of relief from block at -70 mV and -100 mV could be reasonably accounted for on the assumption that capsaicin had to unbind to allow the channels' to close.

Action potential conduction and sodium channel content in the optic nerve of the myelin-deficient rat.

Compound action potential (CAP) conduction and Na+ channel content were studied in optic nerves from control and myelin-deficient (md) rats. Action potential propagation was approximately five times slower in the md rat, but the action potentials propagated securely and had frequency-following and refractory properties equivalent to control myelinated axons. Tritium-labelled saxitoxin ([3H]-STX) binding in md optic nerve was approximately 30% greater, per wet mass of tissue, than in the control optic nerve. However, calculations of channel density per axon based on previously published anatomical data from md and control optic nerves (Dentinger et al. 1985) show an equivalent number of sodium channels per axon, with an average density of 10 channels micron-2 in md and 11 channels micron-2 in control optic nerve axons. The amplitude of the CAP in both control and md optic nerves was significantly attenuated by 50 nM TTX, precluding the possibility that TTX-insensitive channels are responsible for the action potential in myelinated or amyelinated axons. In addition, the amplitudes of voltage-activated Na+ currents in type I and type II astrocytes cultured from control and md optic nerves were similar, suggesting that the glial component of Na+ channels is not abnormal in the optic nerve of the md rat. These results suggest that myelination (or its absence) may not directly regulate the number of axonal Na+ channels.

Leukoerythroblastic anemia in metastatic prostate cancer. Clinical and prognostic significance in patients with hormone-refractory disease.

BACKGROUND: The clinical and prognostic significance of leukoerythroblastic anemia (LKEA) in patients with metastatic prostate cancer and, in general, patients with disseminated solid tumors is poorly understood. Therefore, the authors studied a population of patients with metastatic prostate cancer refractory to hormonal therapy to assess the incidence, clinical features, and prognostic implications of LKEA. METHODS: The medical records of 106 patients with hormone-refractory prostate cancer metastatic to bone seen at the Tucson Veterans Affairs Medical Center between 1985 and 1991 were reviewed retrospectively. The clinical and laboratory data, number of packed erythrocyte transfusions required, and length of survival from the time of diagnosis of hormone-refractory disease until last follow-up visit or death were investigated in 91 identified patients. RESULTS: Twenty-six of 91 patients (28.6%) were found to have LKEA. LKEA developed before or at the time of diagnosis of hormone-refractory disease in 8 patients and after diagnosis of hormone-refractory disease in 18 patients. The presence of LKEA was associated with significantly lower hemoglobin levels and platelet (Plt) counts and significantly higher total bilirubin, lactic dehydrogenase (LDH), and alkaline phosphatase values (P < 0.05). Leukopenia (< 4.0 x 10(9)/l leukocytes), thrombocytopenia (< 150 x 10(9)/l Plt), elevated LDH levels (> 220 U/l), and laboratory evidence of disseminated intravascular coagulation (DIC) were more common in patients with LKEA than in those without LKEA (P < 0.01). Microangiopathic hemolysis was seen in only 2 of 91 patients (2.1%). Patients with LKEA had significantly greater transfusion requirements compared with patients without LKEA (P < 0.0001), but the median survival length was not significantly different (9 months versus 11 months, respectively). The presence of DIC and LDH levels of 500 U/l or greater in patients with LKEA was associated with a poor prognosis. CONCLUSIONS: LKEA is a relatively common finding in patients with hormone-refractory metastatic prostate cancer and is associated with greater transfusion requirements. Its presence, however, does not affect survival significantly.

Two types of 4-aminopyridine-sensitive potassium current in rabbit Schwann cells.

1. Delayed rectifier K+ currents were studied in Schwann cells cultured from neonatal rabbit sciatic nerves with the whole-cell patch-clamp technique. 2. Depolarizing voltage steps (40 ms duration) activated two types of K+ current: type I, whose apparent activation threshold was about -60 mV (half-maximal conductance at -40 +/- 1 mV, n = 10); and type II, whose apparent activation threshold was about -25 mV (half-maximal conductance at + 11 +/- 1 mV, n = 9). 3. Type I current was blocked by alpha-dendrotoxin (alpha-DTX) with an apparent equilibrium dissociation constant (KD) of 1.3 nM, whereas the type II current was unaffected by exposure to 500 nM toxin. The action of alpha-DTX on the type I current was reversible. 4. Most cells exhibited both types of current, but occasionally some cells displayed just type I or just type II. 5. Type I current activated rapidly and then showed a much slower fade, which became more noticeable with larger depolarizations. Activation of type II current was slower than that of type I and depended less steeply on voltage. The time constants of activation for type I and type II currents were derived with a Hodgkin-Huxley formalism (based on second-power activation and deactivation kinetics). The longest activation time constant for type II gating was more than twice the corresponding time constant for type I; however, the time constants determined from tail current decays at potentials more negative than -60 mV were shorter for the type II currents than for the type I currents. 6. Both type I and type II currents were sensitive to micromolar concentrations of 4-aminopyridine (4-AP). The KD for 4-AP blockade of type II current was 630 microM (pH 7.2, membrane potential (Em) = -10 mV), which is about 6 times higher than the corresponding value for 4-AP blockade of type I current at negative membrane potentials. The differential sensitivity of the type I and type II currents to 4-AP may account for the apparent voltage dependence of 4-AP block of delayed rectifier K+ currents. 7. In addition to types I and II, a third type of outward K+ current (type III) was generated in most cells at positive membrane potentials. This latter current was insensitive to millimolar concentrations of 4-AP. 8. Similarities between Schwann cell and neuronal potassium channels are discussed.