Intensity matters: effects of cadence and power output on corticospinal excitability during arm cycling are phase and muscle dependent.

The present study investigated the effects of cadence and power output on corticospinal excitability to the biceps (BB) and triceps brachii (TB) during arm cycling. Supraspinal and spinal excitability were assessed using transcranial magnetic stimulation (TMS) of the motor cortex and transmastoid electrical stimulation (TMES) of the corticospinal tract, respectively. Motor-evoked potentials (MEPs) elicited by TMS and cervicomedullary motor-evoked potentials (CMEPs) elicited by TMES were recorded at two positions during arm cycling corresponding to mid-elbow flexion and mid-elbow extension (i.e., 6 and 12 o'clock made relative to a clock face, respectively). Arm cycling was performed at combinations of two cadences (60 and 90 rpm) at three relative power outputs (20, 40, and 60% peak power output). At the 6 o'clock position, BB MEPs increased ~11.5% as cadence increased and up to ~57.2% as power output increased ( P < 0.05). In the TB, MEPs increased ~15.2% with cadence ( P = 0.013) but were not affected by power output, while CMEPs increased with cadence (~16.3%) and power output (up to ~19.1%, P < 0.05). At the 12 o'clock position, BB MEPs increased ~26.8% as cadence increased and up to ~96.1% as power output increased ( P < 0.05), while CMEPs decreased ~29.7% with cadence ( P = 0.013) and did not change with power output ( P = 0.851). In contrast, TB MEPs were not different with cadence or power output, while CMEPs increased ~12.8% with cadence and up to ~23.1% with power output ( P < 0.05). These data suggest that the "type" of intensity differentially modulates supraspinal and spinal excitability in a manner that is phase- and muscle dependent. NEW & NOTEWORTHY There is currently little information available on how changes in locomotor intensity influence excitability within the corticospinal pathway. This study investigated the effects of arm cycling intensity (i.e., alterations in cadence and power output) on corticospinal excitability projecting to the biceps and triceps brachii during arm cycling. We demonstrate that corticospinal excitability is modulated differentially by cadence and power output and that these modulations are dependent on the phase and the muscle examined.

Automatic contour propagation using deformable image registration to determine delivered dose to spinal cord in head-and-neck cancer radiotherapy.

To determine delivered dose to the spinal cord, a technique has been developed to propagate manual contours from kilovoltage computed-tomography (kVCT) scans for treatment planning to megavoltage computed-tomography (MVCT) guidance scans. The technique uses the Elastix software to perform intensity-based deformable image registration of each kVCT scan to the associated MVCT scans. The registration transform is then applied to contours of the spinal cord drawn manually on the kVCT scan, to obtain contour positions on the MVCT scans. Different registration strategies have been investigated, with performance evaluated by comparing the resulting auto-contours with manual contours, drawn by oncologists. The comparison metrics include the conformity index (CI), and the distance between centres (DBC). With optimised registration, auto-contours generally agree well with manual contours. Considering all 30 MVCT scans for each of three patients, the median CI is [Formula: see text], and the median DBC is ([Formula: see text]) mm. An intra-observer comparison for the same scans gives a median CI of [Formula: see text] and a DBC of ([Formula: see text]) mm. Good levels of conformity are also obtained when auto-contours are compared with manual contours from one observer for a single MVCT scan for each of 30 patients, and when they are compared with manual contours from six observers for two MVCT scans for each of three patients. Using the auto-contours to estimate organ position at treatment time, a preliminary study of 33 patients who underwent radiotherapy for head-and-neck cancers indicates good agreement between planned and delivered dose to the spinal cord.

Image-guided radiotherapy of the prostate using daily CBCT: the feasibility and likely benefit of implementing a margin reduction.

OBJECTIVE: To investigate whether planning target volume (PTV) margins may be safely reduced in radiotherapy of localized prostate cancer incorporating daily online tube potential-cone beam CT (CBCT) image guidance and the anticipated benefit in predicted rectal toxicity. METHODS: The prostate-only clinical target volume (CTV2) and rectum were delineated on 1 pre-treatment CBCT each week in 18 randomly selected patients. By transposing these contours onto the original plan, dose-volume histograms (DVHs) for CTV2 and the rectum were each calculated and combined, for each patient, to produce a single mean DVH representative of the dose delivered over the treatment course. Plans were reoptimized using reduced CTV2 to PTV2 margins and the consequent radiobiological impact modelled by the tumour control probability (TCP) and normal tissue complication probability (NTCP) of the rectum. RESULTS: All CBCT images were deemed of sufficient quality to identify the CTV and rectum. No loss of TCP was observed when plans using the standard 5-mm CTV2 to PTV2 margin of the centre were reoptimized with a 4- or 3-mm margin. Margin reduction was associated with a significant decrease in rectal NTCP (5-4 mm; p < 0.05 and 5-3 mm; p < 0.01). CONCLUSION: Using daily online image guidance with CBCT, a reduction in CTV2 to PTV2 margins to 3 mm is achievable without compromising tumour control. The consequent sparing of surrounding normal tissues is associated with reduced anticipated rectal toxicity. ADVANCES IN KNOWLEDGE: Margin reduction is feasible and potentially beneficial. Centres with image-guided radiotherapy capability should consider assessing whether margin reduction is possible within their institutes.

The value of image-guided intensity-modulated radiotherapy in challenging clinical settings.

OBJECTIVE: To illustrate the wider potential scope of image-guided intensity-modulated radiotherapy (IG-IMRT), outside of the "standard" indications for IMRT. METHODS: Nine challenging clinical cases were selected. All were treated with radical intent, although it was accepted that in several of the cases the probability of cure was low. IMRT alone was not adequate owing to the close proximity of the target to organs at risk, the risk of geographical miss, or the need to tighten planning margins, making image-guided radiotherapy an essential integral part of the treatment. Discrepancies between the initial planning scan and the daily on-treatment megavoltage CT were recorded for each case. The three-dimensional displacement was compared with the margin used to create the planning target volume (PTV). RESULTS: All but one patient achieved local control. Three patients developed metastatic disease but benefited from good local palliation; two have since died. A further patient died of an unrelated condition. Four patients are alive and well. Toxicity was low in all cases. Without daily image guidance, the PTV margin would have been insufficient to ensure complete coverage in 49% of fractions. It was inadequate by >3 mm in 19% of fractions, and by >5 mm in 9%. CONCLUSION: IG-IMRT ensures accurate dose delivery to treat the target and avoid critical structures, acting as daily quality assurance for the delivery of complex IMRT plans. These patients could not have been adequately treated without image guidance. ADVANCES IN KNOWLEDGE: IG-IMRT can offer improved outcomes in less common clinical situations, where conventional techniques would provide suboptimal treatment.

Tumour shrinkage and contour change during radiotherapy increase the dose to organs at risk but not the target volumes for head and neck cancer patients treated on the TomoTherapy HiArt™ system.

AIMS: To quantify the changes in contours of the target and organs at risk and the differences between planned and delivered doses to the target and organs at risk during the course of radiotherapy in head and neck cancer patients treated with intensity-modulated radiotherapy on the TomoTherapy HiArt™ system. MATERIALS AND METHODS: Five patients with squamous cell carcinoma of the head and neck treated with radical chemoradiotherapy using the TomoTherapy HiArt system were included in the study. The target volumes were treated to three different dose levels depending on the level of clinical risk for harbouring disease. Patient positions were assessed daily with megavoltage computed tomography (MVCT) and positional correction made before each treatment when necessary. MVCTs were superimposed on to the planning kilovoltage computed tomography images for each patient and target volumes and organ at risk volumes were re-outlined on MVCT images. Doses to clinical target volumes and organs at risk were recalculated to show the actual delivered doses. RESULTS: There was shrinkage in the volume of the parotid glands during treatment in all cases. The mean volume reduction in the ipsilateral parotid gland was more marked at 30.2%, compared with the contralateral parotid glands. However, the mean percentage dose per fraction increase was higher in the contralateral parotid glands at 24%, compared with the ipsilateral parotids. The calculated doses were higher than the planned doses in all CTV-54, CTV-60 and CTV-68, but the mean dose differences were modest, in the range 1.3-2.4%. CONCLUSIONS: We have shown that there were considerable changes in the volume and dose to the parotids during treatment. The changes in volume and dose to the clinical target volume were more modest in comparison. Adaptive radiotherapy planning can be helpful in improving the dose to the parotid glands. However, its role in the optimisation of the dosage to the clinical target volume is less likely to result in a significant clinical benefit.

A systematic review of the role of imaging before salvage radiotherapy for post-prostatectomy biochemical recurrence.

A substantial proportion of patients who have undergone a radical prostatectomy for localised prostate cancer will have either persistently detectable prostate-specific antigen (PSA) levels or a delayed rise in PSA. The optimum treatment for these situations is not known. The key question is whether the PSA is reflective of local or distant progression. For salvage radiotherapy to be most effective, treatment should be considered before the PSA level is allowed to rise too high, when disease is more likely to be confined to the prostate bed. However, at low PSA levels, current imaging techniques are poor at detecting disease, making it difficult to differentiate local and distant recurrences and to target the radiotherapy appropriately. We review current and investigational imaging techniques, including bone scan, computed tomography, magnetic resonance imaging, positron emission tomography and Prostascint, assessing their utility in the situation of biochemical recurrence after radical prostatectomy.

An assessment of action levels in imaging strategies in head and neck cancer using TomoTherapy. Are our margins adequate in the absence of image guidance?

AIMS: To assess the effectiveness of different on-treatment correction strategies on set-up accuracy in patients with head and neck cancer (HNC) treated on a TomoTherapy HiArt system. To assess the adequacy of clinical target volume (CTV) to planning target volume (PTV) treatment planning margins when treating with intensity-modulated radiotherapy without daily image guidance. MATERIALS AND METHODS: The set-up accuracy measured by daily online volumetric imaging was retrospectively reviewed for the first 15 patients with HNC treated on the TomoTherapy unit at Addenbrooke's Hospital. For each fraction, megavoltage computed tomography was carried out, any discrepancy from the planning scan was noted, and corrected, before treatment. These data were used to evaluate imaging correction protocols using three different action levels. The first three fractions were imaged and used to correct for systematic error, using a 5 mm action level (5 mmAL), a 3 mm action level (3 mmAL), and no action level (NAL). All imaging strategies were applied, to assess the number of fractions that would potentially have exceeded a 5 and 3 mm margin. Systematic and random errors were calculated for the population, assuming the NAL protocol had been applied, and minimum CTV-PTV margins, required to allow for errors attributable only to set-up, were calculated using van Herk's formula. RESULTS: In total, 490 fractions were analysed. Using a 5 mmAL imaging protocol, potentially 198/490 fractions (40%) were outside a 5 mm CTV-PTV margin and 400/490 (82%) were outside a 3 mm margin. Using a 3 mmAL imaging protocol, potentially 67/490 fractions (14%) were outside a 5 mm CTV-PTV margin and 253/490 (52%) were outside a 3 mm margin. A small systematic error was identified in the system; once corrected this would improve these results. Using the NAL imaging protocol, potentially 31/490 fractions (6%) were outside a 5 mm CTV-PTV margin and 143/490 fractions (29%) were outside a 3 mm margin. Estimated minimum CTV-PTV margins to account only for set-up errors, with three-fraction image-guided radiotherapy and a NAL protocol, were 2.8, 3.1 and 4.1 mm in the mediolateral, superior-inferior and anterior-posterior directions, respectively. CONCLUSION: Reducing the action level at which the systematic error is corrected improves the probability of treatment delivery accuracy. Using the NAL correction protocol reduces the number of fractions that have set-up displacements outside a 5 mm CTV-PTV margin. Although a 5 mm margin is probably sufficient for standard HNC radiotherapy, change to a 3 mm margin is not favoured at our centre without access to daily image-guided radiotherapy.

Practical issues in the implementation of image-guided radiotherapy for the treatment of prostate cancer within a UK department.

AIMS: To study the feasibility of using implanted gold seeds in combination with a commercial software system for daily localisation of the prostate gland during conformal radiotherapy, and to assess the effect this may have on departmental workload. MATERIALS AND METHODS: Six patients had three gold radio-opaque seeds implanted into the prostate gland before starting a course of radiotherapy. The seeds were identified on daily portal images and an automated online system provided immediate vector analysis of discrepancies between the planned and actual daily position of the intraprostatic seeds. In total, 138 interfractional displacements were analysed. The workload impact for the department was assessed using the basic treatment equivalence model, by comparing measurements of daily treatment session durations with a control group of patients receiving standard conformal radiotherapy, matched for treatment complexity. RESULTS: No acute complications of seed insertion were observed. A number of developmental issues required solutions to be identified before clinical implementation was possible. The standard deviations of the set-up and organ motion systematic errors in the left-right, superior-inferior and anterior-posterior directions were 2.4, 3.0 and 2.5 mm, respectively. The standard deviations of the set-up and organ motion random errors calculated were 2.5, 2.9 and 3.7 mm. The mean treatment session duration with this daily prostate localisation system was increased by 3 min compared with matched controls using standard imaging practice. If all radical prostate patients in our department were to receive image-guided radiotherapy in this way, this would increase machine workload time by 2.2 h/day. CONCLUSIONS: The implementation of this image-guided system is feasible. No additional linear accelerator modification is required and standard imaging devices can be used. It would be a useful addition to any department's image-guided radiotherapy developmental strategy.

Years of life lost (YLL) from cancer is an important measure of population burden--and should be considered when allocating research funds.

Recently, cancer mortality has been compared to research spending by the National Cancer Research Institute (NCRI), whose research budget is approximately pound sterling 250 million. The analysis shows a mis-match between mortality and research spending. As well as crude mortality rates, other measures of cancer burden should be considered because they contribute additional information. 'Years of life lost' (YLL) summed over each individual dying after a diagnosis of cancer represents a population-based mortality indicator of the impact of that disease on society. Years of life lost divided by the number of deaths for each cancer site produces an additional statistic, the average years of life lost (AYLL), which is a measure of the burden of cancer to the individual patient. For 17 cancer sites where data are available, four tumour sites have a rather large difference in mortality, comparing YLL to crude mortality. Years of life lost shows the population burden from cancers of the ovary, cervix, and CNS to be rather larger than suggested by crude mortality, despite screening programmes for cervix cancer. Using YLL, the underprovision of funding for lung cancer research is similar to that reported using percentage mortality. Breast cancer and leukaemia receive a relatively higher research spend than the population burden of these cancers, and the spending on leukaemia is quite extreme. Prostate cancer has a low per cent YLL but attracts a moderate amount of research spending. The use of AYLL as an indicator of individual cancer burden considerably changes the ranking of the mortality from different tumours. The mean AYLL is 12.5 years. Prostate cancer has the lowest AYLL, only 6.1 years; brain tumour patients have the highest, at just over 20 years. Comparing AYLL to research spending suggests four 'Cinderella' cancer sites with high individual cancer burden but low research spending: CNS tumours, cervix and kidney cancers, and melanoma. Breast cancer and leukaemia have roughly average AYLL but a considerable excess of research spending. YLL emphasises the discrepancy between research spending and mortality, and may be helpful for decisions concerning research support. Average years of life lost measures the burden to individual patients and may be helpful where individuals' needs are relevant, such as palliative care. As well as crude mortality, more subtle and comprehensive calculations of mortality statistics would be useful in debates on research funding and public health issues.

Accuracy of a relocatable stereotactic radiotherapy head frame evaluated by use of a depth helmet.

In high precision radiotherapy, the more accurately the patient can be relocated, the smaller the clinical to planning target volume margin can be, with reduction in the volume of normal tissue irradiated. The Gill-Thomas-Cosman (GTC) relocatable stereotactic head frame provides immobilization of the patient which is highly reproducible. A depth helmet and measuring probe were used to confirm the accuracy of relocation of 31 patients treated in the GTC frame. The measurements were processed in a spreadsheet developed to calculate the size of the patient's displacement as a vector. Twenty-seven patients received fractionated stereotactically-guided conformal radiotherapy, and 4 single fraction stereotactic radiosurgery, amounting to 564 measurement episodes. The accuracy was extremely good, and considerably more accurate than standard thermoplastic head shells. Ninety-two percent of the displacement vectors were less than 2 mm, and 97% less than 2.5 mm. Considering each dimension separately, the largest mean displacement was 0.4 mm in the superior-inferior direction. Accuracy was constant through a fractionated course for most patients, but prediction based on measurements from the first few fractions was not reliable. Results were dependent on patient selection, with worse reproducibility in patients with neurological deficits, or difficulty cooperating. The depth helmet measurements detected a loosened mouth bite in one patient and allowed repositioning to be verified without the need for the simulator. Total treatment time, including use of the depth helmet to verify treatment position, is quicker (mean 15.7 min) than using portal films. The depth helmet, used in conjunction with the vector displacement spreadsheet, provides a simple way to define the CTV-PTV margin. For fractionated stereotactic radiotherapy we use a 3 mm CTV-PTV margin. This system could assist technology transfer to centres starting stereotactic radiotherapy using the GTC frame.

An audit of clinic consultation times in a cancer centre: implications for national manpower planning.

A departmental audit was conducted to assess the frequency, extent and causes of late completion of oncology clinics. Data were collected prospectively from clinical, medical, haematological and multidisciplinary oncology clinics. The data recorded included: clinic start and finish times, number of patients seen, type of consultation, number of doctors in each clinic, time spent by the doctor with the patient, and other factors that may have contributed to the late completion of clinics. A total of 848 patient consultations were recorded in 81 clinics. Of 67 clinics in which the finish time was recorded, 19 (28%) were completed on time, while 48 (72%) were late by a mean time of 49 minutes. The mean time spent by consultants with new, follow-up and chemotherapy patients was 37, 21 and 22 minutes respectively. This did not include time spent reviewing notes, dictating or ordering investigations. There was no significant difference in the time spent by specialist registrars compared with consultants, or clinical oncologists compared with medical oncologists and haematologists. The incidence of unforeseen problems such as difficult consultations, missing information, unplanned interruptions, late starts and overbooking of patients were not significantly different in those clinics that finished late compared with those that finished on time. The mean overrun of multidisciplinary clinics was longer than for non-multidisciplinary clinics (59 and 31 minutes respectively), despite a higher ratio of doctors to patients in the former (1:5.4 and 1:7 respectively). This audit showed that the main cause of late finishes in clinics in our department was the longer than anticipated time spent by doctors with patients. Consultations are taking longer because of the increasing complexity of non-surgical cancer treatments and the greater emphasis placed on patient information and informed consent. The Royal College of Radiologists (RCR) has calculated that, if a consultant oncologist sees a maximum of 315 new patients per year, the time available for each follow-up consultation would be 10 minutes. Our audit showed that follow-up consultations took an average of 21 minutes. These results suggest that the RCR recommendations for consultant expansion substantially underestimates the true number of consultants required for the treatment of cancer patients in the UK.

Radiation-induced malignant melanoma of the cervix.

We report the occurrence of a malignant melanoma of the uterine cervix in a patient previously irradiated for a squamous cell carcinoma of the cervix. The management of malignant melanoma of the cervix is reviewed. The risk of second cancers in general, malignant melanoma in particular, after radiotherapy for cervical cancer is also discussed.

Alcohol consumption and the risk of cirrhosis.

OBJECTIVE: To determine risk levels for the development of cirrhosis in Australian men who consume alcohol and to compare this risk with that of Australian women. DESIGN AND SETTING: A case-control study using an interview technique, based in two major tertiary referral hospitals in Sydney. PATIENTS: Forty-three men with newly diagnosed cirrhosis of the liver and a total of 115 male control subjects, age matched with the case subjects. The data for women (36 cases and 99 controls) have been reported previously. RESULTS: The risk of men developing cirrhosis increases significantly above the baseline when the alcohol intake exceeds 40 g per day. The risk to women is significant at a similar intake level. Dietary intake and past major illnesses appear to have no role in determining risk. CONCLUSION: The recommended safe drinking level for men and women should be 40 g per day, as suggested by the National Health and Medical Research Council.

Newly synthesized calsequestrin, destined for the sarcoplasmic reticulum, is contained in early/intermediate Golgi-derived clathrin-coated vesicles.

We have examined the possible role of clathrin-coated vesicles (CVs) in the genesis of the sarcoplasmic reticulum (SR) in developing chick skeletal myotubes. Calsequestrin (CSQ) a luminal Ca2+ binding protein of the terminal SR cisternae, is contained within the vesicle lumen of skeletal muscle CVs in substantial amounts, approximately four molecules/CV. Employing 3-day cultures of chick skeletal myotubes we demonstrate that after a 30-min labeling with [35S]methionine and cysteine, radioactivity in CSQ remains high in the CVs 45 min later and then declines, while labeled CSQ in the SR continues to rise. No CSQ appears to be secreted. All of the CSQ in both the CVs and SR is sensitive to the activity of endoglycosidase H, and a significant fraction also binds to wheat germ agglutinin. Based on these results, we discuss the hypothesis that a selective CV-mediated pathway exists in developing skeletal muscle cells for the transport of CSQ from the early/intermediate Golgi apparatus to the SR.

Intracellular transport, sorting, and turnover of acetylcholinesterase. Evidence for an endoglycosidase H-sensitive form in Golgi apparatus, sarcoplasmic reticulum, and clathrin-coated vesicles and its rapid degradation by a non-lysosomal mechanism.

Tissue-cultured muscle cells synthesize several oligomeric forms of acetylcholinesterase (AChE) destined for the cell surface or secretion. Previous studies on the biogenesis of AChE polypeptide chains have shown that only a small fraction become assembled into catalytically active oligomers which transit the Golgi apparatus and acquire endoglycosidase H (endo H) resistance. Most of the AChE polypeptides remain endo H-sensitive and are rapidly degraded intracellularly. We now show that all newly synthesized AChE polypeptides are transported from the rough endoplasmic reticulum to the Golgi apparatus where they acquire N-acetylglucosamine. However, approximately 80% of these AChE polypeptides remain endo H-sensitive and are degraded intracellularly with a half-life of about 1.5 h by a mechanism which is insensitive to lysosomotropic agents. These endo H-sensitive AChE molecules can be chased into clathrin-coated vesicles and/or the sarcoplasmic reticulum prior to degradation. Pulse-chase studies of isotopically labeled or catalytically active AChE molecules suggest that there are at least two discreet populations of clathrin-coated vesicles which leave the Golgi, one whose origin is cis/medial and one whose origin is trans. These studies indicate the existence of a post-rough endoplasmic reticulum, non-lysosomal degradative pathway for intra-luminal proteins and suggest that post-translational events at the levels of protein sorting and degradation may play a role in regulating the abundance of exportable proteins.

Binding of rabies virus to purified Torpedo acetylcholine receptor.

The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

An acetylcholinesterase-mediated density shift technique demonstrates that coated vesicles from chick myotubes may contain both newly synthesized acetylcholinesterase and acetylcholine receptors.

Coated vesicles isolated from 17 d chick embryo skeletal muscle contain acetylcholine receptors (AChRs) as shown by the presence of specific, latent binding sites for 125I-alpha bungarotoxin (125I-alpha-BTX). Since these coated vesicles also contain AChE (Benson et al., 1985), we hypothesized that a coated vesicle could carry both molecules: one an integral membrane protein, the other a secreted protein. An AChE-mediated density shift technique was used to obtain data that indicate that most isolated coated vesicles contain AChE and that some contain AChRs as well. Similar results were obtained with coated vesicles isolated from cultured chick embryo myotubes treated briefly with diisopropylfluorophosphate (DFP) to inactivate all preexisting AChE and allowed to synthesize AChE for 2 1/2 hr. These data are compatible with the hypothesis that both an integral plasma membrane protein, AChR, and a secretory protein, AChE, traverse the identical pathway after synthesis, as proposed by Rotundo and Fambrough (1980a). We suggest that coated vesicles are important intermediates in the exocytotic pathway, and that the large percentage of coated vesicles utilized for exocytotic transport can explain the rapid net increase in surface area achieved during myotube development. We also discuss the potential utility of the AChE-mediated density shift in studying the exocytotic and endocytotic pathways in other cell types, and possible pitfalls associated with its use.

Biochemical and cytochemical evidence indicates that coated vesicles in chick embryo myotubes contain newly synthesized acetylcholinesterase.

We have isolated highly purified coated vesicles from 17-d-old chick embryo skeletal muscle. These isolated coated vesicles contain acetylcholinesterase (AChE) in a latent, membrane-protected form as demonstrated enzymatically and morphologically using the Karnovsky and Roots histochemical procedure (J. Histochem. Cytochem., 1964, 12:219-221). By the use of appropriate inhibitors the cholinesterase activity can be shown to be specific for acetylcholine. It also can be concluded that most of the AChE represents soluble enzyme since it is rendered soluble by repeated freeze-thaw cycles. To determine the origin of the coated vesicle-associated AChE, we have isolated coated vesicles from cultured chick embryo myotubes which have been treated with diisopropylfluorophosphate, an essentially irreversible inhibitor of both intra- and extracellular AChE, and have been allowed to recover for 3 h. This time is not enough to allow any newly synthesized AChE to be secreted. These coated vesicles also contain predominantly soluble AChE. These data are compatible with the hypothesis that coated vesicles are important intermediates in the intracellular transport of newly synthesized AChE.