Development of osteoarthritis in patients with degenerative meniscal tears treated with exercise therapy or surgery: a randomized controlled trial.

OBJECTIVE: To evaluate progression of individual radiographic features 5 years following exercise therapy or arthroscopic partial meniscectomy as treatment for degenerative meniscal tear. DESIGN: Randomized controlled trial including 140 adults, aged 35-60 years, with a magnetic resonance image verified degenerative meniscal tear, and 96% without definite radiographic knee osteoarthritis. Participants were randomized to either 12-weeks of supervised exercise therapy or arthroscopic partial meniscectomy. The primary outcome was between-group difference in progression of tibiofemoral joint space narrowing and marginal osteophytes at 5 years, assessed semi-quantitatively by the OARSI atlas. Secondary outcomes included incidence of radiographic knee osteoarthritis and symptomatic knee osteoarthritis, medial tibiofemoral fixed joint space width (quantitatively assessed), and patient-reported outcome measures. Statistical analyses were performed using a full analysis set. Per protocol and as treated analysis were also performed. RESULTS: The risk ratios (95% CI) for progression of semi-quantitatively assessed joint space narrowing and medial and lateral osteophytes for the surgery group were 0.89 (0.55-1.44), 1.15 (0.79-1.68) and 0.77 (0.42-1.42), respectively, compared to the exercise therapy group. In secondary outcomes (full-set analysis) no statistically significant between-group differences were found. CONCLUSION: The study was inconclusive with respect to potential differences in progression of individual radiographic features after surgical and non-surgical treatment for degenerative meniscal tear. Further, we found no strong evidence in support of differences in development of incident radiographic knee osteoarthritis or patient-reported outcomes between exercise therapy and arthroscopic partial meniscectomy. TRIAL REGISTRATION: www.clinicaltrials.gov (NCT01002794).

Anterior knee pain following anterior cruciate ligament reconstruction does not increase the risk of patellofemoral osteoarthritis at 15- and 20-year follow-ups.

OBJECTIVE: To prospectively evaluate the relationship between the presence or persistence of anterior knee pain (AKP) during the first 2-years following anterior cruciate ligament reconstruction (ACLR) and patellofemoral osteoarthritis (PFOA) at 15- and 20-years. DESIGN: This study was ancillary to a long-term prospective cohort study of 221 participants following bone-patellar-tendon-bone ACLR. AKP was assessed at 1- and 2-years post-ACLR using part of the Cincinnati knee score with an additional pain location question (persistence defined as presence at both follow-ups). Radiographic PFOA (definite patellofemoral osteophyte) and symptomatic PFOA (patellofemoral osteophyte, with knee pain during past 4 weeks) was assessed at 15- and 20-years follow-up. We used generalized linear models with Poisson regression to assess the relationship between AKP and PFOA. RESULTS: Of the 181 participants (82%) who were assessed at 15-years post-ACLR (age 39 ± 9 years; 42% female), 36 (24%) and 33 (22%) had AKP at 1- and 2-years, respectively, while 14 (8%) reported persistent AKP. Radiographic and symptomatic PFOA was observed at 15-years in 130 (72%) and 70 (39%) participants, respectively, and at 20-years in 115 (80%) and 60 (42%) participants, respectively. Neither the presence nor persistence of AKP at 1- and/or 2-years post-ACLR was associated with significantly higher risk of radiographic or symptomatic PFOA at 15- or 20-years (risk ratios <2.1). CONCLUSIONS: Although AKP and PFOA were prevalent, AKP does not appear to be associated with long-term PFOA following ACLR.

Evaluation of a home treatment program for cold hypersensitivity using a classical conditioning procedure in patients with hand and arm injuries.

STUDY DESIGN: Case series. INTRODUCTION: A home treatment program using a classical conditioning procedure to decrease cold hypersensitivity has potential to reduce symptoms. PURPOSE: To evaluate a home treatment program for cold hypersensitivity using a classical conditioning procedure in patients who are cold hypersensitive after hand and arm injuries. METHODS: A series of 22 patients followed a classical conditioning procedure consisting of exposing the body to cold outdoor temperatures and immersing the hands in warm water, every other day, for five weeks. The McCabe Cold Sensitivity Severity scale (CSS) was used to measure cold hypersensitivity twice before treatment, at four weeks, and at one year after treatment; Likert scales was used for the patients ratings of improvements. A cold stress test was performed to evaluate rewarming capacity in injured fingers. RESULTS: From the 20 patients, who returned questionnaires at all assessment points, 9 reported a small and three reported a moderate improvement in cold hypersensitivity after treatment. There was a trend toward improvement in the CSS (median 36; interquartile range--19 to 60) and in the rewarming pattern of fingers that were initially slow to rewarm. The improvements were sustained or increased at one-year follow-up. CONCLUSION: These preliminary results suggest that the classical conditioning procedure to treat cold hypersensitivity has potential and should be further explored in a trial with more rigorous design.

Peripheral blood gene expression signature differentiates children with autism from unaffected siblings.

Autism spectrum disorder (ASD) is one of the most prevalent neurodevelopmental disorders with high heritability, yet a majority of genetic contribution to pathophysiology is not known. Siblings of individuals with ASD are at increased risk for ASD and autistic traits, but the genetic contribution for simplex families is estimated to be less when compared to multiplex families. To explore the genomic (dis-) similarity between proband and unaffected sibling in simplex families, we used genome-wide gene expression profiles of blood from 20 proband-unaffected sibling pairs and 18 unrelated control individuals. The global gene expression profiles of unaffected siblings were more similar to those from probands as they shared genetic and environmental background. A total of 189 genes were significantly differentially expressed between proband-sib pairs (nominal p < 0.01) after controlling for age, sex, and family effects. Probands and siblings were distinguished into two groups by cluster analysis with these genes. Overall, unaffected siblings were equally distant from the centroid of probands and from that of unrelated controls with the differentially expressed genes. Interestingly, five of 20 siblings had gene expression profiles that were more similar to unrelated controls than to their matched probands. In summary, we found a set of genes that distinguished probands from the unaffected siblings, and a subgroup of unaffected siblings who were more similar to probands. The pathways that characterized probands compared to siblings using peripheral blood gene expression profiles were the up-regulation of ribosomal, spliceosomal, and mitochondrial pathways, and the down-regulation of neuroreceptor-ligand, immune response and calcium signaling pathways. Further integrative study with structural genetic variations such as de novo mutations, rare variants, and copy number variations would clarify whether these transcriptomic changes are structural or environmental in origin.

High-intensity interval training improves peak oxygen uptake and muscular exercise capacity in heart transplant recipients.

Heart transplant (HTx) recipients usually have reduced exercise capacity with reported VO(2peak) levels of 50-70% predicted value. Our hypothesis was that high-intensity interval training (HIIT) is an applicable and safe form of exercise in HTx recipients and that it would markedly improve VO(2peak.) Secondarily, we wanted to evaluate central and peripheral mechanisms behind a potential VO(2peak) increase. Forty-eight clinically stable HTx recipients >18 years old and 1-8 years after HTx underwent maximal exercise testing on a treadmill and were randomized to either exercise group (a 1-year HIIT-program) or control group (usual care). The mean ± SD age was 51 ± 16 years, 71% were male and time from HTx was 4.1 ± 2.2 years. The mean VO(2peak) difference between groups at follow-up was 3.6 [2.0, 5.2] mL/kg/min (p < 0.001). The exercise group had 89.0 ± 17.5% of predicted VO(2peak) versus 82.5 ± 20.0 in the control group (p < 0.001). There were no changes in cardiac function measured by echocardiography. We have demonstrated that a long-term, partly supervised and community-based HIIT-program is an applicable, effective and safe way to improve VO(2peak) , muscular exercise capacity and general health in HTx recipients. The results indicate that HIIT should be more frequently used among stable HTx recipients in the future.

The association between radiographic knee osteoarthritis and knee symptoms, function and quality of life 10-15 years after anterior cruciate ligament reconstruction.

BACKGROUND: There are conflicting results in the literature regarding the association between radiographic knee osteoarthritis (OA) and symptoms and function in subjects with previous anterior cruciate ligament (ACL) reconstruction. AIM: To investigate the associations between radiographic tibiofemoral knee OA and knee pain, symptoms, function and knee-related quality of life (QOL) 10-15 years after ACL reconstruction. STUDY DESIGN: Cross-sectional study. MATERIAL AND METHODS: 258 subjects were consecutively included at the time of ACL reconstruction and followed up prospectively. The authors included the Knee Injury and Osteoarthritis Outcome Score to evaluate knee pain, other symptoms (symptoms), activities of daily living and sport and recreation (Sport/Rec) and QOL. The subjects underwent standing radiographs 10-15 years after the ACL reconstruction. The radiographs were graded with the Kellgren and Lawrence (K&L) classification (grade 0-4). RESULTS: 210 subjects (81%) consented to participate in the 10-15-year follow-up. Radiographic knee OA (K&L ≥ grade 2) was detected in 71%, and 24% showed moderate or severe radiographic knee OA (K&L grades 3 and 4). No significant associations were detected between radiographic knee OA (K&L grade ≥ 2) and pain, function or QOL, respectively, but subjects with radiographic knee OA showed significantly increased symptoms. Severe radiographic knee OA (K&L grade 4) was significantly associated with more pain, symptoms, impaired Sport/Rec and reduced QOL. CONCLUSION: Subjects with radiographic knee OA showed significantly more symptoms than those without OA, and subjects with severe radiographic knee OA had significantly more pain, impaired function and reduced quality of life than those without radiographic knee OA 10-15 years after ACL reconstruction.

Preoperative quadriceps strength is a significant predictor of knee function two years after anterior cruciate ligament reconstruction.

OBJECTIVE: To identify preoperative predictive factors for knee function two years after reconstructive surgery of the anterior cruciate ligament (ACL). The main hypothesis was that preoperative quadriceps strength would be the most significant predictor for knee function two years after reconstructive surgery. DESIGN: Cohort study. SETTING: ACL injured individuals treated at a University Hospital and an outpatient clinic in Oslo, Norway. PARTICIPANTS: Seventy-three individuals with complete unilateral rupture of the ACL scheduled for reconstruction with a bone-patellar-bone autograft were included in the study, from where 60 were available for two-year follow up and included in the final analyses. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASUREMENTS: Identification of baseline independent variables that may predict knee function assessed with the Cincinnati Knee Score as dependent variable two years after ACL reconstruction. RESULTS: Quadriceps muscle strength, meniscus injury and the Short-Form-36 Bodily Pain sub score were identified as significant predictors for knee function assessed from the Cincinnati Knee Score two years after ACL reconstruction. Individuals with preoperative quadriceps strength deficits above 20% also had persistent significantly larger strength deficits two years after surgery. CONCLUSIONS: Preoperative quadriceps muscle strength deficits and meniscus injuries have significant negative consequences for the long-term functional outcome after ACL reconstruction. From our findings we suggest that ACL reconstruction should not be performed before quadriceps muscle strength deficits of the injured limb is less than 20% of the uninjured limb.

Rehabilitation after anterior cruciate ligament injury influences joint loading during walking but not hopping.

OBJECTIVE: The purpose of this study was to identify changes in clinical outcome and lower extremity biomechanics during walking and hopping in ACL-injured subjects before and after a 20-session neuromuscular and strength training programme. STUDY DESIGN: Pre and post experimental design. SETTING: Outpatient clinic, primary care. PATIENTS: 32 subjects with unilateral ACL injury, mean 60 (SD 35) days after injury, with a mean age of 26.2 (5.4) years. INTERVENTION: The rehabilitation programme consisted of neuromuscular and strength exercises. MAIN OUTCOME MEASUREMENTS: Outcome measurements assessed before and after a 20-session rehabilitation programme were: self-assessment questionnaires (KOS-ADL, IKDC2000, Global function), four single-leg hop tests, and isokinetic muscle strength tests. Lower extremity kinematics and kinetics were captured during the stance phase of gait and landing after a single leg hop, synchronised with three force plates. RESULTS: These ACL-injured individuals significantly improved their clinical outcome after rehabilitation. Gait analysis disclosed a significantly improved knee extension moment after rehabilitation, but no change in hip or knee excursions. During landing after hop no change in knee excursion or knee moment was recorded. CONCLUSION: After rehabilitation the ACL-injured subjects showed a significantly improved clinical outcome, but lower extremity biomechanics were still significantly impaired during both walking and hopping. The rehabilitation programme influenced knee joint loading during walking, but not during hopping. Longer rehabilitation should be considered before ACL-injured individuals return to jumping activities.

Comparison of gait with and without ankle-foot orthoses after lower limb surgery in children with unilateral cerebral palsy.

PURPOSE: Children with spastic unilateral cerebral palsy (SUCP) frequently undergo lower limb surgery to improve gait. Postoperatively, ankle-foot orthoses (AFOs) are used to maintain the surgical corrections and provide adequate mechanical support. Our aim was to evaluate changes in gait and impacts of AFOs one-year postoperatively. METHODS: In all, 33 children with SUCP, 17 girls and 16 boys, mean age 9.2 years (5 to 16.5) were measured by 3D gait analysis walking barefoot preoperatively and walking barefoot and with AFOs one-year postoperatively. Changes in Gait Profile Scores (GPS), kinematic, kinetic and temporal spatial variables were examined using linear mixed models, with gender, gross motor function and AFO type as fixed effects. RESULTS: The results confirm significant gait improvements in the GPS, kinematics and kinetics walking barefoot one year after surgery. Comparing AFOs with barefoot walking postoperatively, there was additionally reduced ankle plantarflexion by an average of 5.1° and knee flexion by 4.7° at initial contact, enhanced ankle moments during loading response, increased velocity, longer steps and inhibited push-off power generation. Stance and swing phase dorsiflexion increased in children walking with hinged AFOs versus children walking with ground reaction AFOs. Changes in the non-affected limbs indicated less compensatory gait postoperatively. CONCLUSION: Major changes were found between pre- and postoperative barefoot conditions. The main impact of AFOs was correction of residual drop foot and improved prepositioning for initial contact, which could be considered as indications for continued use after the one-year follow-up. LEVEL OF EVIDENCE: Level II - Therapeutic.

The role of delayed bone age in the evaluation of stature and bone health in glucocorticoid treated patients with Duchenne muscular dystrophy.

BACKGROUND: Low bone mineral density and an increased risk of appendicular and vertebral fractures are well-established consequences of Duchenne muscular dystrophy (DMD) and the risk of fractures is exacerbated by long-term glucocorticoid treatment. Monitoring of endocrine and skeletal health and timely intervention in at-risk patients is important in the management of children with DMD. METHODS: As part of the Norwegian Duchenne muscular dystrophy cohort study, we examined the skeletal maturation of 62 boys less than 18 years old, both currently glucocorticoid treated (n = 44), previously treated (n = 6) and naïve (n = 12). The relationship between bone age, height and bone mineral density (BMD) Z-scores was explored. RESULTS: The participants in the glucocorticoid treated group were short in stature and puberty was delayed. Bone age was significantly delayed, and the delay increased with age and duration of treatment. The difference in height between glucocorticoid treated and naïve boys was no longer significant when height was corrected for delayed skeletal maturation. Mean BMD Z-scores fell below - 2 before 12 years of age in the glucocorticoid treated group, with scores significantly correlated with age, duration of treatment and pubertal development. When BMD Z-scores were corrected for by retarded bone age, the increase in BMD Z-scores was significant for all age groups. CONCLUSION: Our results suggest that skeletal maturation should be assessed in the evaluation of short stature and bone health in GC treated boys with DMD, as failing to consider delayed bone age leads to underestimation of BMD Z-scores and potentially overestimation of fracture risk.

Alzheimer disease-like clinical phenotype in a family with FTDP-17 caused by a MAPT R406W mutation.

We report clinical, molecular, neuroimaging and neuropathological features of a Danish family with autosomal dominant inherited dementia, a clinical phenotype resembling Alzheimer's disease and a pathogenic mutation (R406W) in the microtubule associated protein tau (MAPT) gene. Pre-symptomatic and affected family members underwent multidisciplinary (clinical, molecular, neuroimaging and neuropathological) examinations. Treatment with memantine in a family member with early symptoms, based on the clinical phenotype and the lack of specific treatment, appears to stabilize the disease course and increase the glucose metabolism in cortical and subcortical areas, as determined by serial [F(18)]FDG-PET scanning before and after initiation of treatment. Neuropathological examination of a second affected and mutation-positive family member showed moderate atrophy of the temporal lobes including the hippocampi. Microscopy revealed abundant numbers of tau-positive neurofibrillary tangles in all cortical areas and in some brainstem nuclei corresponding to a diagnosis of frontotemporal lobe degeneration on the basis of a MAPT mutation. The clinical and genetic heterogeneity of autosomal dominant inherited dementia must be taken into account in the genetic counselling and genetic testing of families with autosomal dominantly inherited dementia in general.

Cold hypersensitivity after hand injuries. A prospective 7-year follow-up.

We studied changes in cold hypersensitivity from 3 to 7 years following severe hand injuries. Data was collected using postal questionnaires 7 years after injury in 71 patients who had participated in a 3-year follow-up from the time of injury. There was no change in cold sensitivity measured using the McCabe Cold Sensitivity Severity scale (CSS) from 3 to 7 years after injury. However, there was a trend toward decreased severity measured using a five-level scale of self-reported cold hypersensitivity. Compared to the 3-year follow-up, fewer respondents rated their condition as severe and two patients had recovered from their cold hypersensitivity at the 7-year follow-up. Furthermore, 21 (30%) of the respondents stated a decrease in cold hypersensitivity during the last 2 years. Limitations in cold associated activities and the importance of being less limited in leisure activities (NRS 0-10) did not change between the two follow-ups. In conclusion, the CSS-scores did not change from 3 to 7 years after injury. Several patients experienced improvements in cold hypersensitivity, but few recovered completely from the condition.

Physical function and health-related quality of life in young adults with unilateral congenital lower-limb deficiencies.

PURPOSE: The primary aim was to examine if there were differences in physical function and health-related quality of life (HRQoL) between young adults (18 to 35 years) with unilateral congenital lower-limb deficiency (CLLD) who had been surgically lengthened (Surg) and those using lengthening prostheses (Pros). Second, we wanted to compare their health status with an age- and gender-matched reference group (Ref) without CLLD. METHODS: Cross-sectional study including a study-specific questionnaire, clinical examination, two field tests evaluating physical function (the six-minute walk test and the Stair test) and HRQoL questionnaires (Short Form (SF)-36 and EuroQol (EQ)-5D-3L). RESULTS: Physical function and HRQoL did not differ between the two treatment groups. The odds for having painful or disfiguring scars were 18 times higher in the Surg group (n = 16) compared with the Pros group (n = 14). The CLLD group showed significantly reduced physical function compared with the Ref group. HRQoL, measured by the EQ-5D-3L visual rating scale, was significantly reduced in the CLLD group compared with the Ref group, as was the SF-36 physical function domain in both genders. Men with CLLD also showed increased bodily pain and reduced general health (SF-36), while we found a reduction in the emotional role domain in women compared with Ref. CONCLUSION: There were no significant differences in physical function and quality of life in young adults with CLLD treated with surgical lengthening compared with those using lengthening prostheses. Compared with the general Norwegian population, young adults with CLLD had significantly lower physical function and reduced HRQoL in some domains.

Curative effect of DL-2-difluoromethylornithine on mice bearing mutant L1210 leukemia cells deficient in polyamine uptake.

The objective of the present investigation was to determine to what extent polyamine uptake from the host contributes to the ability of tumor cells in overcoming the antiproliferative effect of a polyamine synthesis inhibitor. A mutant L1210 leukemia cell line deficient in polyamine transport was isolated by selection for resistance to methylglyoxal bis(guanylhydrazone), an extremely cytotoxic agent which is taken up by the same transport system as the polyamines. C57BL/6 x DBA/2 F1 mice inoculated with mutant L1210 cells survived on the average 60 to 70% longer than mice inoculated with the parental cells. The therapeutic effect of a polyamine synthesis inhibitor, DL-2-difluoromethylornithine (3% in the drinking water), was much greater on mice bearing mutant L1210 cells (87% increase in median survival time; 13 of 40 mice cured) than on mice inoculated with parental cells (22% increase in median survival time). Similar results, although not as striking, were obtained using athymic nude mice, indicating that the therapeutic difference is not merely due to increased immunogenicity of the mutant cells.

Development of resistance to hydroxyurea during treatment of human myelogenous leukemia K562 cells with alpha-difluoromethylornithine as a result of coamplification of genes for ornithine decarboxylase and ribonucleotide reductase R2 subunit.

alpha-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), was used to select two very highly drug-resistant cell lines, designated K562-DFMOr and V79-DFMOr. Both DFMO-resistant cell lines exhibited elevated ODC expression due to gene amplification. Moreover, the K562-DFMOr cells, but not the V79-DFMOr cells, had an elevated level of ribonucleotide reductase subunit R2 (R2) mRNA and an increased R2 gene copy number. By analysis of their electron paramagnetic resonance spectra, an increased level of the R2 protein was observed in the K562-DFMOr cells as compared to the wild type K562 cells. This is the first description of a DFMO-induced mutant cell line exhibiting coamplification of the genes for ODC and R2, and overexpression of their products. There was no coamplification of the N-myc protooncogene, which is located close to the ODC and R2 genes on human chromosome 2. The alterations exhibited by the K562-DFMOr cell line were shown to be stable for many passages and to convey resistance not only to DFMO but also to hydroxyurea, an inhibitor of ribonucleotide reductase and thus DNA replication. In the absence of the selective pressure exerted by DFMO, the V79-DFMOr cell line produced revertants by loss of ODC gene amplification within three passages. Coamplification of linked genes may turn out to be an important mechanism in the development of cross-resistance and should be considered when designing therapeutic strategies.

Small-to-moderate decreases in cold hypersensitivity up to 3 years after severe hand injuries: A prospective cohort study.

BACKGROUND: The natural course and predictors for decreased cold hypersensitivity were studied in 85 patients with severe hand injuries involving nerve lesions. METHODS: Questionnaires including the McCabe Cold Sensitivity Severity scale (CSS 0-400) were collected after injury, and at 6-month, 12-month, 2-year, and 3-year follow-ups. RESULTS: Between the 12-month and 3-year follow-up, there was a small decrease in cold hypersensitivity as measured by the CSS (median = 24; Q1-Q3 = -11-75; n = 85). Five of the patients recovered from cold hypersensitivity, and ∼ 40% of the patients were less affected by cold hypersensitivity in daily life. Little or no pain early after injury and higher CSS-scores 12 months after primary surgery were weakly associated with the reduced CSS-scores (R(2) = 0.20) at the 3-year follow-up. Six patients had changed work or did not work due to cold hypersensitivity, but the majority of the patients had kept their cold-exposed work. CONCLUSION: Cold-hypersensitive patients may have a reasonable chance for decreased cold sensitivity and cold-associated activity limitations over time, although the majority of the patients will experience persistent problems. Tools to predict improvement remain insufficient.

Genetic variation among 82 pharmacogenes: The PGRNseq data from the eMERGE network.

Genetic variation can affect drug response in multiple ways, although it remains unclear how rare genetic variants affect drug response. The electronic Medical Records and Genomics (eMERGE) Network, collaborating with the Pharmacogenomics Research Network, began eMERGE-PGx, a targeted sequencing study to assess genetic variation in 82 pharmacogenes critical for implementation of "precision medicine." The February 2015 eMERGE-PGx data release includes sequence-derived data from ∼5,000 clinical subjects. We present the variant frequency spectrum categorized by variant type, ancestry, and predicted function. We found 95.12% of genes have variants with a scaled Combined Annotation-Dependent Depletion score above 20, and 96.19% of all samples had one or more Clinical Pharmacogenetics Implementation Consortium Level A actionable variants. These data highlight the distribution and scope of genetic variation in relevant pharmacogenes, identifying challenges associated with implementing clinical sequencing for drug treatment at a broader level, underscoring the importance for multifaceted research in the execution of precision medicine.

On the translational control of ornithine decarboxylase expression by polyamines.

Ornithine decarboxylase (ODC, EC 4.1.1.17) expression is subject to negative feedback regulation by the polyamines. The results of previous studies favor either translational or post-translational regulation. To facilitate further analysis of the mechanism by which polyamines affect ODC expression we have used a cell line (L1210-DFMOr) that overproduces ODC. This cell line was isolated by selection for resistance to the antiproliferative effect of the ODC inhibitor alpha-difluoromethylornithine (DFMO). These cells respond similarly to polyamine depletion and repletion as do their wild-type counterparts. When L1210-DFMOr cells were grown in the presence of 20 mM DFMO (i.e., when their polyamine content was reduced to an extent that still permitted a normal growth rate) ODC represented 4-5% of the soluble protein synthesized. After transfer of the cells to a medium lacking DFMO (i.e., when their polyamine pools were repleted), the rate of incorporation of [35S]methionine into ODC was one order of magnitude lower. Since this difference in incorporation of radioactivity into ODC remained the same irrespective of the pulse-label time used (between 2 and 20 min) it is likely to represent a true difference in ODC synthesis rate. Consequently, the pulse-label experiments cannot be explained by rapid degradation of the enzyme during the labeling period. The difference in ODC synthesis rate was not accompanied by a corresponding difference in the steady-state level of ODC mRNA. Analyses of the distribution of ODC mRNA in polysome profiles did not demonstrate any major difference between cells grown in the absence or presence of DFMO, even though the ODC synthesis rate differed by as much as 10-fold. However, the distribution of the ODC mRNA in the polysome profiles indicated that the message was poorly translated. Thus, most of the ODC mRNA was present in fractions containing ribosomal subunits or monosomes. Inhibition of elongation by cycloheximide treatment resulted in a shift of the ODC mRNA from the region of the gradient containing ribosomal subunits to that containing mono- and polysomes, indicating that most of the ODC mRNA was accessible to translation. Taken together these data lend support to a translational control mechanism which involves both initiation and elongation.

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spermine.

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [35S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescine, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.

Down-regulation of ornithine decarboxylase by an increased degradation of the enzyme during gastrulation of Xenopus laevis.

The present study was designed to analyze the regulation of the levels of the polyamines and their biosynthetic enzymes during embryonic development of Xenopus laevis. The activity of ornithine decarboxylase (ODC), a rate-controlling enzyme in polyamine biosynthesis, is elevated until, during gastrulation, there is a precipitous drop in activity. This is not attributable to a decrease in ODC mRNA content and polysome profiles reveal no apparent decrease in ODC message associated with polysomes. ODC synthesis seems to be maintained at a low, relatively constant rate until neurulation whereupon ribosome loading of ODC mRNA increases. During gastrulation the rate of ODC degradation increases dramatically, which can account for the decrease in ODC. S-Adenosylmethionine decarboxylase (AdoMetDC), another rate-controlling enzyme in polyamine biosynthesis, shows a low and constant activity from cleavage to neurulation. Subsequently, the AdoMetDC activity increases dramatically. The changes in AdoMetDC activity parallel the changes in AdoMetDC mRNA levels, suggesting a transcriptional control of AdoMetDC expression during this development period. The activities of ODC and AdoMetDC produce a steady increase in putrescine and spermidine content of the embryo. The spermine content also increases until gastrulation, but then decreases until the tailbud stage.