Physiotherapy rehabilitation for osteoporotic vertebral fracture-a randomised controlled trial and economic evaluation (PROVE trial).

The trial compared three physiotherapy approaches: manual or exercise therapy compared with a single session of physiotherapy education (SSPT) for people with osteoporotic vertebral fracture(s). At 1 year, there were no statistically significant differences between the groups meaning there is inadequate evidence to support manual or exercise therapy. INTRODUCTION: To evaluate the clinical and cost-effectiveness of different physiotherapy approaches for people with osteoporotic vertebral fracture(s) (OVF). METHODS: >Prospective, multicentre, adaptive, three-arm randomised controlled trial. Six hundred fifteen adults with back pain, osteoporosis, and at least 1 OVF participated. INTERVENTIONS: 7 individual physiotherapy sessions over 12 weeks focused on either manual therapy or home exercise compared with a single session of physiotherapy education (SSPT). The co-primary outcomes were quality of life and back muscle endurance measured by the QUALEFFO-41 and timed loaded standing (TLS) test at 12 months. RESULTS: At 12 months, there were no statistically significant differences between groups. Mean QUALEFFO-41: - 1.3 (exercise), - 0.15 (manual), and - 1.2 (SSPT), a mean difference of - 0.2 (95% CI, - 3.2 to 1.6) for exercise and 1.3 (95% CI, - 1.8 to 2.9) for manual therapy. Mean TLS: 9.8 s (exercise), 13.6 s (manual), and 4.2 s (SSPT), a mean increase of 5.8 s (95% CI, - 4.8 to 20.5) for exercise and 9.7 s (95% CI, 0.1 to 24.9) for manual therapy. Exercise provided more quality-adjusted life years than SSPT but was more expensive. At 4 months, significant changes above SSPT occurred in endurance and balance in manual therapy, and in endurance for those ≤ 70 years, in balance, mobility, and walking in exercise. CONCLUSIONS: Adherence was problematic. Benefits at 4 months did not persist and at 12 months, we found no significant differences between treatments. There is inadequate evidence a short physiotherapy intervention of either manual therapy or home exercise provides long-term benefits, but arguably short-term benefits are valuable. TRIAL REGISTRATION: ISRCTN 49117867.

Men's experiences of having osteoporosis vertebral fractures: a qualitative study using interpretative phenomenological analyses.

Men and women with vertebral fractures due to osteoporosis are treated differently by society and health care professionals. This can lead to inequalities in health care and affects how men with fractures view themselves as people. We need to raise awareness that men get these fractures as well as women. INTRODUCTION: There is a lack of research exploring the experience of osteoporosis from the male perspective. This study was undertaken to explore and describe the experiences of men with vertebral fractures due to osteoporosis, including their perceptions of diagnosis, treatment and changes in their sense of self. METHODS: The study consists of in-depth semi-structured interviews with nine male participants of the PROVE (Physiotherapy Rehabilitation for Osteoporotic Vertebral Fracture) study. Interviews were digitally audio recorded and fully transcribed. Data were coded in accordance with an interpretative phenomenological analysis approach to analyses. RESULTS: Three main themes are presented. (i) Osteoporosis is considered an old women's disease. (ii) Men are diagnosed and treated differently than women in the NHS. Health care inequalities exist. (iii) Changes in self can occur in men after vertebral fracture/s due to osteoporosis. CONCLUSIONS: Greater awareness that men get this condition is needed in both society in general and also by health care professionals who often do not expect osteoporosis to affect men. Approaches to diagnosis and treatment need to be considered and improved to ensure that they become appropriate and effective for men as well as women.

Is the timed loaded standing test a valid measure of back muscle endurance in people with vertebral osteoporosis?

Timed loaded standing (TLS) is a suggested measure of back muscle endurance for people with vertebral osteoporosis. Surface electromyography revealed back muscles work harder and fatigue during TLS. The test end-point and total time were associated with back fatigue. The findings help demonstrate the concurrent validity of the TLS test. INTRODUCTION: The TLS test is suggested as a measure of back muscle endurance for patients with vertebral osteoporosis. However, to date, no study has demonstrated that TLS does measure back extensor or erector spinae (ES) muscle endurance. We used surface electromyography (sEMG) to investigate the performance of the thoracic ES muscles during TLS. METHODS: Thirty-six people with vertebral osteoporosis with a mean age of 71.6 (range 45-86) years participated. sEMG recordings were made of the ES at T3 and T12 bilaterally during quiet standing (QS) and TLS. The relative (%) change in sEMG amplitude between conditions was compared. Fatigue was evaluated by analysing the change in median frequency (MF) of the sEMG signal during TLS, and the correlation between maximal TLS time and rate of MF decline was examined. RESULTS: Activity in the ES increased significantly during TLS at all electrode locations. During TLS, the MF declined at a mean rate of -24.2% per minute (95% C.I. -26.5 to -21.9%). The MF slope and test time were strongly correlated (r2 = 0.71), and at test end, the final MF dropped to an average 89% (95% C.I. 85 to 93%) of initial MF. Twenty-eight participants (78%) reported fatigue was the main reason for stopping, and for eight (22%), it was pain. CONCLUSIONS: This study demonstrates that TLS challenges the ES muscles in the thoracic region and results in ES fatigue. Endurance time and the point at which the TLS test ends are strongly related to ES fatigue.

Blind outcome assessment: the development and use of procedures to maintain and describe blinding in a pragmatic physiotherapy rehabilitation trial.

OBJECTIVES: (1) To develop trial protocols which promote the achievement of blind outcome assessment. (2) To report outcome assessor beliefs regarding group allocation at follow-up assessments. (3) To document and describe instances of unblinding occurring during the trial to assist and inform rehabilitation researchers and clinicians. DESIGN: Prospective longitudinal observational study. SETTING: An NHS Hospital Trust specializing in orthopaedic surgery. SUBJECTS: One hundred and seven patients participating in a prospective pragmatic randomized controlled trial investigating physiotherapy rehabilitation following total knee arthroplasty, plus three outcome assessors. INTERVENTIONS: A protocol was developed using available research and designed to minimize instances of unblinding during a physiotherapy rehabilitation trial. Administrative, office, patient and research staff procedures were included. MAIN MEASURES: Trial questionnaires measured blind outcome assessment responses at 3 and 12 months post surgery. The outcome assessor kept a field diary recording the events surrounding instances of unblinding. Data underwent descriptive and content analysis. RESULTS: Blind outcome assessment was believed successful for n = 74 (81.32%) assessments at 3-month follow-up, and n = 83 (91.21%) at 12 months. Forty instances (n = 28 participants) of unblinding were described in the field diary. While the main cause of unblinding was participants telling the outcome assessor, in 12.5% of events the assessor drew the wrong conclusion regarding group allocation. Not all unblinding events were remembered at subsequent assessments, even in this relatively small trial. CONCLUSIONS: Blind outcome assessment was considered achievable in this trial. Specific trial protocols enabled blinding beliefs to be reported and instances of unblinding to be described.

Measuring step count: why it is important not to assume measures are reliable.

BACKGROUND: The validity and reliability of pedometer software Apps versus the previously investigated spring lever and piezoelectric pedometers is unknown. PURPOSE: To evaluate the validity and reliability (in adults aged 18-65) of two pedometer software Apps, the Walk Star and the Accupedo, with spring lever (Digi-Walker CW 700) and piezoelectric (Omron HJ-720ITC and Tanita PD-724) pedometers in the measurement of step count. The criterion for comparison was researcher tallied direct observation of step count using an electronic E3-EHT hand held tally counter. METHODS: Eighteen participants walked for 5minutes on a treadmill at slow (2miles per hour) moderate (3miles per hour) and fast walking (4miles per hour) speeds and on urban streets and upon grass at a perceived "comfortable" walking speed. RESULTS: Bland and Altman plots show wide limits of agreement observed for the Yamax CW 700, Accupedo App and Walk Star App, suggesting these pedometers are unsuitable for measuring step counts in individuals due to high random error (indicating low reliability). Narrow limits of agreement were observed for the Omron HJ-720ITC and the Tanita PD-724 pedometers compared against Tally count and were considered suitable for use. CONCLUSION: The validity and reliability of pedometers cannot be assumed but must be tested and ensured before use in measuring step count.

Development of a functional rehabilitation intervention for post knee arthroplasty patients: COmmunity based Rehabilitation post Knee Arthroplasty (CORKA) trial.

Knee osteoarthritis is a common cause of disability in older people and knee arthroplasty surgery in the UK is increasing. The CORKA trial is a randomised controlled trial of rehabilitation targeted at patients identified as being at risk of a poor outcome after knee arthroplasty. This paper describes the development and delivery of the CORKA intervention. It was informed by current evidence, relevant guidelines, expert and patient opinion, practical considerations and a pilot study. The intervention is a multicomponent rehabilitation programme with the main component being an exercise programme delivered to participants in their own home. It includes functional task practice, strategies to improve adherence and where appropriate the provision of appropriate aids and equipment.

Reliability and validity of the Four Square Step Test in patients with hip osteoarthritis before and after total hip replacement.

OBJECTIVE: To determine the validity and inter- and intra-rater reliability of the Four Square Step Test (FSST) in assessing gait performance, balance and physical function for patients with hip osteoarthritis before and after total hip replacement (THR). DESIGN: Observational, repeated measures. SETTING: A specialist orthopaedic hospital. PARTICIPANTS: Fifty-eight participants with moderate to severe hip osteoarthritis scheduled to receive primary hip replacement within 4 months from recruitment. MAIN OUTCOME MEASURE: Time to complete the FSST, time and steps to complete the Figure of 8 Walk Test (F8W) and Berg Balance Scale score (BBS). RESULTS: The Bland and Altman limits of agreement for intra-rater measurements of the FSST were -3.2s to 3.5seconds before THR and -1.5 to 2.0seconds after THR. Limits of agreement for two different raters were -2.2 to 3.4seconds, all with small mean differences indicating little bias between raters or replications. Concurrent validity was assessed, and the FSST correlated highly with the F8W (r=0.7, P<0.001) and moderately with the BBS (r=0.6, P<0.001). Only one participant was rated as being at moderate risk of falls on the BBS, with the other participants scoring low; only one participant failed to complete the F8W. This is in contrast to the FSST, which 21 people failed to complete pre-operatively. CONCLUSIONS: The FSST is a valid and reliable measure of multi-directional stepping speed and balance, giving a more informative measure of gait performance than the F8W and BBS, and is feasible for use in a clinical population of patients both before and after THR.

Does arthroplasty type influence knee joint proprioception? A longitudinal prospective study comparing total and unicompartmental arthroplasty.

Proprioception was assessed after knee replacement to compare total (TKA) to unicompartmental (UKA) knee arthroplasty. Thirty-four patients were recruited; seventeen patients underwent TKA and seventeen patients underwent UKA. The patient's age was similar in both groups. Two measures of proprioception, joint position sense (JPS) and postural sway (PS) were measured. Function was assessed using the Oxford Knee Score (OKS). Measurements were taken pre-operatively and 6 months post-operatively on both the operated and contralateral leg. Pre-operatively, no differences in JPS or PS were found between groups or between limbs in either group. Post-operatively, both groups had significant improvement of JPS in the operated limb only (20% increase). The improvement in JPS was similar in both groups. PS also improved significantly in both groups although the improvement of PS in the UKA group was twice that for the TKA group. The OKS improved by a similar amount in both groups. Both UKA and TKA result in a significant improvement in proprioception. Dynamic aspects of proprioception improve more after UKA than TKA, which may explain, in part, why UKA patients have superior functional outcome to that of TKA patients.

A systematic review of lateral wedge orthotics--how useful are they in the management of medial compartment osteoarthritis?

Studies on the use of lateral wedge orthotics in the conservative management of medial compartment osteoarthritis are widely quoted. This approach, however, does not consider the disruption of the interaction between lower limb and foot and ankle function that lateral wedges would produce. This comprehensive, systematic review was therefore undertaken to evaluate all available literature to determine whether evidence exists to support their use. MEDLINE, EMBASE, CINAHL, Allied and Complimentary Medicine, PubMed, EBSCO HOST and PEDro, Abstracts of Reviews of Effects in the National Electronic Library for Health for Cochrane Reviews and manual searching were used to identify studies. was searched for trials in progress. Data extraction was performed by the three authors using a paper data extraction form which was based on the CONSORT statement and Critical Skills Appraisal Programme (CASP) guidelines. Overall, the results of this review suggest that, based on current evidence there are no major or long-term beneficial effects with the use of lateral wedges.

A qualitative systematic review of patients' experience of osteoporosis using meta-ethnography.

We aimed to systematically review qualitative studies exploring the experience of living with osteoporosis to develop new conceptual understanding. We identified themes about the invisibility/visibility of osteoporosis, the experience of uncertainty of living with osteoporosis (OP) and living with an ageing body and the place of gender. PURPOSE: The aim of this review was to systematically review the body of qualitative studies exploring the experience of living with either osteoporosis or osteopenia and to use meta-ethnography to develop new conceptual understanding. METHODS: We systematically reviewed and integrated the findings of qualitative research from four bibliographic databases (Medline, Embase, Cinahl, Psychinfo) to September 2015 in order to increase our conceptual understanding of the lived experience of osteoporosis and osteopenia. Articles were appraised for quality; each was independently read by two researchers to identify concepts which were compared and developed into a conceptual model. RESULTS: Our findings demonstrate that coming to terms with a diagnosis of osteoporosis is linked to its relative visibility or invisibility. For some, OP has not become manifest and self-identity is intact (biographical integrity). For others, OP is profoundly manifest and self-identity is no long intact (biographical fracture). We also demonstrate that overwhelming uncertainty pervades the experience of OP. Our final theme demonstrates how the experience of OP is set within a cultural context with certain views about ageing and gender. CONCLUSIONS: Our synthesis has highlighted the wealth of qualitative data about osteoporosis and osteopenia. Despite the increasing body of literature on the subject, there remains a need to adjust our interactions with patients. This will allow clinicians to understand how patients can be helped to receive and understand their diagnosis and move forward in partnership with healthcare providers to promote optimal management of the disease.

Effect of estradiol and progesterone on long chain fatty acyl-coenzyme A levels in the rat uterus.

Fatty acyl-CoAs are potential in vivo inactivators of glucose-6-phosphate dehydrogenase (G6PD). Ovariectomized mature rats (n = 74) were given 5 micrograms of estradiol intravenously, then killed 0, 24, 36, 48 and 72 h later. Control levels of myristoyl-, palmitoyl-, stearoyl-, arachidonoyl-, oleoyl- and linoleoyl-CoA were 0.6, 3.2, 4.7, 3.4, 2.4 and 3.0 micrograms/uterus and were increased 39, 110, 146, 100, 84 and 69% at 36-48 h, respectively. Levels of fatty acyl-CoAs in the rat uterus become elevated 36 h after estradiol treatment. At the same time G6PD changes from a stable enzyme to one that is irreversibly inactivated, possibly due to being rapidly degraded. Progesterone (2 mg subcutaneously every 12 h, n = 30), administered beginning at either 24 or 36 h after estradiol treatment, had no effect on estradiol-induced changes in myristoyl-, palmitoyl-, or stearoyl-CoA. Compared to the groups of rats treated with estradiol alone, animals treated with combinations of estradiol and progesterone exhibited higher levels of arachidonoyl-CoA after 48 h, and oleoyl-CoA and linoleoyl-CoA were greater after 72 h. Progesterone increased the estradiol-induced levels of unsaturated fatty acyl-CoAs suggesting that progesterone may induce uterine fatty acid desaturase activity and/or uptake of dietary fatty acids. Addition of fatty acyl-CoAs, at concentrations seen in vivo at 36-48 h after estradiol, to purified G6PD, causes irreversible G6PD inactivation.

Isolation of a precursor and a nascent chain form of glucose-6-phosphate dehydrogenase from rat uterus and regulation of precursor processing by estradiol.

SDS-polyacrylamide gel electrophoresis of anti-glucose-6-phosphate dehydrogenase immunoprecipitates from radiolabeled uterine tissue extracts previously revealed three proteins: A, B and C, which were tentatively identified as a 60-64 kDa precursor form, a 57 kDa predominant form, and a 40-42 kDa nascent peptide form of the enzyme, respectively. A peptide-mapping technique was used to examine structural homologies among A, B and C. Following the labeling of uterine proteins with [35S]methionine, labeled proteins A, B and C were isolated by immunoprecipitation and electrophoresis. Each protein was individually co-digested with authentic, [3H]methionine-labeled glucose-6-phosphate dehydrogenase using papain, the resulting peptides were resolved by isoelectric focusing and the peptides from the two sources on each gel were compared using double-label counting methods. Proteins A, B and C had at least eight peptides in common, both proteins A and C had two additional peptides in common that were not present in protein B, and B protein had two peptides that were either absent or present in reduced amounts in digests of proteins A and C. The extensive structural homology and immunoreactivity of these proteins indicated that proteins A, B and C were all related to glucose-6-phosphate dehydrogenase. The presence of two extra peptides in proteins A and C suggested that these peptides may be derived from a common NH2-terminal leader sequence which was present in both the precursor and nascent peptide chains. The presence of two peptides that were present in protein B and absent from proteins A and C is easiest to explain if they are derived from the two ends of the molecule, with the corresponding peptides in proteins A and C containing additional peptide sequences that are 'normally' removed by endogenous proteolytic processing enzymes. Based on the relative time-course of synthesis of the three glucose-6-phosphate dehydrogenase-related proteins in control and estrogen-treated uteri, it appears that estradiol promotes an increase in the relative rate of transfer of label from protein A into B by stimulating the rate of processing of the precursor to the predominant form of the enzyme and enhances the rate of translational conversion of protein C into higher molecular weight forms.

Effect of sodium fluoride on glucose-6-phosphate dehydrogenase activity in the rat uterus.

Intrauterine administration of 50 mumol of NaF to the ovariectomized mature rat causes a 2--3-fold increase in the total uterine glucose-6-phosphate dehydrogenase activity within 24 h. The response is characterized by a 4--6 h lag with a maximum effect from 24 to 36 h after a single treatment. Uterine glucose-6-phosphate dehydrogenase activity continues to increase with daily administration of NaF through 4 days. The NaF-induced response is blocked by prior intrauterine administration of cycloheximide but not actinomycin D suggesting that the enzyme activity increases by a post-transcriptional effect of NaF on de novo enzyme synthesis. Direct measurement of the effect of NaF on the rate of incorporation of [14C] leucine into immunoprecipitable uterine glucose-6-phosphate dehydrogenase indicates that NaF causes a 9-fold increase in the rate of enzyme synthesis during the interval from 12 to 16 h after treatment. The half-life of the enzyme as measured by the rate of loss of [1-14C] glutamate from previously labeled utreine glucose-6-phosphate dehydrogenase is decreased from 27 to 10 h by NaF. The NaF response does not seem to be mediated by activation of uterine adenylyl cyclase since theophylline does not potentiate the response and since intrauterine application of cyclic AMP does not mimic the response. The increase in enzyme activity is preceded by an increase in the rate of utilization of the hexose monophosphate shunt pathway as determined by the ratio of the the rates of oxidation of [1-14C]glucose to [6-14C] glucose to CO2 by uterine slices in vitro. The action of NaF on this pathway most likely resutls from inhibition of the glycolytic enzyme, enolase, and increased pathway utilization may be the factor which controls enzyme synthesis. When given in combination with other known inducers of uterine glucose-6-phosphate dehydrogenase such as estradiol and NADP+, NaF acts synergistically.

Isolation and characterization of an estrogen-regulated ribosome-associated inactivator of tRNA aminoacylation in the uterus.

Estradiol (E2) induces an increase in the peptide elongation rate of isolated uterine ribosomes assayed in a cell-free protein synthesis system. An inhibitory factor, extracted from ribosomes of E2-deprived rats, was found to inhibit the peptide elongation reaction by acting on certain tRNAs to render them incapable of binding to aminoacyl-tRNA synthetases, thus reducing the availability of specific aminoacylated tRNAs required for the sequential translation of the codons in mRNA. The uterine ribosome-associated tRNA inactivator (RATI) has been partially purified and monoclonal antibodies (MABs) to RATI have been prepared. Specificity of the MABs for RATI was indicated by the inactivation of RATI in vitro by the anti-RATI MABs. RATI selectively inactivates deacylated, but not acylated, tRNAs and the inactivation does not appear to involve nuclease cleavage of the tRNA. Within 1 h after E2 treatment 50% of both RATI activity and immunoreactivity were lost from the uterine ribosome extracts, suggesting that E2 regulation of tRNA reutilization may occur through dissociation of RATI from the ribosomal site of tRNA deacylation or alteration in the structure of RATI resulting in inactivation both biologically and immunologically. We propose that RATI may function as an E2-regulatable 'switch' mechanism which inactivates, delays or defers the aminoacylation of certain tRNAs in the absence of E2 and which participates in the regulation of protein synthesis at the translational level by creating rate-limiting levels of certain tRNAs in the E2-deprived uterus.

Estradiol regulation of the synthesis of uterine proteins with clusters of proline- and glycine-rich peptide sequences.

Estradiol (E2) regulates the synthesis of uterine proteins at both the transcriptional and translational levels. E2 induces an increase in the specific amino acid acceptor activity of uterine tRNA, with the largest increases seen for proline, glycine and methionine. The synthesis of three uterine proteins that are rich in proline and glycine, estrogen receptor, progesterone receptor and glucose-6-phosphate dehydrogenase, is induced by E2. E2-induced increases in these proteins were preceded by an correlated with stimulation of tRNA acceptor activity for proline and glycine and these responses were specifically and simultaneously inhibited by prior azaserine treatment, which inhibits the E2-induced repair and synthesis of the 3'-CCA acceptor terminus of tRNAs. The high frequency and clustering of proline and glycine residues in estrogen receptor, progesterone receptor and glucose-6-phosphate dehydrogenase suggests that the translating ribosomes may slow down during synthesis of these proteins due to limiting levels of these tRNAs in E2-deprived uteri.

Glucose-6-phosphate dehydrogenase: partial characterization of the rat liver and uterine enzymes.

Some properties of rat liver and uterine glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) have been determined. A procedure has been used for the purification of rat liver glucose-6-phosphate dehydrogenase to homogeneity (spec. act. 210-225 units/mg protein) from large amounts of liver (0.5-2 kg) with yields of up to 30%. Uterine glucose-6-phosphate dehydrogenase was obtained by immunoprecipitation methods and the properties of radioactively-labeled forms of this enzyme were then determined. The amino acid composition of the liver enzyme was found to be similar to that for the enzyme from other mammalian tissues. The liver and uterine enzymes have a subunit molecular weight of 57000 and a pI of 6.5. The NH2-terminal amino acid of both enzymes was found to be pyroglutamate.

Efficacy of an accelerated recovery protocol for Oxford unicompartmental knee arthroplasty--a randomised controlled trial.

Unicompartmental knee arthroplasty (UKA) is appropriate for one in four patients with osteoarthritic knees. This study was performed to compare the safety, effectiveness and economic viability of a new accelerated protocol with current standard care in a state healthcare system. A single blind RCT design was used. Eligible patients were screened for NSAID tolerance, social circumstances and geographical location before allocation to an accelerated recovery group (A) or standard care group (S). Primary outcome was the Oxford Knee Assessment at 6 months post operation, compared using independent Mann-Whitney U-tests. A simple difference in costs incurred was calculated. The study power was sufficient to avoid type 2 errors. Forty-one patients were included. The average stay for Group A was 1.5 days. Group S averaged 4.3 days. No significant difference in outcomes was found between groups. The new protocol achieved cost savings of 27% and significantly reduced hospital bed occupancy. In addition, patient satisfaction was assessed as greater with the accelerated discharge than with the routine discharge time. The strict inclusion criteria meant that 75% of eligible patients were excluded. However, a large percentage of these were due to the distances patients lived from the hospital.

Regulation of glucose-6-phosphate dehydrogenase activity in uterine tissue in organ culture.

An organ culture technique, employing a totally defined medium, was developed, in which the levels of glucose-6-P dehydrogenase (G6PD) activity in uterine tissue from estradiol-treated ovariectomized mature rats continued to increase in vitro for 18 h at a rate similar to that seen in vivo. Uterine G6PD levels did not increase in vitro in tissues from estrogen-deprived rats even if estradiol (10(-6)m) was added in vitro, but the administration of estradiol (5 mug/rat) in vivo for as little as 2 min permitted G6PD levels to increase by 0.03 units/uterus (from 0.075 to 0.10, units/uterus) after the 18 h incubation. The maximum increase of 0.08 units/uterus (from 0.147 to 0.230 units/uterus) was seen in uteri from rats which were given estradiol 12 h prior to sacrigice. Tissues from animals given estradiol for greater than 30 h exhibit a net decrease in G6PD levels under the in vitro conditions. The in vitro increase in G6PD activity is inhibited by the addition of either actinomycin D (5 mug/ml), cycloheximide (5 mug/ml), or cordycepin (150 mug/ml), or by the intrauterine injection of actinomycin D (10 mug/rat). Intrauterine administration of cycloheximide (100 mug/rat) inhibited the in vivo increase in enzyme activity; however, the enzyme levels increased after placement of uterine tissues from these animals into organ culture. The removal of cycloheximide, which was added at the beginning of incubation after the 12th h, restores the ability of the tissues to increase the G3PD activity, and this restoration is not blocked by the addition of actinomycin D, suggesting that the mRNA activity for uterine G6PD accumulated during in vitro inhibition of protein synthesis by cycloheximide. The in vitro increase in uterine G6PD is due to an increase in immunologically identifiable G6PD protein and this increase is due, at least in part, to an increase in the de novo synthesis of the enzyme, as measured by the incorporation of [14C]leucine into immunochemically isolated G6PD protein. These results suggest that once initiated in vivo by estradiol, the uterus is capable of continuing in vitro those events, including the synthesis of both RNA and protein, which result in an increased rate of synthesis of uterine G6PD.

Ribosome-associated estradiol-binding components in the uterus and their relationship to the translational capacity of uterine ribosomes.

Estradiol transiently increases the rate of peptide elongation on uterine ribosomes from ovariectomized mature rats during the first 2 h after hormone injection, suggesting the existence of direct or indirect estradiol receptor interaction with ribosomes. Characterization of estradiol-binding components on isolated uterine ribosomes, microsomes, and cytosol under identical assay conditions indicated that microsomes and cytosol contain estradiol-binding components with similar affinities for estradiol (Kd = 0.5 nM) and sucrose gradient sedimentation characteristics (3.8S and 5.2S for preparations incubated at 0 and 30 C for 1 h, respectively). Those on ribosomes exhibited a higher affinity for estradiol (Kd = 0.14 nM) and had heterogeneous and more dense sedimentation characteristics (5.5-6.0S). The ribosome-associated estradiol binder was clearly different from transformed cytosol and nuclear estradiol receptors based on sedimentation characteristics under identical conditions. Like cytosol and nuclear receptors, microsomal and ribosomal estradiol binding underwent exchange reactions in vitro at 30 C, but not at 0 C. All in vitro bound, but not all in vivo bound, [3H] estradiol could be exchanged from microsomes or ribosomes by estradiol. [3H]Estradiol could be exchanged from ribosomes by a variety of estrogens, but not by progestins, glucocorticoids, or androgens. The amount of estradiol-binding activity on ribosomes decreased after estradiol administration in vivo and was inversely correlated with the rate of peptide elongation by the ribosomes in a cell-free protein synthesis system. These results suggest that accumulation of an estradiol-binding protein, perhaps a nascent estradiol receptor, on ribosomes in the absence of in vivo estradiol may directly or indirectly inhibit the peptide elongation reaction.