Viscosupplementation in the hip : evaluation of hyaluronic acid formulations This study compares three different hyaluronate formulations and evaluates functionality , time of satisfactory pain relief and also the delay in performing a total hip arthroplasty . One hundred and twenty patients -LRB- 126 hips -RRB- received viscosupplementation with one of the three hyaluronate formulations . All patients were candidate for surgical treatment with a total hip arthroplasty . Three different products were consecutively used : Adant ® , Synocrom ® or Synvisc ® . Patients were assessed 6 weeks after each infiltration using Visual Analogue Scale and Harris Hip Score . The Harris Hip Score increased significantly in two of the three groups compared to baseline , but no statistical significant difference was noted between the groups . Viscosupplementation provides significant pain reduction in two of the three groups . There is no significant difference in duration of the effect of the first infiltration between the three groups . The positive effect was still ongoing at the end point of the study in 46 hips : 51 % of the patients did not undergo total hip arthroplasty , 3 years after viscosupplementation . Introduction Severe osteoarthritis -LRB- OA -RRB- of the hip is characterized by pain and reduced mobility . It affects primarily adults aged over 50 years , but the age of onset of hip OA linked complaints seems to decrease over the last decennia -LSB- 12 -RSB- . First-line treatment consists of pain reduction with analgesics and non steroidal anti inflammatory drugs -LRB- NSAID -RRB- . Rehabilitation treatment , education and the use of walking aids can help providing pain relief and improving mobility . When those therapeutic options fail or the analgesic treatment causes intolerable side effects , joint lavage , hip osteotomy or total hip arthroplasty -LRB- THA -RRB- can be considered . This arthroplasty technique has greatly improved and patients experience pain reduction and improvement of the quality of life . However , the intervention still requires prolonged anaesthesia , and is followed by a long-term rehabilitation program . Though complications are rare and protheses have an increasing duration of life , reinterventions are still needed -LSB- 4 -RSB- . Viscosupplementation -LRB- VS -RRB- is the administration of hyaluronan and/or hyaluronic acid preparations to joint synovial fluid for the treatment of OA in order to restore the biologic properties of normal hyaluronic acid -LRB- HA -RRB- . The use of VS with HA was first described to provide pain relief and to increase mobility of the knee joint . The VS is an effective treatment for OA of the knee with beneficial effects on pain , function and patient global assessment -LSB- 14 -RSB- . HA products have more prolonged effects than intra-articular corticosteroids -LSB- 14 -RSB- . Since 1984 , this technique is also used for the management of OA of the hip joint -LSB- 2 -RSB- . In the available literature there is no consensus on the number of injections , the dosage per injection , the most appropriate formulation of HA , and the optimal method for controlling the needle positioning in the hip joint . Moreover , the patient selection criteria differ from one study to another . To our knowledge there is no documentation available on the possible time gain for patients treated with VS of the hip joint before they have to undergo THA . The results of a trial comparing three formulations of HA were presented . All patients were candidates for THA . The time of satisfactory pain relief , improved functionality obtained with each infiltration of HA and the delay in surgery were evaluated . Patients and methods Patient selection The use of VS in patients with severe OA is common practise in our hospital , thus not requiring approval of the ethical committee . Patients gave their informed consent for this prospective study . Between March 2001 and February 2005 , 120 patients -LRB- 126 hips -RRB- , 49 males and 71 females , responding to the eligibility criteria listed below , received viscosupplementation . Three different products were consecutively used : Adant ® -LRB- Tedec-Meiji Failma , Madrid , Spain -RRB- -LRB- Group 1 -RRB- -LRB- Synthetic hyaluronic acid with an average molecular weight of 0.6 -- 1.2 million Da -RRB- , Synocrom ® -LRB- Croma Pharma , Leobendorf , Austria -RRB- -LRB- Group 2 -RRB- -LRB- Sodium hyaluronate with a average molecular weight of 1.6 million Da -RRB- or Synvisc ® -LRB- Altana Pharma , Konstanz , Germany -RRB- -LRB- Group 3 -RRB- -LRB- Hylan GF 20 with an average molecular weight of 6.0 million Da -RRB- -LRB- Table 1 -RRB- . Table 1Characteristics of infiltrated populationPeriod of treatmentPatientsGenderAgeAdant27-11-02 till 23-02-059135 ♂ and 65 ♀ 61.8 ± 12.8Synocrom19-01-04 till 04-10-04207 ♂ and 13 ♀ 62.1 ± 14.5Synvisc15-03-01 till 24-04-03157 ♂ and 8 ♀ 61.9 ± 15.3 Eligibility criteria Age between 30 and 70 years and suffering idiopathic radiologically confirmed hip OA . Visual Analogue Scale -LRB- VAS -RRB- score for pain greater then 30 -LRB- on a 100-point scale ; 0 no pain and 100 `` the worst pain imaginable '' -RRB- Have persistent pain for longer than 1 month despite use of analgesics or NSAID 's . Be candidate for surgical treatment with a THA , according to the following criteria : continuous hip pain , also during the night , requiring daily intake of NSAID 's or pain medicationdisabled gait pattern and need of walking aid Be able to understand the information relative to viscosupplementation and to give informed consent . Exclusion criteria Pregnancy Contraindications to intra-articular hyaluronic-acid preparations Major hip dysplasia or congenital abnormality of the hip Patients with systemic corticosteroids or intra-articular corticosteroid injections in the last 6 months Contra-lateral THA or hip arthroscopy in the last 6 months Oral or parenteral anticoagulant therapy Previous hyaluronic acid hip infiltrations Skin diseases or infections Signs of haemarthrosis History of allergy or hypersensitivity to iodated contrast Treatment Patients received an intra articular infiltration with one of the three products . The manufacturer 's treatment recommendations were followed . Patients having initially experienced a satisfactory pain relief are offered a second and third infiltration or THA when the condition deteriorates . Injection of the viscosupplementation was performed under sterile conditions by the same experienced orthopaedic surgeon -LRB- MM -RRB- in all patients . After skin cleaning a lumbar puncture needle was inserted in a lateral approach . Layer by layer local anaesthesia was performed using lidocaine 1 % . Iodinated contrast agent Ultravist ® -LRB- Schering , Berlin , Germany -RRB- was injected . The needle positioning into the joint cavity was fluoroscopically controlled -LRB- Fig. 1 -RRB- . Arthrocentesis was carefully performed prior to each injection to remove any effusion . Fig. 1a Needle placement for injection of the viscosupplementation . b Fluoroscopic image of the needle insertion in the hip joint space After resting for 2 h , the patient was allowed to walk and to return home . The patient was advised to rest at home until the next morning . Oral symptomatic slow acting drugs for osteoarthritis were authorized if they were taken at a stable dose for more than 3 months prior to inclusion in the study . These analgesics were continued at a stable dose during the VS treatment . Evaluation All patients were assessed at baseline and 6 weeks after each infiltration . During this consultation in the outpatient clinic , the pain and functionality were evaluated using the VAS pain during walking score -LRB- 100-point scale -RRB- and the Harris Hip Score -LRB- HHS -RRB- . The latter is a clinical scoring system on a total of 100 points whereby the following subscales are rated : function -LRB- 47 points -RRB- , pain -LRB- 44 points -RRB- , range of motion of the hip -LRB- 5 points -RRB- and absence of muscle contractures and length discrepancy -LRB- 4 points -RRB- . All side effects and complications of viscosupplementation were noted . In April 2005 , all patients were contacted for follow-up assessment over the phone VAS and HHS . Statistical analysis Software -LRB- MS Excel -RRB- ® was used to collect the data . Statistical analyses were of two kinds , first the differences in HHS and VAS pre-infiltration and post-infiltration -LRB- in the three groups -RRB- were compared using the paired t test and with the Wilcoxon paired test in the Synvisc group because this group is too small to analyse with the paired t test . The second comparison was made between the three groups -LRB- Synvisc , Synocrom and Adant -RRB- . The differences in HHS and VAS evolution between the three groups were checked by the analysis of variance statistics with Tukey extension , which also was used for controlling if the groups were initially homogeneous . The duration of the viscosupplementation effect of the first infiltration was analysed using the Kaplan-Meier survival curves . The delay for the need of hip surgery was also analysed using a Kaplan-Meier survival curve . The statistical significance was set on P ≤ 0.05 . Results The mean pre-infiltration HHS was comparable for the three groups and varied from 64.8 points in the Adant group to 66.8 points in the Synocrom-group . The post-infiltration HHS increased with 6.3 points in the Adant group -LRB- P < 0.001 -RRB- , with 10.6 points in the Synocrom group -LRB- P < 0.05 -RRB- and with 6.1 points in the Synvisc-group -LRB- P > 0.05 ; Table 2 -RRB- . There was no statistical significant difference in the evolution of the HHS between the three groups -LRB- Table 2 -RRB- . Table 2Evolution in average HHS scoreHHS preHHS postDifferencePAdant64 .8 ± 13.871.1 ± 15.7 + 6.3 < 0.001 Synocrom66 .8 ± 13.877.4 ± 14.7 + 10.6 < 0.05 Synvisc66 .3 ± 13.572.4 ± 14.5 + 6.1 > 0.05 Viscosupplementation provided a highly significant pain reduction in the Adant-group -LRB- P < 0.0001 -RRB- , a significant pain reduction in the Synocrom-group -LRB- P < 0.05 -RRB- and a pain reduction that did not reach significance in the Synvisc-group -LRB- P > 0.05 -RRB- . There was no significant difference in pain relief between the three treatment products -LRB- Fig. 2 , Table 3 -RRB- . Fig. 2Pain relief in the three treatment groups a Adant , b Synocrom and c Synvisc The duration of the effect of the first infiltration in the three groups is shown in a Kaplan-Meier curve -LRB- Fig. 3 -RRB- . The first infiltration was the starting point . Endpoints were the second infiltration or operation of the afflicted hip , or when these were not applicable the latest patient contact , which can be considered as ongoing effect . Fig. 3Kaplan-Meier survival curve of the duration of effect of the first infiltration in days for the three different treatment groups There is no significant difference between the three groups -LRB- χ2 = 0.988 and P = 0.61 -RRB- . Figure 4 illustrates the duration of effect of the three groups globally . Fig. 4Kaplan-Meier survival curve of the duration of effect of the first infiltration in days -LRB- three treatment groups together -RRB- The positive effect was still ongoing in 46 hips , while in 80 hips patients had either received a second infiltration or THA at , the end of the study -LRB- Table 4 -RRB- . Table 3Evolution in average VAS pain during walking scoreVAS preVAS postDifference PAdant51 ± 2339 ± 27 − 12 < 0.0001 Synocrom43 ± 2229 ± 23 − 14 < 0.02 Synvisc47 ± 2630 ± 29 − 17 > 0.05 Pre : at first infiltrationPost : at follow-up The delay in performing a hip operation is analysed using a Kaplan-Meier survival curve -LRB- Fig. 4 -RRB- . After 3 years , 51 % of the patients have not undergone surgery . Because of the comparable outcome achieved with the three HA formulations , we did not differentiate between the three products in this survival analysis -LRB- Fig. 5 -RRB- . Fig. 5Kaplan-Meier survival curve for the delay to surgery in days for the three groups confounded Discussion The patients attending our outpatient clinic , who were judged eligible for THA , were offered the possibility of a VS therapy . Hundred twenty patients choose this treatment option and 126 hips were treated with Adant , Synocrom or Synvisc . All studies , published up till now , studying the effect of VS in the treatment of hip OA were performed in small patient groups -LRB- 12 -- 57 patients -RRB- -LSB- 6 , 9 -RSB- . Table 4Duration of treatment effect after first infiltrationPatientsAverage duration -LRB- days -RRB- Effect of 1st infiltration still going on46 352 ± 258 -LRB- range 6 -- 886 -RRB- Effect of 1st infiltration terminated80174 ± 163 -LRB- range 13 -- 724 -RRB- The three preparations provided a significant pain relief and improvement of the HHS . The isolated Synvisc group never reached statistical significance in HHS score evolution and VAS during walk test after VS treatment ; possibly due to the small number of patients -LRB- N = 15 -RRB- in this group . As in the study of Tikiz -LSB- 13 -RSB- , we found no significant difference between lower and higher molecular weight hyaluronic acids . We could identify two groups of responders , the ongoing responders who last on average for 352 days after the first infiltration . The second group , where patients either received a second infiltration or THA had an average duration of effect of 174 days . The VS method is widely used for OA of the knee joint , but there are only a few studies about its use in OA of the hip . Most authors agree that there should be a role for viscosupplementation in the treatment of hip OA . -LSB- 1 -- 3 , 6 , 7 , 9 -- 11 -RSB- Our findings confirm the effect of VS in patients suffering OA of the hip . To our knowledge , this is the largest series of patients with hip osteoarthritis treated with viscosupplementation . There are no guidelines regarding the optimal number of injections needed to obtain optimal clinical response , which seems to be different -LSB- 1 , 2 , 8 , 10 , 11 -RSB- between products and dependent on the molecular weight -LRB- MW -RRB- -LSB- 7 -RSB- . We evaluated the time to relapse of one infiltration . Other authors advocated the use of ultrasound for controlling the needle position in the hip joint . -LSB- 9 , 3 -RSB- In our series , we performed the infiltrations under fluoroscopy , in experienced hands this approach has proven to be accurate and safe . We use a very low amount of contrast liquid to avoid dilution of the product . Of all the reports published , we have the longest follow-up , up to approximately 3 years . The analysis by means of the Kaplan-Meier survival curve learns that there is a 50 % probability of long-term effect -LRB- more than 2 years -RRB- . We saw no infectious adverse events and no serious systemic reactions , but all the interventions are performed in the operating theatre under strict aseptic conditions . The adverse events rates ranged from 10 to 30 % which is slightly higher than the rates reported in VS treatment of knee OA -LSB- 7 -RSB- . Repeated injections did not increase the risk of adverse events -LSB- 7 -RSB- . Some patients experienced transient hip pain after the infiltration but made a full recovery in the next days . In the study of Conrozier -LSB- 6 -RSB- , transient hip pain was reported after 10.1 % of the injections . The products used in this study were all well tolerated . Complications are rare but a single case of septic arthritis was reported after multiple injections of hyaluronate and glucocorticoid -LSB- 5 -RSB- . Gout , pseudogout and chondrocalcinosis have not been reported after hip infiltrations . Intra articular injection of hyaluronic acid can provide long-term pain relief and improvement of joint function even in patients eligible for THA . Despite the use of fluoroscopy , this technique can be performed in an outpatient clinic , allowing patients to return home on the day of the intervention . At present , viscosupplementation therapy for OA of the hip is only palliative . It can be an alternative for young THA candidates , patients with surgical contra-indications and patients in whom NSAID use is not appropriate . There is only 1 randomised , double blind , placebo controlled trial of patients with hip OA -LSB- 15 -RSB- . The study was designed with a three-armed parallel-group -LRB- Hyalgan vs. methylprednisolone vs. lidocaine -RRB- . One hundred and one were treated . There was no statistically significant difference between the three products on any outcome measure including the primary outcome measure , i.e. , ` Pain on walking ' , at three 3 months of follow-up . The results of our study should be considered in the light of the limitations of the design of this study . It is a non placebo controlled non randomised prospective study . It is known from experience with knee OA that the placebo effect of VS tends to be substantial -LSB- 8 -RSB- . The dimension of the groups treated with the three different products differs from 15 to 91 patients . Viscosupplementation with hyaluronic acid seems to be a valuable technique for the management of painful OA of the hip that may delay the need for surgical intervention . Further prospective randomised placebo controlled studies are necessary to draw definite conclusions .

B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hyaluronic acid
hyaluronan
injections
viscosupplementation
hip osteoarthritis


Measuring access to primary care appointments : a review of methods Background Patient access to primary care appointments is not routinely measured despite the increasing interest in this aspect of practice activity . The generation of standardised data -LRB- or benchmarks -RRB- for access could inform developments within primary care organisations and act as a quality marker for clinical governance . Logically the setting of targets should be based on a sound system of measurement . The practicalities of developing appropriate measures need debate . Therefore we aimed to search for and compare methods that have been published or are being developed to measure patient access to primary care appointments , with particular focus on finding methods using appointment system data . Background Primary care is under scrutiny along with other public services to improve access to its users . Access in primary care is typically conceptualised as the achievable access to appointments with clinical professionals , although it is not routinely measured in most practices . It is widely recognised that it represents an important dimension in determining the quality of care -LSB- 1,2 -RSB- . Nevertheless , it is also known that waiting times can vary widely in differing localities and countries , from patients being seen the same day to a wait of several weeks . Most primary care organisations have no more than a perception of variable demand and no method of comparing fluctuating levels of access to appointments within or between practices . Measuring patient access could generate useful information for patients , clinicians and practice managers . Demand management initiatives and ways to optimise access could then be audited bearing in mind the impact of such initiatives on opportunity costs -LSB- 3-5 -RSB- . There are potential difficulties , recognised by a recent discussion document -LSB- 6 -RSB- , that have been accentuated by policy and political influences . The NHS Plan in the UK -LSB- 7 -RSB- suggested that patients should have access to primary care services within 48 hours , but the concept lacked sufficient definition . The proposed new GMS contract includes an optional 48 hour target for access to GP appointments . But what exactly should we measure ? Access can be measured at many different interfaces , from the wait for traditional services such as appointments with a clinician to the alternative solutions of nurse triage , nurse led clinics , telephone advice -LRB- including NHS Direct -RRB- or electronic mail responses . Differing interpretation of terms can also cause confusion . How soon for example should a problem that is defined by a patient as urgent be seen in general practice ? The perception of urgent differs between patients themselves -LSB- 8 -RSB- and between doctors and patients . The concept of routine appointments is easier to define and quantify . A proposed measure is the waiting time for the next available routine appointment but this provokes debate about whether this should be practice-based or specified for individual clinicians . If the latter , factors such as part-time working , practitioner popularity , the creation of multiple review appointments are likely to rapidly diminish a clinician 's accessibility . How transparent would a practice want to be about such data and how useful or acceptable would it be to publish information at the clinician level ? -LSB- 9 -RSB- Recognising the complex nature of this issue , we set out to review the literature . Our main aim was to search the international primary care literature for methods that had been , or were being developed to measure access to GP appointments , focusing on measures using appointment system data . Once identified , existing methods -LRB- tools , scales or other instruments -RRB- would be compared , with specific attention given to the type and levels of access they aimed to assess . Method Preliminary searches indicated that this area did not have an extensive or long-standing research literature ; most articles had been published after 1998 . A broad but systematic search process was designed to allow for a poorly indexed publication pool . Medline , PubMed , Clin Psyc and ASSIA were searched for relevant publications between 1990 and 2001 . The following MeSH terms were used : family practice , health service accessibility -LRB- organisation and administration , statistics and numerical data , standards , trends , methods , manpower -RRB- , appointments and schedules -LRB- waiting lists -RRB- , research design , health service needs and demand , weights and measures , quality of healthcare , management audit , patient satisfaction , health service needs and demand . In addition the following keywords were used : general practice , access , appointments -LRB- same day , urgent , routine -RRB- appointment systems , measurement , measures , tools , scales , demand , availability , audit and waiting times . Terms were used both singly and in combination . Title searches were used to increase the sensitivity . All citations and abstracts were appraised for relevance and full articles selected for examination by two researchers independently -LRB- GE and WJ -RRB- . Key authors were contacted directly -LSB- 1,12,16,18,21,25,27 -RSB- and searches conducted on their previous work . Departments of General Practice in Universities in the United Kingdom were also asked to send details of any relevant research . Conference literature -LSB- 21 -RSB- and non-peer reviewed literature obtained from websites was also appraised . Relevant websites were identified using and the search terms health service delivery , access to primary care and general practice . The following sites were reviewed : the Royal College of General Practitioners -LSB- 6 -RSB- , the National Primary Care Research and Development Centre -LSB- 1 -RSB- , The National Primary Care Development Team -LSB- 10 -RSB- and the Centre for Innovation in Primary Care -LSB- 11 -RSB- . Studies or articles were included in the review if they described tools , scales , questionnaires or other methods of measuring actual patient access to appointments . We also included descriptions of methods that were currently being developed in this field , provided they had undertaken pilot studies and had completed one data collection exercise . Articles were excluded if they were purely editorial . Results A total of 1763 citations were initially identified and 38 articles retrieved for detailed assessment from the Pubmed and the Medline searches . Clin Psych and ASSIA searches provided some overlap but no new relevant material . The most helpful pointers to relevant publications were obtained from the website searches and personal communications rather than the traditional search engines . Two broad approaches to the measurement of patient access were identified . Firstly , appointment systems in organisations were analysed in differing ways to provide numerical data and , secondly , patient perceptions -LRB- reports -RRB- of access were evaluated using survey techniques . Methods using appointment system data Table 1 summarises the six identified methods that were based on appointment system data . Table 1 Comparison of methods based on appointments systems to measure access to primary care Third appointment -LSB- 21 -RSB- NEMAS -LSB- 15 -RSB- Ledlow -LSB- 14 -RSB- Access Response Index AROS -LSB- 22 -RSB- Campbell -LSB- 12 -RSB- Kendrick -LSB- 13 -RSB- Measurement The 3rd available routine appointment at 12 midday , one day per week , for every clinician Date of patient call and appointment provided . GP requested and GP allocated . Appointments demanded but not available in US style primary care clinics compared to community clinics . Demand versus availability gap coded into 4 categories . Number of days until next available routine appointment -LRB- with any clinician -RRB- at 4 pm , every working day Number of appointments provided at the beginning of the day and the number still available . Total number of patients seen during the day , noting the number of ` extras ' Number of appointments available at the start of the day and the number of patients seen as ` extras ' at the end of the day Frequency of data collection Once a week Continuous Daily Once a day Twice a day Twice a day Weighted for part time staff Yes No No No No No Named clinician access measured Yes Yes No No No No Data analysis Weekly median score and monthly average Computerised Demand versus availability gap Computer to work out 5 day moving average Data related to practice list size , with rates given per 1000 patients Daily tally Results Weekly snapshot of patient access profile Complete computerised analysis of practice appointment system Feedback reports generated to clinic staff Trends across weekly schedules . Bar charts represent number of appointments offered versus number of patients seen . Start of day appointment availability categorised as low , medium and high Graphical display of extras versus number of free appointments during the day Extent of and reason for use Primary Care Collaborative in England . To inform implementation of advanced access 145 teaching practices Audit US Military Clinic Study 10 practices To inform improvement 19 practices Research Study 1 practice Research Study Co-ordination National Primary Care Development Team Department of General Practice , University of Glasgow . Healthcare Programs Central Michigan University . University department of General Practice University Department of General Practice , Edinburgh . Department of Primary Health Care , University of Southampton . Three methods determine appointment availability and/or the satisfaction of demand on a daily basis but do not measure the days wait for appointments . Campbell measured clinician availability by recording the number of provided appointments at the beginning of each day plus the number of these un-booked at this time -LSB- 12 -RSB- . At the end of the day numbers seen and numbers of extras were counted , and adjusted for practice list size . Kendrick and Kerry recorded the number of available appointments at the beginning of the day and the number of extras seen at the end -LSB- 13 -RSB- . Ledlow also suggested a categoriations system for recording differing levels of unmet daily demand in a military medical service -LSB- 14 -RSB- Three methods measure access as days waited by representing appointment system data . A computerised package , NEMAS -LSB- 15,16 -RSB- enables practices to audit four areas : practice service provision -LRB- including ` appointment availability ' and patient satisfaction -RRB- , chronic disease management , drug monitoring and significant event analysis . The method calculates the mean time waited in days plus the minimum and maximum patient waits . Data can be presented for the whole practice or for individual GPs . Data entry involves using electronic forms to record the date of appointment request , the date the patient was seen , whether it was an elective , forced -LRB- i.e. next available clinician -RRB- or urgent appointment , the clinician requested and the clinician actually consulted . The costs of collecting data has varied considerably depending on which staff member is employed for the task -LSB- 17 -RSB- . Transfer of data from the practice system can be automatic with compatible systems -LRB- personal communication -RRB- . The National Primary Care Development Team is measuring access as part of the adoption of the system of ` Advanced Access ' , a system developed in the USA -LSB- 18,19 -RSB- , as a response to patient waiting times of 4 -- 6 weeks for routine appointments in primary care -LSB- 20 -RSB- . This method collects data on one day per week -LRB- which can vary -RRB- at 12 noon . The number of days to the third available routine appointment for each clinician is recorded and a median figure calculated to represent an access score for the specified week . Over a month , the average of four median values is taken to represent a monthly access score . The third appointment is chosen , rather than the first , in order to negate the effect of sudden cancellations which otherwise could give false impressions of availability if at the moment of measuring there is one sudden cancellation in a schedule that is otherwise booked for several days ahead . The third appointment has been found by trial and improvement to best represent the actual waits involved -LRB- personal communication -RRB- . Embargoed appointments are not included . A system of weighting has been developed -LSB- 21 -RSB- for part time workers to enable their scores to be incorporated . The Access Response Index -LRB- AROS -RRB- was developed as a rapidly calculable measure of organisational access -LSB- 22 -RSB- . This index is derived by counting the number of days until the next available routine appointment , with any clinician , once during every normal working day . The data is recorded at four pm -- a time chosen to avoid the influence of embargoed appointments that many organisations use to maintain urgent same day availability . The results are plotted on a graph , the daily fluctuation represented by the raw data is smoothed to a demonstrate trends by calculating a 5-day moving average -LRB- a data point which is the mean of every successive 5-day group -RRB- . An example of the data produced by this measure is shown in Figure 1 . Figure 1 AROS scores for routine appointment availability -LRB- data from 11 practices -RRB- Methods using patient questionnaires Four patient experience questionnaires were found that contained access assessments and the relevant items are outlined in Table 2 . The General Practice Assessment Survey -LRB- GPAS -RRB- -LSB- 23 -RSB- was developed by the National Primary Care Research and Development Centre , by adapting the Family Practice Assessment Survey -LRB- FPAS -RRB- -LSB- 24 -RSB- . A second survey named Europep -LSB- 25,26 -RSB- has been validated in 10 European countries . Thirdly Baker describes the use of a validated surgery satisfaction questionnaire -LRB- SSQ -RRB- that he developed -LSB- 27,28 -RSB- and finally Grogan 's patient Satisfaction Questionnaire contains two sections that ask about access and appointment availability -LSB- 29 -RSB- . Table 2 Patient survey instruments : items used to determine access perceptions Survey items Response ratings GPAS -LSB- 23 -RSB- 6 . Thinking of times when you want to see a particular doctor : a -RRB- 5 point scale , 1 = same day , 5 = more than 5 days a -RRB- How quickly do you get an appointment ? b -RRB- How do you rate this ? b -RRB- Range from 1 = very poor , 6 = excellent 7 . Thinking of times when you are willing to see any doctor : a -RRB- How quickly do you get an appointment ? 8 . Yes / No / Not applicable / Do n't know b -RRB- . How do you rate this ? 8 . If you need an urgent appointment to see your GP can you normally get one on the same day ? EUROPEP -LSB- 25,26 -RSB- What is your opinion of the general practitioner and/or the practice over the past 12 months with respect to : 5 point scale -LRB- poor to excellent -RRB- 19 -RRB- Getting an appointment to suit you ? 23 -RRB- Providing quick services for urgent health problems ? Baker -LSB- 28 -RSB- 10 -RRB- It can sometimes be difficult to get an appointment with my doctor at this surgery . 5 point agreement scale 14 -RRB- It can be hard to get an appointment for medical care right away . Grogan -LSB- 29 -RSB- 33 -RRB- Getting an appointment at a convenient time is easy . 5 point agreement scale 34 -RRB- Appointments are easy to make whenever I need them . 35 -RRB- It is often difficult to get an appointment with a doctor . 36 -RRB- It is easy to see a doctor of my choice . Discussion Principal findings This review of access measurement reveals the heterogeneous nature of the methods and the lack of any widely accepted conceptualisation of patient access . Identified measures are either practice centred using appointment data or patient orientated via surgery satisfaction questionnaires . It is clear that these two methods represent entirely different aims . It is not possible for episodic patient surveys to provide data that has enough currency or accuracy to inform organisational responses to patient demand . Also demonstrated are inherent problems over definition of terms -- defining what is to be measured and setting of targets . There appear to be three appointment categories over and above emergencies -LRB- which by definition can not be given ` appointments ' -RRB- . Appointments for healthcare could be categorised into urgent , soon , and elective . Urgent appointments are typically seen as requests for same-day consultations . The soon category would fit problems that should be seen within two or three days to prevent escalation or symptom prolongation . Finally , routine or elective appointments suit individuals who value an agreed time window over other factors . The methods that represent patient waiting times for ` routine ' , i.e. elective appointments seem to hold promise as they avoid the definitional problems that surround ` urgent ' appointments , and the different views that patients , clinicians and others have about ` urgency ' . The task of deciding whether to represent access profiles for organisations or for individual clinicians also needs careful consideration . It may be more feasible -LRB- and less threatening -RRB- to routinely measure organisational access , especially if the data is to be used for benchmarking purposes . To attempt to measure access means to obtain meaningful data from a dynamic system that is not always in equilibrium . The access experienced by an individual varies determined by demand , adequate appointment provision , sudden cancellations and block release of held appointments . We have distinguished two approaches to overcome this , either for a full statistical analysis using compatible systems or data retrieval software , or to use a simple snapshot method , deciding whether data-smoothing methods such as daily moving averages or aggregated weekly median scores are the best portrayal of overall access patterns . Strengths and weaknesses of the study Multiple search methods were used to ensure that the breadth of literature and online resources were examined as systematically as possible . The searches proved difficult and reflect the emergent status and the diversity of terms used in this area . We may have overlooked methods developed in other healthcare systems . Implications of the findings to healthcare services and research The lack of a widely agreed measurement method to represent patient access to primary care services will make it impossible for practices to compare their response to patient demand with any degree of certainty . The ` third appointment ' system is the most widely used method and is currently supported by the National Primary Care Development Team in England -LRB- but has no equivalent support in Scotland , Wales or Northern Ireland -RRB- . It is however a relatively complex manipulation of appointment system data , and incorporates individual clinician availability . It seems from first principles that the important requirements of a tool designed to measure a dynamic concept such as patient access is simplicity and ease of regular data collection , so that longitudinal data patterns capable of indicating trends in organisations can be generated rather than data on individual clinician availability . A recent survey compared mechanisms used to manage requests for same day appointments -LSB- 30 -RSB- . Murray 's proposal of doing today 's work today -LRB- Advanced Access -RRB- eliminates appointment categories and the work involved in negotiating urgency by dealing with virtually all demand on the day it arises -LSB- 31 -RSB- . Access is an important determinant of healthcare quality but what are the implications of this approach to the balance of overall quality ? Continuity of care , whilst not important for some problems is desirable for others -LSB- 32,33 -RSB- . Too drastic a shift in favour of access is likely to be at the cost of reduced continuity and a diminution of other services , such as screening and chronic disease management . Measures of organisational quality need to be aware of the dangers of focusing too much on one dimension , and should work towards the creation of measures that balance scores across internal and external requirements -LSB- 34 -RSB- . An effective consultation with a well trained clinician who knows the patient and who has access to a well structured longitudinal record will probably remain a gold standard and the issue of immediate ` access ' should not be elevated above all the other components in this equation . Nevertheless , it seems prudent to develop measures that provide a better understanding of patient access to organisations with similar resources . Measuring the interval to the next available routine appointment , whilst measuring the measurable -LSB- 35 -RSB- will , if a consistent standard can be agreed , provide benchmark data , and mark an important step towards a compendium of methods to assess quality in primary care . Conclusion The two approaches of either using patient questionnaires or appointment system data to measure access are methods that represent entirely different aims . The latter method when used to represent patient waiting times for ` routine ' elective appointments seems to hold promise as a useful tool and this avoids the definitional problems that surround ` urgent ' appointments . The purpose for which the data is being collected needs to be borne in mind and will determine the chosen methods of data retrieval and representation Competing Interests The authors of this article have piloted the AROS Index . Authors ' Contributions Peter Edwards , Melody Emmerson and Glyn Elwyn were involved in the development of the Aros index . Wendy Jones and Glyn Elwyn conducted the Aros pilot study and the literature review with contributions from Adrian Edwards and Richard Hibbs . Pre-publication history The pre-publication history for this paper can be accessed here :

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O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O 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access
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Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria We previously reported on the secretion of Streptomyces mobaraensis transglutaminase by Corynebacterium glutamicum ATCC13869 -LRB- formerly classified as Brevibacterium lactofermentum -RRB- . In the present work , we investigated whether any other coryneform bacteria showed higher productivity than C. glutamicum ATCC13869 . We found that most coryneform species secreted pro-transglutaminase efficiently . Moreover , we confirmed that Corynebacterium ammoniagenes ATCC6872 produced about 2.5 g/l pro-transglutaminase over a 71-h period in a jar fermentor . Our findings suggest that some other coryneform bacteria , especially C. ammoniagenes ATCC6872 , are potential hosts for industrial scale protein production . Introduction The secretion of heterologous proteins into culture medium is a potentially useful method of production -LRB- Choi and Lee 2004 -RRB- , although the amounts obtained through this approach are generally small . Thus , there remains a need to develop an efficient secretion system for industrial proteins . Streptomyces mobaraensis transglutaminase -LRB- TG -RRB- has been used in the food industry to modify proteins -LRB- Yokoyama et al. 2004 -RRB- . It is utilized in binding meat and fish and in gelled food products such as jelly , yogurt , and cheese . Moreover , it has great potential for use in manufacturing the materials found in cosmetics , thermostable microcapsules , and carriers for immobilized enzymes -LRB- Yokoyama et al. 2004 -RRB- . However , so far , an efficient production system for TG has been lacking . A large number of different coryneform bacteria have been isolated from soil , animals , and plants -LRB- Liebl 2005 -RRB- . Some are used in biotechnological production processes ; for example , Corynebacterium glutamicum and Corynebacterium ammoniagenes are well-known industrial producers of amino acids and nucleotides , respectively -LRB- Liebl 2005 -RRB- . C. glutamicum is a Gram-positive , non-sporulating bacterium with about 53.8 % guanine -- cytosine -LRB- GC -RRB- DNA content and its genome has been sequenced -LRB- Ikeda and Nakagawa 2003 ; Kalinowski et al. 2003 -RRB- . This species is extensively used in the industrial production of amino acids , such as glutamate and lysine , which have been applied in human food , animal feed , and pharmaceutical products for several decades -LRB- Hermann 2003 ; Krämer 1994 ; Liebl 2005 -RRB- . However , there had been only few reports concerning heterologous protein secretion in C. glutamicum -LRB- Billman-Jacobe et al. 1995 ; Liebl et al. 1992 ; Salim et al. 1997 -RRB- . Recently , we demonstrated that TG could be efficiently secreted in an active form using a signal peptide derived from a cell-surface protein of coryneform bacteria in C. glutamicum ATCC13869 -LRB- Date et al. 2003 , 2004 ; Kikuchi et al. 2003 -RRB- . We showed that C. glutamicum ATCC13869 could also secrete an active form of human epidermal growth factor , which consists of 53 amino acid residues , including six cysteine residues that form three disulfide bonds -LRB- Date et al. 2006 -RRB- . More recently , it has been shown that the twin-arginine translocation -LRB- Tat -RRB- pathway , which is a recently detected protein secretion pathway , is active in C. glutamicum ATCC13869 and that the Chryseobacterium proteolyticum pro-protein glutaminase , which is not secreted via the Sec pathway in C. glutamicum ATCC13869 , is efficiently secreted by the Tat pathway -LRB- Kikuchi et al. 2006 , 2007 -RRB- . These results demonstrate that protein production using C. glutamicum might be useful on an industrial scale . However , little is known about heterologous protein production by other species of coryneform bacteria . In this report , we show that most other coryneform bacteria also secrete pro-TG efficiently and that C. ammoniagenes ATCC6872 is the best producer species , accumulating about 2.5 g/l pro-TG over a period of 71 h in a jar fermentor . Materials and methods Bacterial strains , plasmids , and culture medium The bacterial strains used are shown in Table 1 . The plasmids were as follows : pPSPTG1 -LRB- Kikuchi et al. 2003 -RRB- , which contains the cspB promoter derived from C. glutamicum ATCC13869 ; the CspA signal peptide sequence derived from a cell-surface protein of C. ammoniagenes ATCC6872 ; and the coding region of pro-TG , derived from S. mobaraensis , and pPKPTG1 -LRB- Kikuchi et al. 2003 -RRB- , in which the signal peptide for pro-TG secretion is replaced by the CspB signal peptide sequence from C. glutamicum ATCC13869 . The coryneform bacteria were aerobically grown in CM2G medium -LRB- Kikuchi et al. 2003 -RRB- at 30 °C in a shaking test tube . Plasmids were introduced by electroporation with a Gene Pulser -LRB- Bio-Rad -RRB- using the standard protocol for Corynebacterium species . For pro-TG production in a test tube culture , we used MMTG medium containing 60 g glucose , 1 g MgSO4 · 7H2O , 30 g -LRB- NH4 -RRB- 2SO4 , 1.5 g KH2PO4 , 0.01 g FeSO4 · 7H2O , 0.01 g MnSO4 · 4H2O , 450 μg thiamine hydrochloride , 450 μg biotin , 0.15 g dl-methionine , and 50 g CaCO3 per l distilled water , adjusted to pH 7.5 -LRB- Kikuchi et al. 2003 -RRB- . Kanamycin -LRB- 25 mg/l -RRB- was added to the culture medium when required . Table 1Pro-TG accumulation by coryneform bacteria carrying pPSPTG1 in MMTG medium at 30 °C for 72 hStrainPro-TG -LRB- mg/l -RRB- Corynebacterium ammoniagenes ATCC6872454Corynebacterium glutamicum ATCC13032147Brevibacterium taipei ATCC13744201Corynebacterium glutamicum -LRB- Micrococcus glutamicus -RRB- ATCC1376166Brevibacterium roseum ATCC13825269Corynebacterium glutamicum -LRB- Brevibacterium flavum -RRB- ATCC13826314Corynebacterium herculis ATCC1386854Corynebacterium glutamicum -LRB- Brevibacterium lactofermentum -RRB- ATCC13869118Corynebacterium acetoacidophilum ATCC13870369Corynebacterium glutamicum -LRB- Brevibacterium divaricatum -RRB- ATCC14020391Brevibacterium saccharolyticum ATCC14066238Brevibacterium immariophilium ATCC14068257Microbacterium ammoniaphilum ATCC15354423Corynebacterium glutamicum -LRB- Corynebacterium lilium -RRB- ATCC15990246Corynebacterium callunae ATCC15991759Brevibacterium thiogenitalis ATCC19240156 Jar fermentation Fermentor growth was carried out in a 1-l jar fermentor -LRB- 300 ml working capacity -RRB- . Glycerol stock suspensions of bacteria were stored at − 80 °C in 20 v/v glycerol . Glycerol stock suspension -LRB- 50 μl -RRB- was inoculated into 50 ml CM2G medium in a 500-ml Sakaguchi flask , and cultured at 27 °C for 24 h. Then , 15 ml of the culture was inoculated into 300 ml MMTG-J medium containing 120 g glucose , 1 g MgSO4 · 7H2O , 30 g -LRB- NH4 -RRB- 2SO4 , 1.5 g KH2PO4 , 0.01 g FeSO4 · 7H2O , 0.01 g MnSO4 · 4H2O , 450 μg thiamine hydrochloride , 450 μg biotin , 0.15 g dl-methionine , and 0.2 g total nitrogen of soybean hydrolyzate per l distilled water , adjusted to pH 7.2 . The jar fermentor operating conditions were as follows : agitation rate = 600 rpm , airflow rate = 1/2 vvm ; temperature = 27 °C . The culture was automatically maintained at pH 7.2 by the controlled addition of ammonia gas with airflow . The glucose concentrations were analyzed using an AS-210 glucose analyzer -LRB- Sakura Seiki , Tokyo , Japan -RRB- , and the optical densities were measured at 625 nm with a DU640 photometer -LRB- Beckman Coulter -RRB- . Protein analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis -LRB- SDS-PAGE -RRB- in a 4 -- 20 % gradient polyacrylamide gel was carried out as described by Laemmli -LRB- 1970 -RRB- , with Coomassie brilliant blue R-250 staining . The secreted pro-TG was measured by reverse-phase high-performance liquid chromatography , using purified native TG as a standard -LRB- Yokoyama et al. 2002 -RRB- . For processing the pro-TG to convert to an active form , the secreted pro-TG in a culture supernatant was incubated with purified SAM-P45 -LRB- Suzuki et al. 1997 -RRB- at 30 °C for 2 h , at a 100:1 ratio of pro-PG to SAM-P45 . The purified SAM-P45 was a gift from Dr. S. Taguchi . The active-form TG was purified according to the method described previously -LRB- Yokoyama et al. 2002 -RRB- . TG activity was measured by the calorimetric hydroxamate procedure using N-carbobenzoxy-L-glutaminylglycine as described previously -LRB- Yokoyama et al. 2002 -RRB- . For determining the N-terminal amino acid sequence of the pro-TG , after separation by SDS-PAGE , proteins were transferred to polyvinylidene difluoride membrane by electroblotting . The membrane carrying the pro-TG was directly subjected to a gas phase protein sequencer -LRB- model PSQ ; Shimadzu , Kyoto , Japan -RRB- equipped with an in-line amino acid analyzer -LRB- model RF-550 ; Shimadzu -RRB- as described previously -LRB- Kikuchi et al. 1997 -RRB- . Results and discussion Secretion of pro-TG by coryneform bacteria using C. ammoniagenes ATCC6872 CspA signal peptide We transformed the bacteria listed in Table 1 with pPSPTG1 , cultured each transformant in MMTG medium at 30 °C for 72 h , and then subjected the supernatants to SDS-PAGE . As shown in Fig. 1 , each transformant could stably secrete the pro-TG , and high levels of pro-TG with the anticipated molecular weight -LRB- 42 kDa -RRB- were detected in the supernatants for all of the bacteria tested -LRB- Table 1 -RRB- . The pro-TG was most efficiently secreted by C. ammoniagenes ATCC6872 , Corynebacterium acetoacidophilum ATCC13870 , C. glutamicum ATCC14020 -LRB- formerly classified as Brevibacterium divaricatum -RRB- , Microbacterium ammoniaphilum ATCC15354 , and Corynebacterium callunae ATCC15991 . Fig. 1SDS-PAGE analysis of pro-TG secretion by coryneform bacteria carrying pPSPTG1 . Each track contained a 10-μl aliquot of supernatant mixed with an equal volume of sample buffer . After electrophoresis , the gel was stained with Coomassie brilliant blue R-250 . aLane 1 , molecular weight markers ; lanes 2 and 3 , B. saccharolyticum ATCC14066 ; lanes 4 and 5 , B. roseum ATCC13825 ; lanes 6 and 7 , B. immariophilium ATCC14068 ; lanes 8 and 9 , C. glutamicum ATCC13826 ; lanes 10 and 11 , C. glutamicum ATCC15990 ; lanes 12 and 13 , C. callunae ATCC15991 ; lanes 14 and 15 , C. herculis ATCC13868 ; lanes 16 and 17 , C. acetoacidophilum ATCC13870 ; lanes 18 , C. ammoniagenes ATCC6872 . bLanes 1 and 2 , C. glutamicum ATCC13761 ; lane 3 , molecular weight markers ; lanes 4 and 5 , C. glutamicum ATCC14020 ; lanes 6 and 7 , M. ammoniaphilum ATCC15354 ; lanes 8 and 9 , B. taipei ATCC13744 ; lanes 10 and 11 , B. thiogenitalis ATCC19240 ; lanes 12 and 13 , C. glutamicum ATCC13869 ; lanes 14 and 15 , C. glutamicum ATCC13032 ; lane 16 , C. ammoniagenes ATCC6872 We tested 16 strains of coryneform bacteria for the secretion of S. mobaraensis pro-TG . As shown in Fig. 1 and Table 1 , all of the bacteria secreted pro-TG . We have previously shown that the C. glutamicum system has two advantages for heterologous protein secretion : a lack of proteolytic activity in C. glutamicum culture supernatants and a small amount of protein secretion by the bacteria themselves -LRB- Date et al. 2006 ; Kikuchi et al. 2003 -RRB- . As a result , the purification of secreted heterologous proteins is relatively easy . From the results shown in Fig. 1 , it is clear that the other coryneform bacteria share these advantages for heterologous protein secretion : their culture supernatants contain little endogenous protein and no degradation of pro-TG is detected in the supernatants . Effect of signal peptide sequence on pro-TG secretion Next , to investigate whether the secretion efficiency depended on the signal peptide sequence , we examined the effect of using the CspA signal peptide from C. ammoniagenes ATCC6872 -LRB- Fig. 2a -RRB- or the CspB signal peptide from C. glutamicum ATCC13869 -LRB- Fig. 2a -RRB- for the pro-TG secretion . C. ammoniagenes ATCC6872 , C. acetoacidophilum ATCC13870 , C. glutamicum ATCC14020 , M. ammoniaphilum ATCC15354 , and C. callunae ATCC15991 could efficiently secrete the pro-TG using the CspA signal peptide from C. ammoniagenes ATCC6872 in this study . These strains and C. glutamicum ATCC13869 were transformed with pPKPTG1 harboring the CspB signal peptide from C. glutamicum ATCC13869 . As shown in Fig. 2b , the substitution of the CspB signal peptide had little effect on pro-TG accumulation in C. acetoacidophilum ATCC13870 , C. glutamicum ATCC14020 , and M. ammoniaphilum ATCC15354 . However , in C. callunae ATCC15991 , the pro-TG accumulation -LRB- 403 mg/l -RRB- was approximately halved -LRB- 746 mg/l -RRB- while in C. ammoniagenes ATCC6872 , it was about 2.8-fold higher . Fig. 2Effect of signal peptide sequence on pro-TG secretion in coryneform bacteria . a Amino acid sequence alignment of C. ammoniagenes CspA signal peptide and C. glutamicum CspB signal peptide using Vector NTI software . Identical and similar residues are shown on black and gray backgrounds , respectively . b Accumulation of secreted pro-TG by coryneform bacteria carrying pPSPTG1 or pPKPTG1 . The solid bars indicate pro-TG accumulation using the CspA signal peptide from C. ammoniagenes ATCC6872 , and the open bars indicate pro-TG accumulation using the CspB signal peptide from C. glutamicum ATCC13869 The amount of pro-TG accumulation by C. ammoniagenes ATCC6872 using the CspB signal peptide from C. glutamicum ATCC13869 was about 2.8-fold higher than with its own CspA signal peptide -LRB- Fig. 2b -RRB- . Interestingly , the difference between the effects of the two signal peptides was the opposite in C. glutamicum ATCC13869 : the amount of pro-TG accumulation using the CspA signal peptide from C. ammoniagenes ATCC6872 was about 1.5-fold higher than with the CspB signal peptide from C. glutamicum ATCC13869 -LRB- Kikuchi et al. 2003 -RRB- . We found the same to be true for all of the heterologous proteins examined so far -LRB- data not shown -RRB- . The reason for the differing effects of these signal peptides is unclear . Cultivation of C. ammoniagenes ATCC6872 carrying pPKPTG1 in a jar fermentor As C. ammoniagenes ATCC6872 carrying pPKPTG1 was the best producer strain in our study , we subjected it to a fermentation test in a jar fermentor under the conditions described in the `` Materials and methods '' section . The MMTG-J medium contained 120 g/l glucose ; after the consumption of glucose for 71 h under these conditions , the optical density at 625 nm reached approximately 140 , and the dry cell concentration was approximately 50 g/l . During the exponential growth phase , little pro-TG accumulated in the culture supernatant ; however , the rate of accumulation increased linearly after entry into the stationary phase , and approximately 2.5 g/l pro-TG was produced -LRB- Fig. 3 -RRB- . The N-terminal amino acid of the secreted pro-TG was Asp , as in native pro-TG -LRB- Kikuchi et al. 2003 -RRB- , demonstrating that the CspB signal peptide had been correctly processed in C. ammoniagenes ATCC6872 . We then incubated the culture supernatant with purified SAM-P45 , which is a subtilisin-like protease from Streptomyces albogriseolus , for 2 h at a 100:1 ratio of pro-TG to SAM-P45 in order to process the pro-domain -LRB- Kikuchi et al. 2003 -RRB- . The pro-TG secreted by C. ammoniagenes ATCC6872 carrying pPKPTG1 was cleaved by purified SAM-P45 to the C-side of 41-Ser of the pro-domain and converted to an active form and the conversion yield of this enzymatic process was approximately 100 % . The specific activity of the purified active-form TG , with additional Phe-Arg-Ala-Pro residues , was similar to that of the native TG -LRB- about 23 U/mg ; Kikuchi et al. 2003 -RRB- . Fig. 3Jar fermentation experiment employing C. ammoniagenes ATCC6872 carrying pPKPTG1 . Growth -LRB- optical density at 625 nm -RRB- , glucose concentration , and pro-TG accumulation are indicated by open circles , open squares , and closed triangles , respectively The results of the fermentation experiments in the 1-l jar fermentors showed that the pro-TG secreted by C. ammoniagenes ATCC6872 was correctly folded . Thus , protein production using C. ammoniagenes ATCC6872 could be useful for industrial scale protein production .

O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

corynebacterium glutamicum
corynebacterium ammoniagenes
transglutaminase
protein secretion