Factors affecting the species of Campylobacter colonizing chickens reared for meat.

AIM: To investigate factors influencing Campylobacter spp. colonization of broiler chickens. METHODS AND RESULTS: Campylobacters were isolated from caeca from 319 flocks of two different breeds (199 Cobb and 120 Hubbard), reared as standard (199), Freedom Food/corn fed (57), free-range (47) or organic (16). The standard category exclusively used Cobb birds slaughtered at 38-41 days. The Freedom Food/corn-fed and free-range Hubbard birds were slaughtered at 49-56 days and the organic flocks at 70 days. Campylobacters were picked at random from direct plates. Both breed of chicken (Hubbard) and age at slaughter were independently associated with increased likelihood of colonization by Campylobacter coli rather than Campylobacter jejuni, but breed could not be separated from other aspects of husbandry with the data available. CONCLUSIONS: Chickens are frequently colonized by C. jejuni and C. coli and most human infections originate from poultry. In most developed countries approximately 90% of human infections are caused by C. jejuni, but fewer than 10% by C. coli. This might be due to C. coli being less pathogenic than C. jejuni to humans, and/or to chicken meat carrying fewer C. coli than C. jejuni. More investigations are needed into these aspects before it can be concluded that slaughtering older birds from slower-growing breeds would reduce the risk of human Campylobacter disease. SIGNIFICANCE AND IMPACT OF THE STUDY: Meat from certain breeds of poultry are predominantly colonized by C. coli rather than C. jejuni. More research is needed to understand the impact this may have on the number and severity of human campylobacter infections.

Impacts of selective logging on inbreeding and gene flow in two Amazonian timber species with contrasting ecological and reproductive characteristics.

Selective logging in Brazil allows for the removal of up to 90% of trees above 50 cm diameter of a given timber species, independent of a species' life history characteristics or how quickly it will recover. The genetic and demographic effects of selective logging on two Amazonian timber species (Dipteryx odorata Leguminosae, Jacaranda copaia Bignoniaceae) with contrasting ecological and reproductive characteristics were assessed in the same forest. Genetic diversity and gene flow were characterized by genotyping adults and seed sampled before and after logging, using hypervariable microsatellite markers. Overall, there were no short-term genetic impacts on the J. copaia population, with commercial application of current Brazilian forest management regulations. In contrast, for D. Odorata, selective logging showed a range of genetic impacts, with a 10% loss of alleles, and reductions in siring by pollen from trees within the 546-ha study area (23-11%) and in the number of pollen donors per progeny array (2.8-1.6), illustrating the importance of the surrounding landscape. Asynchrony in flowering between D. odorata trees led to trees with no breeding partners, which could limit the species reproduction and regeneration under current regulations. The results are summarized with other published studies from the same site and the implications for forest management discussed. The different types and levels of impacts associated with each species support the idea that ecological and genetic information by species, ecological guild or reproductive group is essential in helping to derive sustainable logging guidelines for tropical forests.

Remnant Pachira quinata pasture trees have greater opportunities to self and suffer reduced reproductive success due to inbreeding depression.

Habitat fragmentation is extensive throughout the world, converting natural ecosystems into fragments of varying size, density and connectivity. The potential value of remnant trees in agricultural landscapes as seed sources and in connecting fragments has formed a fertile area of debate. This study contrasted the mating patterns of bat-pollinated Pachira quinata trees in a continuous forest to those in pasture through microsatellite-based paternity analysis of progeny. The breeding system was determined by analysis of pollen tube growth and seed production from controlled pollinations. Fitness of selfed and outcrossed seed was compared by germination and seedling growth. There was more inbreeding within pasture trees (outcrossing=0.828±0.015) compared with forest trees (0.926±0.005). Pasture trees had fewer sires contributing to mating events, but pollen dispersal distances were greater than those in the forest. Paternity analysis showed variation in outcrossing rates among pasture trees with high proportions of external and self pollen sources detected. A leaky self-incompatibility system was found, with self pollen having reduced germination on stigmas and slower growth rate through the style. Controlled pollinations also showed a varied ability to self among trees, which was reflected in the selfing rates among pasture trees shown by the paternity analysis (0-80% selfing). Self pollination resulted in lower seed set, germination and seedling growth compared with outcrossing. While remnant trees in agricultural landscapes are involved in broader mating patterns, they show increased but varied levels of inbreeding, which result in reduced fitness.

Long-term impacts of selective logging on two Amazonian tree species with contrasting ecological and reproductive characteristics: inferences from Eco-gene model simulations.

The impact of logging and subsequent recovery after logging is predicted to vary depending on specific life history traits of the logged species. The Eco-gene simulation model was used to evaluate the long-term impacts of selective logging over 300 years on two contrasting Brazilian Amazon tree species, Dipteryx odorata and Jacaranda copaia. D. odorata (Leguminosae), a slow growing climax tree, occurs at very low densities, whereas J. copaia (Bignoniaceae) is a fast growing pioneer tree that occurs at high densities. Microsatellite multilocus genotypes of the pre-logging populations were used as data inputs for the Eco-gene model and post-logging genetic data was used to verify the output from the simulations. Overall, under current Brazilian forest management regulations, there were neither short nor long-term impacts on J. copaia. By contrast, D. odorata cannot be sustainably logged under current regulations, a sustainable scenario was achieved by increasing the minimum cutting diameter at breast height from 50 to 100 cm over 30-year logging cycles. Genetic parameters were only slightly affected by selective logging, with reductions in the numbers of alleles and single genotypes. In the short term, the loss of alleles seen in J. copaia simulations was the same as in real data, whereas fewer alleles were lost in D. odorata simulations than in the field. The different impacts and periods of recovery for each species support the idea that ecological and genetic information are essential at species, ecological guild or reproductive group levels to help derive sustainable management scenarios for tropical forests.

Peptide synthesis and self-assembly.

Peptides and proteins are the most diverse building blocks in biomolecular self-assembly in terms of chemistry, nanostructure formation and functionality. Self-assembly is an intrinsic property of peptides. In this chapter, we attempt to address the following issues: How can we synthesize a self-assembling peptide? What are the fundamental physical and chemical principles that underpin peptide self-assembly? How can we learn to finely control peptide self-assembly? The merits of answering these questions are inspiring both for biology and medicine in terms of new opportunities for understanding, preventing and curing of diseases, and for nanotechnology in terms of new prescribed routes to achieving peptide-based nanostructures with a range of properties appropriate for specific applications.

Influence of season and geography on Campylobacter jejuni and C. coli subtypes in housed broiler flocks reared in Great Britain.

Geographical and seasonal variation in the incidence and prevalence of Campylobacter jejuni and C. coli in housed broiler flocks reared in Great Britain in 2004 to 2006 was investigated in this study. Ceca (30) from 797 flocks, not subject to prior partial depopulation and reared on 211 farms, were examined individually for the presence of Campylobacter spp. The best-fitting climatic factors explained approximately 46% of the prevalence of Campylobacter-colonized flocks at slaughter and consisted of a combination of temperature at slaughter, number of sunshine hours in placement month, and millimeters of rainfall in placement month. Positive flocks were more likely to be slaughtered between June and November than during the rest of the year and to be reared in northern Great Britain than in central or southern Great Britain. C. jejuni was identified in approximately 90% of flocks, and C. coli was present in 10% of flocks. The most common clonal complexes identified in 226 isolates typed by multilocus sequence typing (MLST) were ST-45, ST-21, ST-574, ST-443, and ST-828. Flocks slaughtered at the same time were more likely to have similar complexes, and ST-45 had a seasonal pattern, with the highest prevalence in June, and was also more likely to be present in flocks reared in northern Great Britain.

Sporophytic self-incompatibility in Senecio squalidus (Asteraceae): S allele dominance interactions and modifiers of cross-compatibility and selfing rates.

Understanding genetic mechanisms of self-incompatibility (SI) and how they evolve is central to understanding the mating behaviour of most outbreeding angiosperms. Sporophytic SI (SSI) is controlled by a single multi-allelic locus, S, which is expressed in the diploid (sporophyte) plant to determine the SI phenotype of its haploid (gametophyte) pollen. This allows complex patterns of independent S allele dominance interactions in male (pollen) and female (pistil) reproductive tissues. Senecio squalidus is a useful model for studying the genetic regulation and evolution of SSI because of its population history as an alien invasive species in the UK. S. squalidus maintains a small number of S alleles (7-11) with a high frequency of dominance interactions. Some S. squalidus individuals also show partial selfing and/or greater levels of cross-compatibility than expected under SSI. We previously speculated that these might be adaptations to invasiveness. Here we describe a detailed characterization of the regulation of SSI in S. squalidus. Controlled crosses were used to determine the S allele dominance hierarchy of six S alleles and effects of modifiers on cross-compatibility and partial selfing. Complex dominance interactions among S alleles were found with at least three levels of dominance and tissue-specific codominance. Evidence for S gene modifiers that increase selfing and/or cross-compatibility was also found. These empirical findings are discussed in the context of theoretical predictions for maintenance of S allele dominance interactions, and the role of modifier loci in the evolution of SI.

Differential sympathetic neural control of oxygenation in resting and exercising human skeletal muscle.

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.

A simple physical description of DNA dynamics: quasi-harmonic analysis as a route to the configurational entropy.

It has become increasingly apparent that the dynamic as well as the structural properties of biological macromolecules are important to their function. However, information concerning molecular flexibility can be difficult to obtain experimentally at the atomic level. Computer modelling techniques such as molecular dynamics (MD) have therefore proved invaluable in advancing our understanding of biomolecular flexibility. This paper describes how a combination of atomistic MD simulations and quasi-harmonic analysis can be used to describe the dynamics of duplex DNA, with a particular emphasis on methods for calculating differences in configurational entropies. We demonstrate that DNA possesses remarkably simple mechanical properties relative to globular proteins, making it an ideal system for exploring biomolecular flexibility in general. Our results also highlight the importance of solvent viscosity in determining the dynamic behaviour of DNA in aqueous solution.

Induced fit DNA recognition by a minor groove binding analogue of Hoechst 33258: fluctuations in DNA A tract structure investigated by NMR and molecular dynamics simulations.

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.

Estimated aortic stiffness is independently associated with cardiac baroreflex sensitivity in humans: role of ageing and habitual endurance exercise.

We hypothesised that differences in cardiac baroreflex sensitivity (BRS) would be independently associated with aortic stiffness and augmentation index (AI), clinical biomarkers of cardiovascular disease risk, among young sedentary and middle-aged/older sedentary and endurance-trained adults. A total of 36 healthy middle-aged/older (age 55-76 years, n=22 sedentary and n=14 endurance-trained) and 5 young sedentary (age 18-31 years) adults were included in a cross-sectional study. A subset of the middle-aged/older sedentary adults (n=12) completed an 8-week-aerobic exercise intervention. Invasive brachial artery blood pressure waveforms were used to compute spontaneous cardiac BRS (via sequence technique), estimated aortic pulse wave velocity (PWV) and AI (AI, via brachial-aortic transfer function and wave separation analysis). In the cross-sectional study, cardiac BRS was 71% lower in older compared with young sedentary adults (P<0.05), but only 40% lower in older adults who performed habitual endurance exercise (P=0.03). In a regression model that included age, sex, resting heart rate, mean arterial pressure (MAP), body mass index and maximal exercise oxygen uptake, estimated aortic PWV (ß±s.e.=-5.76±2.01, P=0.01) was the strongest predictor of BRS (model R(2)=0.59, P<0.001). The 8-week-exercise intervention improved BRS by 38% (P=0.04) and this change in BRS was associated with improved aortic PWV (r=-0.65, P=0.044, adjusted for changes in MAP). Age- and endurance-exercise-related differences in cardiac BRS are independently associated with corresponding alterations in aortic PWV among healthy adults, consistent with a mechanistic link between variations in the sensitivity of the baroreflex and aortic stiffness with age and exercise.

orchard: Paternity program for autotetraploid species.

Advances in molecular marker technology have provided new opportunities to study the population genetics of polyploid taxa. Paternity analysis using microsatellite markers can be used in detection of gene flow between individuals and populations, in mating system analysis, to identify factors that influence fecundity and fertility, to identify behaviour of parent-offspring relationships and in the analysis of the reproductive success of different ecological groups. As there is no specific program for carrying out paternity analysis in tetraploid species, specialized software was designed for the assignment of paternity for autotetraploid species. orchard is a novel implementation of exclusion and likelihood statistics for carrying out paternity analysis of autotetraploids. First, the program performs an exclusion method, and then, a likelihood statistic is used with nonexcluded candidate fathers. Optional features include estimation of allele dosage of known mother trees and the estimation of pollen flow distances. orchard was tested using a data set of microsatellite data of Dipteryx odorata, a tetraploid Amazonian tree species.

Estrogen inhibits interleukin-6 production and gene expression in a human osteoblastic cell line with high levels of estrogen receptors.

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.

Normal human osteoblast-like cells consistently express genes for insulin-like growth factors I and II but transformed human osteoblast cell lines do not.

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)

Development and characterization of a conditionally immortalized human fetal osteoblastic cell line.

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.

Evidence for interleukin-1 beta production by cultured normal human osteoblast-like cells.

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)

Rapid down-regulation of c-jun protooncogene transcription by progesterone in the avian oviduct.

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.

Estrogen regulation of human osteoblastic cell proliferation and differentiation.

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.

Alterations in insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis in transformed osteoblastic cells.

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.

The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signaling peptides in ovariectomized growing rats.

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.