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Senecio squalidus L. (Asteraceae) has been the subject of several ecological and population genetic studies due to its well-documented history of introduction, establishment and spread throughout Britain in the past 300 years. Our recent studies have focused on identifying and quantifying factors associated with the sporophytic self-incompatibility (SSI) system of S. squalidus that may have contributed to its success as a colonist. These findings are of general biological interest because they provide important insights into the short-term evolutionary dynamics of a plant mating system. The number of S-alleles in populations and their dominance interactions were investigated in eight wild British populations using cross-diallel studies. The numbers of S-alleles in British S. squalidus populations are typically low (average of 5.3 S-alleles) and the entire British population is estimated to possess no more than 7-11 S-alleles. Such low numbers of S-alleles are most probably a consequence of population bottlenecks associated with introduction and colonization. Potential evolutionary impacts on SSI caused by a paucity of S-alleles, such as restricted mate availability, are discussed, and we suggest that increased dominance interactions between S-alleles may be an important short-term means of increasing mate availability when S-allele numbers are low.

A comprehensive survey to document the presence of free-living amoebae was conducted along 58 km of James River, near Richmond, Virginia, USA. Sites included tidal and non-tidal freshwater areas, near 40 combined sewer outflows, three municipal wastewater treatment plant release sites, and thermal discharge from a coal-fired power plant. Amoebae were present on all collection dates, spring through autumn, and at all sites ( n=330). Five genera, Naegleria, Vannella, Acanthamoeba, Vahlkampfia, and Hartmannella were present in both the water column and sediment. The most common isolates from the water column were Naegleria and Vannella. Water conditions conducive to the presence of large quantities of fecal coliform bacteria were correlated with the prevalence of free-living amoebae. Some of the amoebae in this complex ecosystem can act as opportunistic pathogens, may play a role in diseases of aquatic organisms in this heavily urbanized river, and may present a risk to human health.

In a follow-up to studies of school-children's medical knowledge among the rural Luo of western Kenya, seven mothers were asked for their knowledge of plant medicine, and the 91 plant remedies mentioned by them were collected, 74 of these remedies were identified as 69 different species (in 13 cases, the material did not allow identification of the species, in two cases, only the family could be identified, and in two, not even this was possible). The results of this survey and some comments on Luo illness concepts are presented below and briefly discussed in relation to the earlier work on school-children and to another survey of Luo plant medicine in the same district. The article concludes that the consensual core of Luo plant medicine is known by ordinary mothers and their children as well as by recognised healers. It is a shared resource, that is used by women, mainly in the care for their children, and it is not an expert domain of knowledge, as is often, in studies of herbal or 'traditional' medicine are studied. The medicinal plants, upon which many mothers as well as healers agree should be examined further pharmacologically in order to assess their efficacy against the common infectious and parasitic diseases found in this area of western Kenya.

The photodissociation dynamics of 1,3-butadiene at 193 nm have been investigated with photofragment translational spectroscopy coupled with product photoionization using tunable VUV synchrotron radiation. Five product channels are evident from this study: C(4)H(5) + H, C(3)H(3) + CH(3), C(2)H(3) + C(2)H(3), C(4)H(4) + H(2), and C(2)H(4) + C(2)H(2). The translational energy (P(E(T))) distributions suggest that these channels result from internal conversion to the ground electronic state followed by dissociation. To investigate the dissociation dynamics in more detail, further studies were carried out using 1,3-butadiene-1,1,4,4-d(4). Branching ratios were determined for the channels listed above, as well as relative branching ratios for the isotopomeric species produced from 1,3-butadiene-1,1,4,4-d(4) dissociation. C(3)H(3) + CH(3) is found to be the dominant channel, followed by C(4)H(5) + H and C(2)H(4) + C(2)H(2), for which the yields are approximately equal. The dominance of the C(3)H(3) + CH(3) channel shows that isomerization to 1,2-butadiene followed by dissociation is facile.

Molecular genetic markers complement archaeological, breeding and geographical investigations of the origins, history and domestication of plants. With increasing access to wild apples from Central Asia, along with the use of molecular genetic markers capable of distinguishing between species, and explicit methods of phylogeny reconstruction, it is now possible to test hypotheses about the origin of the domesticated apple. Analyses of nuclear rDNA and chloroplast DNA (cpDNA) sequences indicate that the domesticated apple is most closely related to series Malus species. Moreover, the occurrence of a shared 18-bp duplication in the cpDNAs of wild and cultivated apple supports the close relationship between them. Hypotheses about the hybridization and the origin of the domesticated apple cannot be rejected completely until more variable, phylogenetically informative markers are found.

Previous analyses of species relationships and polyploid origins in the mimosoid legume genus Leucaena have used chloroplast DNA (cpDNA) restriction site data and morphology. Here we present an analysis of a new DNA sequence data set for the nuclear ribosomal DNA (nrDNA) 5.8S subunit and flanking ITS 1 and ITS 2 spacers, a simultaneous analysis of the morphology, ITS and cpDNA data sets for the diploid species, and a detailed comparison of the cpDNA and ITS gene trees, which include multiple accessions of all five tetraploid species. Significant new insights into species relationships and polyploid origins, including that of the economically important tropical forage tree L. leucocephala, are discussed. Heterogeneous ITS copy types, including 26 putative pseudogene sequences, were found within individuals of four of the five tetraploid and one diploid species. Potential pseudogenes were identified using two pairwise comparison approaches as well as a tree-based method that compares observed and expected proportions of total ITS variation contributed by the 5.8S subunit optimized onto branches of one of the ITS gene trees. Inclusion of putative pseudogene sequences in the analysis provided evidence that some pseudogenes in allopolyploid L. leucocephala are not the result of post-allopolyploidization gene silencing, but were inherited from its putative diploid maternal progenitor L. pulverulenta.

Objective: To compare clinical outcomes and quality of life (QOL) in elderly patients on peritoneal dialysis (PD) and hemodialysis (HD) in the North Thames Dialysis Study., Design: A 12-month prospective cohort study., Setting: Four hospital-based renal units in London, UK., Patients: 174 patients that were 70 years or older at the start of dialysis, separated into two cohorts: 78 new patients (36 PD, 42 HD) that were recruited after 90 days of chronic dialysis; and 96 stock patients (42 PD, 54 HD) that were already on dialysis during the recruitment period., Main Outcome Measures: 12-month survival and hospitalization rate, and QOL assessed at baseline and at 6 and 12 months by the SF-36 and the Symptoms/Problems scale of the Kidney Disease Quality of Life Questionnaire (KDQOL)., Results: Peritoneal dialysis and HD patients were similar for sociodemographic and clinical characteristics. Annual mortality and hospitalization rates in PD versus HD patients were 26.1 versus 26.4 deaths/100 person-years and 1.9 versus 2.0 admissions/person-year, respectively. Adjusted relative risks showed no effect of modality on clinical outcomes. Multiple linear regression analyses of QOL at baseline showed similar SF-36 scores between PD and HD patients, but higher KDQOL scores in PD patients (3.5 points higher, 95% confidence interval 0.3-6.6). There was, however, no effect of dialysis modality on QOL at 6 or 12 months., Conclusions: Clinical outcomes and QOL are similar in elderly people on PD and HD. Peritoneal dialysis is a viable option for more than a carefully selected minority of elderly people requiring dialysis.

Nitrogen oxides (NO(x)) are ubiquitous pollutants in outdoor and indoor air. However, epidemiologic studies that evaluate health effects associated with NO(x) commonly rely upon outdoor concentrations of NO(x), nitrogen dioxide (NO(2)), or residence characteristics as surrogates for personal exposure. In this study, personal exposures (48 h) and corresponding indoor and outdoor concentrations of nitric oxide (NO), NO(2), and NO(x) were measured (July-September) in 39 adults and 9 children from 23 households in Richmond, Virginia, using Ogawa passive NO(x) monitors. Demographic, time-activity patterns, and household data were collected by questionnaire and used to develop exposure prediction models. Adults had higher NO(2), NO, and NO(x) exposures (means: 16, 63, and 79 ppb, respectively) than children (13, 49, and 62 ppb). Measurements taken in bedrooms (18, 57, and 75 ppb) and living rooms (19, 65, and 84 ppb) surpassed measurements taken outdoors (15, 21, and 36 ppb). In indoor locations, NO(x) concentrations were influenced largely by NO, and consequently, personal exposure prediction models for NO(x) were reflective of models for NO. Statistical models that best predicted personal exposures included indoor measurements; outdoor measurements contributed relatively little to personal exposure. Close to 70% of the variation in personal NO(2) and NO(x) exposure was explained by two variable models (bedroom NO(2) and time spent in other indoor locations; bedroom NO(x) and time spent in kitchen). Given appropriate resources, measurement error in epidemiologic studies can be reduced significantly with the use of personal exposure measurements or prediction models developed from indoor measurements and survey data.

Epidemiologic studies designed to assess the chronic effects of pesticides are limited by inadequate measurements of exposures. Although cohort studies have been initiated to evaluate the effects of 2,4-dichlorophenoxyacetic acid (2,4-D) and other pesticides in professional turf applicators, they may have limited power to detect significant health risks and may be subject to bias from exposure measurement error. In this study, the doses of 2,4-D, mecoprop [2-(4-chloro-2 methylphenoxy) propionic acid, MCPP] and dicamba (3,6-dichloro-o-anisic acid) were evaluated in a group of 98 professional turf applicators from 20 companies across southwestern Ontario. During a 1-week period (Saturday to Thursday), the volume of pesticide (active ingredient) applied was only weakly related to the total dose of 2,4-D absorbed (R(2)=0.21). Two additional factors explained a large proportion of variation in dose: the type of spray nozzle used and the use of gloves while spraying. Individuals who used a fan-type nozzle had significantly higher doses than those who used a gun-type nozzle. Glove use was associated with significantly lower doses. Job satisfaction and current smoking influenced the dose but were not highly predictive. In the final multiple regression models predicting total absorbed dose of 2,4-D and mecoprop, approximately 63-68% of the variation was explained. The future application of these models for epidemiologic research will depend on the availability of information and records from employers, the feasibility of contacting study subjects and cost.

We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T-antigen (Harris et al. [1995a] J. Bone. Miner. Res. 10:178-1860). To characterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M-FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1-2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2-3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non-transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro.

NMR studies have shown that the minor groove-binding ligand Hoechst 33258 binds to the two T4/A4 tracts within the duplex d(CTTTTCGAAAAG)2 in a highly cooperative manner, such that in titration experiments no intermediate 1:1 complex can be detected. The NMR-derived structures of the free DNA and the 2:1 complex have been obtained, but can shed little light on what the origins of this cooperativity may be. Here we present the results of a series of molecular dynamics simulations on the free DNA, the 1:1 complex, and the 2:1 complex, which have been designed to enable us to calculate thermodynamic parameters associated with the molecular recognition events. The results of the molecular dynamics studies confirm that structural factors alone cannot explain the cooperativity observed, indeed when enthalpic and hydration factors are looked at in isolation, the recognition process is predicted to be slightly anticooperative. However, when changes in configurational entropy are taken into account as well, the overall free energy differences are such that the calculated cooperativity is in good agreement with that observed experimentally. The results indicate the power of molecular dynamics methods to provide reasonable explanations for phenomena that are difficult to explain on the basis of static models alone, and provide a nice example of the concept of "allostery without conformational change".

Extraskeletal Ewings sarcoma is a tumour of neuroectodermal origin sharing close similarities with Ewings sarcoma of bone. We report the case of a 21 year old 16 week pregnant woman presenting with vomiting and weight loss and found to have an extraskeletal Ewings sarcoma of the small bowel. In a review of the literature there are no previous reports of extraskeletal Ewings sarcoma occurring in the small bowel. The diagnosis of extraskeletal Ewings sarcoma and the complicated management of a young pregnant woman with a malignant tumour are described.

The evaluation of absorbed dose of pesticides in humans requires a knowledge of the kinetics and dynamics of the compound. In some circumstances, data that allow for the estimation of dose may be available from human volunteer studies, although often, it will be based on results from animal studies. If human metabolism data are available, estimates of dose may be more accurate, but it should be recognized that pesticide exposure in an occupational setting may differ from that in a controlled laboratory study. In this study, data from previously published studies are used to evaluate the urinary excretion of 2,4-dichlorophenoxyacetic acid (2,4-D), following single dermal applications to human volunteers. These studies are evaluated with the objective of determining the best method of predicting total absorbed dose following multiple and varied exposures in occupational settings. Further, an alternative to laboratory-controlled human volunteer studies is presented. Data from a third previously published biological monitoring study on six professional pesticide applicators over a 2-week period were used to generate estimates of the urinary excretion of the pesticide 2,4-D that would result from a single dose. The method used to estimate the urinary excretion parameters is a variation of an overlay technique used in pharmacology, and may provide information on the kinetics of other pesticides when it is not possible to conduct human studies. The generated estimates of 24-h urinary excretion of 2,4-D over a 6-day period were remarkably similar to those obtained in controlled studies. Finally, a method was developed to use the generated estimates to determine total absorbed dose of pesticides for an independent group of 95 professional pesticide applicators. This method requires information on the amount of pesticide used for 6 days prior to the collection of two, 24-h urine samples.

We model and analyze a short section of a DNA chain with a defect, with the aim of understanding how the frequency, amplitude, and localization of breathing events depend on the strength of the bonds between base pairs, both along the chain and between the chains. Our results show that the presence of a defect in the chain permits the existence of a localized breather mode. The models we analyze are linear and hence solvable, with solvability extending to the statistical mechanics formulation of the problem. Parameter values for the interaction energy of a base with its nearest neighbors are obtained from AMBER. The results indicate good agreement with both the amplitude and the number of base pairs affected by defect-induced breathing motion.

NMR analysis and molecular dynamics simulations of d(GGTAATTACC)(2) and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar-phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving 'lock and key' recognition.

Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutation of the gene encoding the cytoskeletal protein dystrophin. Despite a wealth of recent information about the molecular basis of DMD, effective treatment for this disease does not exist because the mechanism by which dystrophin deficiency produces the clinical phenotype is unknown. In both mouse and human skeletal muscle, dystrophin deficiency results in loss of neuronal nitric oxide synthase, which normally is localized to the sarcolemma as part of the dystrophin-glycoprotein complex. Recent studies in mice suggest that skeletal muscle-derived nitric oxide may play a key role in the regulation of blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to alpha-adrenergic receptor activation. Here we report that this protective mechanism is defective in children with DMD, because the vasoconstrictor response (measured as a decrease in muscle oxygenation) to reflex sympathetic activation was not blunted during exercise of the dystrophic muscles. In contrast, this protective mechanism is intact in healthy children and those with polymyositis or limb-girdle muscular dystrophy, muscle diseases that do not result in loss of neuronal nitric oxide synthase. This clinical investigation suggests that unopposed sympathetic vasoconstriction in exercising human skeletal muscle may constitute a heretofore unappreciated vascular mechanism contributing to the pathogenesis of DMD.

A QSAR study, involving the use of calculated physical properties (TSAR), and the use of a neural network approach (TSAR), has been performed concerning the anti-HIV activity and cytotoxic effects of a series of d4T phosphoramidate derivatives with varying L-alanine esters. Models were obtained which allow reliable predictions for the anti-HIV activity, and cytotoxicity, of these derivatives.

The accurate quantification of the absorbed dose of pesticides following occupational exposures generally requires complete 24-hour urine collections, often over extended periods of time. Difficulty in obtaining volunteer cooperation may result in incomplete urine collections. Traditionally, 24-hour urinary creatinine has been used to identify incomplete urine samples and has been used to standardize pesticide and other chemical dose estimates. More recently, the use of creatinine to standardize dose estimates has been questioned, as has its utility in the identification of incomplete urine collections. This research evaluates the use of personal observation, statistical methods, and published models to predict creatinine excretion to identify and adjust for incomplete urine collections. Based on the use of published creatinine prediction models, an evaluation of the day-to-day creatinine excretion within subjects, and personal observation, a small number of suspected urine samples were identified. Although it is likely that these samples were incomplete, correction of these urine volumes based on the published models did little to improve pesticide dose prediction. Further, results indicate that subjects who report missed urine samples may be able to estimate the missing volumes with some accuracy. In future pesticide exposure studies, the use of self-reported missed volumes may help to increase the accuracy of dose prediction when there is strong cooperation with collection procedures. A statistical model to predict creatinine excretion in professional turf applicators was developed to provide a preliminary screening for urinary completeness for future studies in which compliance with urinary collection is thought to be insufficient.

Many health professionals have received formal training in epidemiology; however, much of it has been limited to introductory courses at the undergraduate level. Further, continuing education for health professionals has focused historically on substantive rather than methodologic issues in epidemiology. A methodologic focus is recommended to improve continuing education for the health of the public. It is crucial to equip educators and health professionals with the necessary tools or resources to understand study design, conduct research, analyze and interpret data, and critically evaluate published research. Thus, in this article, a general overview of epidemiologic study design and some of the most common methodologic issues are presented. Issues such as confounding, effect modification, measurement error, and power and sample size are highlighted. A broader recognition of these issues by educators and health professionals may ultimately help to improve public health by facilitating effective educational interventions, proper study design, analysis, interpretation, and application of epidemiologic research.

Etiologies of shoulder pain in the hemiplegic population, such as glenohumeral subluxation, frozen shoulder, and reflex sympathetic dystrophy (RSD), have been described extensively. We present an 89-year-old woman with right hemiparesis secondary to ischemic lacunar infarction who developed sudden onset of right shoulder pain on the fifth day of inpatient rehabilitation. The pain was severe, limiting range of motion (ROM) and participation in therapy. Extensive investigations to rule out subluxation, fracture, connective tissue disease, RSD, and pulmonary embolism were negative. Ultimately, her shoulder pain and decreased ROM completely resolved with antibiotic treatment for right lower lobe pneumonia. We conclude that her symptoms were possibly referred pain from diaphragmatic irritation transmitted via right C4 sensory axons in the phrenic nerve, which shares the same dermatome as the right acromion area. This case was an unusual presentation of pneumonia in an elderly woman with hemiplegia. We recommend that pneumonia be considered in the differential diagnoses of shoulder pain.

During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) were not significantly changed following spaceflight. Gene-specific reductions in mRNA levels for cytokines and skeletal growth factors were detected in the flight cultures using RNase protection assays. Steady-state mRNA levels for interleukin (IL)-1alpha and IL-6 were decreased 8 h following the flight and returned to control levels at 24 h postflight. Also, transforming growth factor (TGF)-beta(2) and TGF-beta(1) message levels were modestly reduced at 8 h and 24 h postflight, although the change was not statistically significant at 8 h. These data suggest that spaceflight did not significantly affect hFOB cell proliferation, expression of type I collagen, or PGE(2) production, further suggesting that the removal of osteoblastic cells from the context of the bone tissue results in a reduced ability to respond to weightlessness. However, spaceflight followed by return to earth significantly impacted the expression of cytokines and skeletal growth factors, which have been implicated as mediators of the bone remodeling cycle. It is not yet clear whether these latter changes were due to weightlessness or to the transient increase in loading resulting from reentry.

The decrease in cancellous bone formation after estrogen treatment is generally thought to be coupled with a prior decrease in bone resorption. To test the possibility that estrogen has rapid tissue-specific actions on bone metabolism, we determined the time course (1-32 h) effects of diethylstilbestrol on steady-state mRNA levels for immediate-response genes, extracellular matrix proteins, and signaling peptides in the proximal tibial metaphysis and uterus by using Northern blot and RNase protection assays. The regulation of signaling peptides by estrogen, although tissue specific, followed a similar time course in bone and uterus. The observed rapid decreases in expression of insulin-like growth factor I, a growth factor associated with bone formation; decreases in mRNA levels for bone matrix proteins; evidence for reduced bone matrix synthesis; failure to detect rapid increases in mRNA levels for signaling peptides implicated in mediating the inhibitory effects of estrogen on bone resorption (interleukin-1 and -6) as well as other cytokines that can increase bone resorption; and the comparatively long duration of the bone remodeling cycle in rats indicate that estrogen can decrease bone formation by a mechanism that does not require a prior reduction in bone resorption.

It is often difficult to achieve stable fixation of a comminuted fracture associated with a metaphyseal defect. The injection of a resorbable cement into an osseous defect may help to stabilize the fracture and to maintain osseous integrity as the cement is resorbed and replaced by bone. The purpose of the present study was to evaluate the repair of a metaphyseal defect after treatment with an injectable calcium-phosphate cement. The injectable cement undergoes isothermic curing in vivo to form a carbonated apatite (dahllite) with a compressive strength of twenty-five megapascals. Either the cement or allograft bone was placed in proximal tibial metaphyseal and distal femoral metaphyseal defects in seventy-two dogs and was evaluated from twenty-four hours to seventy-eight weeks postoperatively. Histological examination showed that the cement was osteoconductive; nearly the entire surface area was covered with bone two weeks after the injection. The resulting bone-cement composite underwent gradual remodeling over time in a pattern that was qualitatively similar to the remodeling of normal cortical and cancellous bone. Osteoclasts were found to resorb the cement and were usually associated with adjacent new-bone formation. With increasing time in vivo, the cement was penetrated by small blood vessels that became surrounded by circumferential lamellae of bone and that closely resembled evolving haversian systems. This process occurred more rapidly in the cortex than in the medulla. Mechanical testing showed that, by eight weeks, the tibiae that had been treated with cement had reached nearly 100 per cent of the torsional strength of the contralateral, control (intact) tibiae; this finding paralleled the histological observations of bone apposition to the cement and rapid restoration of the cortex. At no time was fibrous tissue present between the cement and the bone, and there was no evidence of acute inflammation. Small particles of cement were present within occasional macrophages during the process of cement resorption, but the macrophages disappeared over time and were not associated with fibrosis or unexpected resorption of bone. Resorption of the cement was incomplete in the medullary area at seventy-eight weeks, but the pattern of cement resorption and bone-remodeling suggested gradual restoration of a physiological proportion of bone and marrow in both the cortical and the medullary region with maintenance of mechanical function.

Background: The purpose of this survey was to describe the natural history of complications in 52 long-surviving haemodialysis patients to obtain a clearer picture of the impact these patients have on the dialysis population. This is important as they are often no longer suitable for transplantation and therefore are destined to remain on dialysis for the rest of their lives., Methods: The patients who survived for more than 10 years on haemodialysis alone were studied. Information was obtained from patients' records and from the renal unit computer., Results: Mean age at start of dialysis was 43 years and mean duration of HD 14.5 years. Renal failure was most commonly due to polycystic kidney disease or glomerulonephritis. Sixty-two per cent of patients developed cardiovascular disease, 78% complained of joint pains, 72% had a parathyroidectomy, and 50% developed carpal-tunnel syndrome. Two hundred and forty-five episodes of infection were recorded, 41% related to vascular access acquired in hospital or on immunosuppression. Only three infections occurred which could be described as opportunistic. Twelve patients were hepatitis C positive. In the 37 patients who have died, cardiovascular disease was the most common cause of death. Compared to other patients who started on dialysis before 1986 but who had a successful transplant the survival of patients on haemodialysis is much worse., Conclusion: Long-term survival on renal replacement therapy is dependent on successful transplantation. Complications, morbidity, and mortality are high after 10 years of dialysis.

Insulin-like growth factors (IGF)-I and IGF-II are produced by osteoblasts and are important paracrine/autocrine regulators of osteoblast proliferation and differentiation. Estrogen has been reported to increase gene expression of IGF-I in rodent osteoblasts. However, because species differences have been demonstrated in expression of various aspects of the IGF system in bone cells, it is not known whether this action also occurs in human osteoblasts. Thus, we assessed the effects of estrogen treatment on IGF-I and IGF-II gene expression in vitro in a recently developed human fetal osteoblast cell line that has high levels of estrogen receptors. As assessed by a quantitative reverse transcriptase-polymerase chain reaction method, treatment of hFOB/ER9 cells with 17beta-estradiol (E2) increased steady state levels of IGF-I mRNA in a time- and dose- dependent fashion with a maximal increase of 319% +/- 33% (P < 0.01) of control occurring after treatment with 10(-7) M E2 for 48 hours. In contrast, E2 did not alter steady state levels of IGF-II mRNA. The pure (type 2) antiestrogens ICI 182,780 (10(-7) M) and ICI 164,384 (10(-6) M) blocked the E2- induced increase in IGF-I mRNA levels. Interestingly, 4-hydroxytamoxifen (10(-7) M), a documented pure antiestrogen in reproductive tissues, also increased IGF-I mRNA to levels similar to those observed in E2-treated cells. Since E2 was shown to mediate its effects on some target genes through a cAMP-dependent pathway, we studied the interaction between E2 and agents that are known to increase intracellular cAMP. Forskolin (10(-8) M) and dibutyryl cAMP (10(-3) M) increased IGF-I mRNA levels sixfold, and cotreatment with E2 did not affect these changes, consistent with a possible mediation of the estrogen effect on IGF-I gene expression by cAMP. We conclude that in human osteoblastic cells, the IGF-I gene is a target for estrogen action, suggesting that IGF-I may mediate part of the effects of estrogen in human bone.

Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completeness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild-type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5 alpha-dihydrotestosterone (5 alpha-DHT) (10(-8) M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 +/- 73 (mean +/- SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 +/- 395 functional hARs/nucleus in hFOB/AR-2 cells, and 3,987 +/- 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5 alpha-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5 alpha-DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.

Estrogen (E2) has been shown to prevent bone loss among postmenopausal women. The molecular mechanism(s) by which this is accomplished is not clear. The discovery of E2 receptor (ER) in osteoblasts and osteoclasts has implicated these cells as direct targets for E2. Previous studies on the effects of E2 on osteoblastic cells in vitro or in organ culture present conflicting results, possibly due to heterogeneity in cell types, stage of differentiation, ER levels, and/or species differences. The effects of E2 on gene expression during various stages of human osteoblast cell differentiation has not been investigated extensively. In this study we employed a newly developed human fetal osteoblastic cell line (hFOB/ER9) that contains high levels of ER to examine the effects of E2 on osteoblast proliferation and differentiation. The basal levels and E2 effects on the expression of various extracellular matrix proteins were also characterized throughout different stages of differentiation. These stages include a proliferative/relatively undifferentiated stage (day 6), a matrix maturation stage (days 10-14), and a mineralization/calcified nodule stage (day 18). During the stage of rapid cell proliferation, E2 treatment of hFOB/ER9 cells resulted in a dose-dependent decrease in [3H]thymidine incorporation to a maximum of 72% compared to the vehicle control value. Treatment of hFOB/ER9 cells with 10(-9) M E2 for 48 h resulted in an increase in alkaline phosphatase (AP) activity throughout cell differentiation. The magnitude of AP induction varied from approximately 200-500%. In contrast, E2 decreased osteocalcin protein levels to a minimum of 54% compared to the vehicle control value. The steady state messenger RNA levels for AP increased and osteocalcin decreased after E2 treatment, similar to the responses observed at the protein level. At all stages, there was little or no effect of E2 on type I collagen protein levels or osteonectin steady state messenger RNA levels. The E2 responses on hFOB/ER9 cell matrix protein expression and cell proliferation were mediated through the ER, as cultures cotreated with a 100-fold molar excess of a type II anti-E2 (ICI 182,780) abrogated these effects. These results support the hypothesis that E2 does have an effect on osteoblastic differentiation by decreasing hFOB/ER9 cell proliferation and differentially regulating extracellular matrix expression.

A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-alpha2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-beta, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous bone loss in the unloaded limb in excess of that induced by gonadal hormone deficiency. This additional bone loss was arrested by estrogen replacement. We conclude from these studies that estrogen alters the expression of signaling peptides believed to mediate skeletal adaptation to changes in mechanical usage and likewise modifies the skeletal response to mechanical unloading.

Metabolic products of skeletal muscle contraction activate metaboreceptor muscle afferents that reflexively increase sympathetic nerve activity (SNA) targeted to both resting and exercising skeletal muscle. To determine effects of the increased sympathetic vasoconstrictor drive on muscle oxygenation, we measured changes in tissue oxygen stores and mitochondrial cytochrome a,a3 redox state in rhythmically contracting human forearm muscles with near infrared spectroscopy while simultaneously measuring muscle SNA with microelectrodes. The major new finding is that the ability of reflex-sympathetic activation to decrease muscle oxygenation is abolished when the muscle is exercised at an intensity > 10% of maximal voluntary contraction (MVC). During high intensity handgrip, (45% MVC), contraction-induced decreases in muscle oxygenation remained stable despite progressive metaboreceptor-mediated reflex increases in SNA. During mild to moderate handgrips (20-33% MVC) that do not evoke reflex-sympathetic activation, experimentally induced increases in muscle SNA had no effect on oxygenation in exercising muscles but produced robust decreases in oxygenation in resting muscles. The latter decreases were evident even during maximal metabolic vasodilation accompanying reactive hyperemia. We conclude that in humans sympathetic neural control of skeletal muscle oxygenation is sensitive to modulation by metabolic events in the contracting muscles. These events are different from those involved in either metaboreceptor muscle afferent activation or reactive hyperemia.

Background: The use of recombinant human erythropoietin in pregnancy has been described in patients with renal impairment. We present a patient with a hypoproliferative anemia with low serum erythropoietin levels and no renal disease or other known cause for this type of anemia, who was treated with recombinant human erythropoietin., Case: A 29-year-old white woman who became pregnant after leuprolide acetate and menopausal gonadotropin therapy developed a moderate anemia (hemoglobin 8.5 g/dL) in early pregnancy. The only important laboratory findings were a hypoproliferative marrow and a low serum erythropoietin level. She was treated successfully with injections of recombinant human erythropoietin. Her pregnancy was otherwise uncomplicated and her pre-delivery hemoglobin level was 12 g/dL., Conclusion: Hypoproliferative anemia in pregnancy with low erythropoietin levels and no renal disease can be treated successfully with recombinant human erythropoietin.

Using a recently developed human osteoblastic cell line (hFOB/ER9) with high levels (approximately 4,000 per nucleus) of estrogen receptors and the characteristic phenotype of the mature osteoblast, we tested the hypothesis that estrogen decreases bone formation by inhibiting the action of the insulin-like growth factor (IGF) paracrine/autocrine system. IGF-II, the predominant IGF produced by osteoblastic cells, was measurable in hFOB/ER9-conditioned medium (approximately 10 ng/mL) and its level did not change significantly after treatment with 17 beta-estradiol (E2) or anti-estrogens. Treatment with E2 at 0.1-100 nM decreased [3H]thymidine uptake to 53% of control (p < 0.001) in a dose-dependent fashion. The predominant IGF-binding proteins (IGFBPs) produced by hFOB/ER9 and by normal trabecular osteoblasts are IGFBP-3 and IGFBP-4, of which IGFBP-4 is consistently inhibitory of IGF action. Treatment with E2 at 0.01-10 nM for 48 h increased IGFBP-4 mRNA to 346% +/- 90% (mean +/- SE) of control (p < 0.05) and IGFBP-4 protein to 278% +/- 75% of control (p < 0.01) in a dose-dependent fashion but did not alter IGFBP-3 mRNA or protein. E2 treatment also attenuated IGF-dependent, IGFBP-4 specific proteolysis to approximately 50% of control. ICI 182,780, a pure anti-estrogen, completely blocked E2-mediated decreases in cell proliferation and increases in levels of IGFBP-4 mRNA and protein. Treatment of the hFOB/ER9 cells with recombinant human IGFBP-4 (200 ng/mL) decreased cell proliferation to 55% of control (p < 0.01). Thus, E2 acts on osteoblastic cells to increase availability of inhibitory IGFBP-4, by both increasing its production and decreasing its degradation, which may oppose the mitogenic effect of the IGFs on osteoblastic cells. This action may mediate, at least in part, the decreases in bone formation that are observed after estrogen treatment in vivo.

Some studies suggest that estrogen acts on bone by decreasing the production of interleukin-6 (IL-6), a cytokine that increases bone resorption, by osteoblasts or bone marrow cells. However, other studies have not confirmed this, possibly because of a low and variable number of estrogen receptors (ER) in the model systems used. Thus, we employed a recently developed human fetal osteoblast cell line with high levels of ER. Treatment (n = 4 experiments) with 0.01 to 10 nM of 17 beta-estradiol had no effect on the constitutive production of IL-6. However, stimulated production, induced by treatment with IL-1 beta plus tumor necrosis factor-alpha (TNF-alpha), was reduced in a dose-dependent manner to 74 +/- 3% (mean +/- SEM) of control (p < 0.01). This response was blocked by cotreatment with the type II antiestrogen ICI 182,780. Treatment with hydrocortisone (1 microM), a known inhibitor of IL-6 production in many cell types, reduced IL-6 production to 17 +/- 1% of control (p < 0.001). As assessed by Northern analysis, treatment (n = 3 experiments) with 0.01-10 nM of 17 beta-estradiol decreased steady-state levels of IL-6 mRNA in a dose-dependent manner. These data support the hypothesis that at least part of the antiresorptive action of estrogen in humans is mediated by decreased production of IL-6 by osteoblastic cells.

The TGF-beta family of growth factors has been extensively studied and found to play major roles in bone physiology and disease. A novel, TGF-beta-inducible early gene (TIEG) in normal human fetal osteoblasts (hFOB) has been identified using differential-display PCR. Using this differentially expressed cDNA fragment of TIEG to screen a hOB cDNA library, a near full-length cDNA for this gene was isolated. Northern analyses indicated that the steady-state levels of the 3.5 kb TIEG mRNA increased within 30 min of TGF-beta treatment of human osteoblasts and reached a maximum of 10-fold above control levels at 120 min post-treatment. This regulation was independent of new protein synthesis. Computer sequence analyses indicates that TIEG mRNA encodes for a 480 amino-acid protein. The TIEG protein contains three zinc finger motifs, several proline-rich src homology-3 (SH3) binding domains at the C-terminal end, and is homologous in this region to the zinc finger-containing transcription factor family of genes. A growth factor/cytokine-specific induction of TIEG has been shown. TIEG expression in hFOB cells was highly induced by TGF-beta and bone morphogenetic protein-2 (BMP-2), with a moderate induction by epidermal growth factor (EGF), but no induction by other growth factors/cytokines was observed. In addition to osteoblastic cells, high levels of TIEG expression were detected in skeletal muscle tissue, while low or no detectable levels were found in brain, lung, liver or kidney. Because TIEG is an early induced putative transcription factor gene, and shows a growth factor induction and tissue specificity, its protein product might play an important role as a signalling molecule in osteoblastic cells.

Universal primers were used for PCR amplification of three noncoding regions of chloroplast DNA in order to study restriction site variation in 12 Mexican pine species. Two length mutations were identified that are of diagnostic value for two subgenera or sections of the genus. Phylogenetic analysis of the restriction site and length variation showed patterns of variation largely consistent with previous arrangements of these pines, except for the position of Pinus nelsonii, indicating that Pinus section Parraya Mayr, as circumscribed by Little and Critchfield (1969) and later authors, is not a monophyletic group.

The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (+/- 1,341) 17beta-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (+/- 2) fmol/mg protein. This subclone was examined further for estradiol (E2) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased approximately 25-fold in hFOB/ER9 cells following 10(-9)M E2 treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10(-9)M E2 resulted in a 2.5-4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function.

Insulin-like growth factors I (IGF-I) and II (IGF-II) are anabolic for osteoblastic cells. Although expression of IGF-I and IGF-II mRNA has been demonstrated in rodent osteoblastic cells, little is known about IGF gene expression in human osteoblastic cell models. In this study we characterized IGF-I and -II mRNA expression in (1) normal human osteoblast-like (hOB) cells, (2) a simian virus 40 immortalized hOB (HOBIT) cell line, and (3) human osteosarcoma cell lines SaOS-2, TE-85, MG-63, and U-2. Since cross-hybridization of IGF cDNA probes with ribosomal RNA obscures detection of some of the multiple IGF transcripts in human cells, we replaced Northern analysis with the more specific ribonuclease protection assay (RPA). We also used the reverse transcriptase-polymerase chain reaction (RT-PCR) to assess whether mRNAs were present at trace levels. IGF-I mRNA expression was consistently observed in normal hOB cells only and by both RT-PCR and RPA. Among IGF-I transcript variants, Ea IGF-I mRNA was more abundant than the Eb mRNA in normal hOB cells. Trace levels of IGF-I mRNA were variably detected in SaOS-2 and U-2 osteosarcoma cells when RT-PCR was performed, but we found no IGF-I mRNA in HOBIT, TE-85, or MG-63 cells. IGF-II mRNA was expressed in normal hOB, HOBIT, TE-85, and U-2 cells as assessed by either method.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is secreted by a variety of osteoblastic cells and appears to be an integral component of bone cell physiology. We have previously reported that normal human osteoblast-like (hOB) cells secrete IGFBP-4 as well as a novel IGFBP-4 protease, which requires IGF for functional activity. In this study we assessed the IGFBP-4/IGFBP-4 protease system in transformed osteoblastic cells by Western ligand blotting and cell-free IGFBP-4 protease assays. Simian virus-40-immortalized hOB cells (HOBIT), human osteosarcoma cells (TE-85), and rat osteosarcoma cells (UMR 106-01, ROS 17/2.8) secrete IGFBP-4. In contrast to the rapid and dramatic proteolysis in hOB medium, medium conditioned by these cells had no apparent IGFBP-4 protease activity when assayed with exogenous IGF-II in culture or under cell-free conditions. Assayed in the presence of exogenous protease. HOBIT cells, but not the osteosarcoma cell lines, appeared to produce a cycloheximide-sensitive inhibitor of the IGFBP-4 proteolytic reaction. Transient cell transformation induced by incubating human osteoblasts transfected with a temperature-sensitive mutant of simian virus-40 T-antigen at the permissive temperature or by treating hOB cells with phorbol ester tumor promoters also resulted in inhibition of IGF-dependent IGFBP-4 proteolysis. Inhibition was observed if phorbol ester was added to the cultures at the time of medium change or after the protease had been expressed and secreted. Differences in IGFBP-4 proteolysis could not be accounted for by changes in IGFBP-4 messenger RNA expression or substrate levels. These data suggest that transformation is associated with alterations in the IGFBP-4/IGFBP-4 protease system in osteoblastic cells. Normal human osteoblasts secrete an IGF-dependent IGFBP-4 protease. The induction of an inhibitor of the IGF-dependent IGFBP-4 proteolytic reaction may be associated with early transformation processes. Fully tumorigenic bone cells expressed neither IGFBP-4 protease nor protease inhibitor activity.

We report the establishment of a human fetal osteoblast cell line derived from biopsies obtained from a spontaneous miscarriage. Primary cultures isolated from fetal tissue were transfected with a gene coding for a temperature-sensitive mutant (tsA58) of SV40 large T antigen along with a gene coding for neomycin (G418) resistance. Individual neomycin resistant colonies were screened for alkaline phosphatase (AP)-specific staining. The clone with the highest AP level, hFOB 1.19, was examined further for other osteoblast phenotypic markers. Incubation of hFOB cells at the permissive temperature (33.5 degrees C) resulted in rapid cell division, whereas little or no cell division occurred at the restrictive temperature (39.5 degrees C). Both AP activity and osteocalcin (OC) secretion increased in a dose-dependent manner following dihydroxyvitamin D3 (1,25-D3) treatment when cultured at either temperature. However, AP and 1,25-D3-induced OC levels were elevated in confluent hFOB cells cultured at 39.5 degrees C compared with 33.5 degrees C. Treatment of hFOB cells with 1-34 parathyroid hormone (PTH) resulted in an increase in cAMP levels. Upon reaching confluence, hFOB cultures went through programmed differentiation and formed mineralized nodules as observed by von Kossa staining. Further, immunostaining of postconfluent, differentiated hFOB cells showed that high levels of osteopontin, osteonectin, bone sialoprotein, and type I collagen were expressed. Therefore, the clonal cell line hFOB 1.19 provides a homogeneous, rapidly proliferating model system to study certain stages of human osteoblast differentiation.

Previous work in this and other laboratories has shown that steroids rapidly regulate the expression of nuclear protooncogenes. In this present study, we have investigated the effect of progesterone (Pg) on the expression of c-jun in the avian oviduct system and its promoter activity in avian liver cells. Pg treatment of estrogen-withdrawn chickens brings about a decrease in the steady state mRNA level of the protooncogene c-jun within 30 min. This decrease is steroid dose dependent and gene specific. Using nuclear run-off transcription analyses, this rapid regulation was shown to occur at the level of gene transcription, as the rate of c-jun transcription decreases by more than 80% within 15 min after progesterone treatment. As expected, ovalbumin gene transcription is increased only after a lag period of 4 h following Pg treatment. In other studies, we have linked the c-jun promoter sequences between -1000 and +192 to chloramphenicol acetyltransferase reporter gene and cotransfected them into transformed avian liver cells along with the expression vector for the Pg receptor. Pg treatment of these cells causes a decrease in chloramphenicol acetyltransferase gene expression, albeit to a lesser extent than Pg inhibition of c-jun gene transcription. These results suggest that the 5'-domain of the chicken c-jun gene contains sequence elements that negatively regulate c-jun promoter activity in response to Pg.

Recent studies have shown that macrophage colony-stimulating factor and its receptor c-fms protein are significantly overexpressed in endometrial and ovarian cancers. In the present study, we analyzed the steady-state levels of c-fms mRNA in benign and malignant endometrial tissues by Northern and slot blot analyses. The relative levels of c-fms mRNA were quantified by using a hybridization signal for each sample on Northern blot analysis. Slot blot analysis was used to further quantitate the relative increase in c-fms mRNA in malignant specimens compared to benign specimens. Correlation of c-fms expression in the endometrial cancers was made with traditional prognostic indicators. Secretory endometrium had low levels of c-fms mRNA, whereas the endometrial cancers had the highest levels. Proliferative and hyperplastic endometrium values were intermediate. Comparative assessment of c-fms expression in endometrial cancer relative to other prognostic factors demonstrated greater expression of c-fms in specimens from patients with abnormal DNA ploidy, high-grade lesions, and possibly extrauterine metastases. Our study confirms the overexpression of c-fms in endometrial cancer and demonstrates a positive correlation between the steady-state mRNA levels of c-fms and other select adverse prognostic indicators.

The sequences required for the maximal expression of the mouse L32 ribosomal protein gene and the binding of nuclear factors to L32 promoter elements were analyzed in mouse myoblasts, fibers, and L cells. Various L32 r-protein promoter sequences were linked to the chloramphenicol acetyltransferase gene (CAT), and the expression of the chimeric genes was measured transiently or after their incorporation into the genome. The sequence requirements for maximal expression of the L32 gene are very similar among the various cells and include the previously identified L32 core promoter from approximately -150 to +75. Only the promoter regions between -45 and +11 displays significant cell type specific differences. Relative to the maximal activity in each cell type, the expression of the L32-CAT gene containing the -45 to +11 region is greater in L cells than in myoblasts or fibers. This difference is correlated with the increased activity of an L cell nuclear factor(s) that binds to this fragment. In addition, our results show that deletion of sequences between -981 and -141 causes a 50-70% reduction of the expression of the L32-CAT gene in myoblasts, fibers and L cells. The transcription of all the L32-CAT genes examined decrease after myoblasts differentiate into fibers in a manner similar to the endogenous L32 gene, but we were unable to distinguish between sequences involved in controlling the expression of the L32 gene during myoblast differentiation and those sequences required for maximal promoter activity. However, gel mobility shift assays showed differences in the binding of myoblast and fiber factors to the four promoter fragments examined. The possible role of these factor binding differences in controlling L32 transcription is discussed.

The percutaneous penetration of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-D dimethylamine salt (DMA) was evaluated separately in five male volunteers who participated in both experiments. Urine samples collected for 144 h following dermal applications of 10 mg to the dorsum of the hand (9 cm2) were analyzed for 2,4-D. Following the acid application, an average of 4.46 +/- 0.849% was recovered in the urine and a significantly lower amount of 1.76 +/- 0.568% following the DMA application. Significantly higher amounts of 2,4-D DMA (7.68 +/- 0.493 mg) were washed off the hand 6 h following application as compared with 2,4-D acid (5.35 +/- 0.384 mg). These results indicate that, in addition to the differences in physical and chemical properties of the two compounds that will affect absorption, the amount of the chemical absorbed is related inversely to the amount of washed off. Urinary excretion of 2,4-D was not complete in all volunteers 144 h following either application, but in all cases it was approaching the limit of detection. An average of 84.8 +/- 2.55% and 76.8 +/- 8.05% of the total recovered in 144 h was recovered in the urine 96 h following 2,4-D acid and 2,4-D DMA application, respectively. Average, approximated half-lives for excretion were 39.5 +/- 8.1 h for the acid application and 58.5 +/- 13.2 h for the DMA application.

Total body dose received in home gardeners applying 2,4-D and bystanders living within the household, but not applying the pesticide was measured. Levels of 2,4-D were monitored in air samples both inside the home and downwind of the application site. Homeowners were divided into protective and non-protective apparel groups and applied both a granular and liquid formulation of 2,4-D on two separate dates. Analyses of urine collected from homeowners for 96 hours following applications found total body doses ranging from non-detectable to 0.0071 mg/kg of body weight. The highest exposures occurred in the non-protected group and were consistently associated with spills of the liquid concentrate or excessive contact with the dilute mixture on the hands or forearms. Residues of 2,4-D were not detected in urine samples supplied by bystanders to home applicators. Residues of 2,4-D were detected in five of the 76 air samples taken during the home applications. Two of these air samples coincided with measurable applicator exposure but it is unlikely that this was a major route of exposure.

Total body dose of 2,4-D was determined in 10 volunteers following exposure to sprayed turf 1 hour following application and in 10 volunteers exposed 24 hours following application. Each group of 10 volunteers was divided in half and five wore long pants, a short-sleeved shirt, socks and closed footwear. The other five wore shorts and a short-sleeved shirt and were barefoot. All volunteers were exposed to a 2 by 15 m area of turf for 1 hour during which they alternated between walking and sitting or lying on the turf surface for intervals of 5 minutes. Dislodgeable residues of 2,4-D taken during the exposure sessions showed a rapid decline from 1 hour following application (8%) to 24 hours following application (1%). No detectable residues were found in 4-day urine samples supplied by volunteers except for 3 people who were barefoot and wearing shorts and contacted the turf 1 hour following 2,4-D application. The highest dose was measured in a volunteer who removed his shirt for 30 minutes of the exposure session (426 micrograms). Exposure levels of the other two volunteers who wore the prescribed clothing were lower (153 and 103 micrograms). No detectable residues were found in urine samples supplied by volunteers exposed to sprayed turf 24 hours following application. These results indicate that at the doses measured, exposure to sprayed turf should present little risk in humans. However, people can reduce exposure to non-detectable levels by remaining off treated turf for a period of 24 hours or until after rainfall or irrigation so that dislodgeable residues and therefore potential exposure are essentially zero.

To determine if bone cells produce interleukin-1 beta (IL-1 beta), a potent bone resorption-stimulating agent, we studied well-characterized, nearly homogeneous cultures of normal human osteoblast-like (hOB) cells. With four strains of such cells, vehicle-treated cultures produced minimal IL-1 beta (mean +/- SEM, 1.3 +/- 0.3 pg/ml per 10(6) cells per 24 h) and showed dose-dependent (r = 0.99) increases to 2.2 +/- 0.7, 5.0 +/- 0.9, or 17.8 +/- 6.7 pg/ml, respectively, after treatment with lipopolysaccharide (LPS) at 3, 10, or 30 micrograms/ml (for increases after 10 and 30 micrograms/ml treatments, P less than 0.05). After treatment with tumor necrosis factor alpha (TNF-alpha) at 10 U/ml, IL-1 beta increased to 16.2 +/- 3.7 pg/ml (P less than 0.05). Neither 17 beta-estradiol nor bovine parathyroid hormone(1-34) (each at 10 nM), alone or in combination with LPS or TNF-alpha, affected IL-1 beta release. Northern blot analysis of total cellular RNA preparation revealed a single hybridization band at 1.9 kb when probed with a partially deleted cDNA for human IL-1 beta. The steady-state IL-1 beta mRNA levels showed a significant increase with LPS treatment and a lesser increase with TNF-alpha treatment in hOB cells. Moreover, TNF-alpha produced an even greater increase in IL-1 mRNA in HOBIT cells, a well-differentiated clonal cell line derived from normal hOB cells transfected with the SV40 large T antigen. We conclude that human cells of the osteoblast lineage produce IL-1 beta in response to well-recognized stimuli for IL-1 release from responsive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of 22 patients, most of whom had spinal injuries with detrusor sphincter dyssynergia, have had Medinvent Wallstents placed across the distal mechanism. All but 8 patients had undergone outflow surgery and 11 had had repeated unsuccessful sphincterotomies. The proximal end of the stent was placed over the verumontanum unless fertility was contemplated, when it was placed immediately below the verumontanum. Fifteen patients achieved complete voiding after placement of the first stent; 3 developed bladder neck obstruction after stenting, but in 1 of these cases resolution occurred after bladder neck incision. The 3 patients with artificial urinary sphincters failed to improve after stenting. Use of the urethral stent for patients with detrusor sphincter dyssynergia and failed sphincterotomy is a major advance. It should probably be the primary treatment in selected cases. Its effect on fertility is currently under assessment.