[Pedicle bone grafting in treatment of bone union disorders after femoral neck fracture]. Clinical and radiological assessment of 8 patients operated because of femoral neck pseudoarthrosis or delayed union proved the pedicle bone grafting to be very useful method, especially in young patients. Surgical reconstruction of femoral neck with the use of pedicular graft (Judet--Chacha) resulted in all the patients in absence of pain; satisfactory range of movement has been restored in operated hip. Bone union has been achieved in 7 from the 8 operated patients that is in all the cases of posttraumatic union disorders. However, we failed in total reconstruction of anatomy and the radiographs revealed osteoarthritis of the joint.


[Tactics in management of coccygodynia]. The results of treatment of 160 patients with coccygodynia were analysed. The condition was caused by contusion, fracture or dislocation of coccygeal vertebrae or excess of osteochondral tissue around the coccyx. The patients were treated conservatively and surgically, 56 and 104 respectively. In 94 cases (50,3%) of surgically treated complete recovery was achieved. Surgery should be undertaken in cases not responding to conservative measures.


[The issue of lower limb amputations in vascular diseases]. A series of 363 amputations in 335 patients treated between 1979 and 1988 because of chronic peripheral vascular disease was reviewed. In years from 1979 to 1988 3500 vascular surgical procedures were carried out. In 217 cases (6,2%) subsequent amputation was necessary. Previously, from 1962 to 1978 it had occurred in 8,5%. The authors experience is indicative of limb vascularization as decisive factor in choosing the level of amputation. In high aortoiliac arterial occlusion a vascular procedure is carried out, resulting in good blood supply of the stump allowing lowering the level of amputation and shortening recovery period.


[Stresses in self-compressing plates used for joining bone fragments]. The analysis of the state of stresses in the auto-compression plates after different stages of joining has been carried out. Distinct differences in the values of stresses in the specific zones of plates were detected. The highest level of stress was located near the holes, in the areas where prebending takes place. The main reason of the loss of the ability to carry introduced compressing forces in the joint and possibility of cracks initiation in plates during the operation has been clarified.


[Myocutaneous flap from the tensor fasciae latae in treatment of trochanteric decubitus ulcers]. The paper presents paraplegic patient after L1-L9 subluxation due to the spine injury. Excision of trochanteric decubitus ulcer covered with the tensor fasciae latae myocutaneous flap gave permanent result.


[Avulsion injury of spinal nerve roots in part of the lumbosacral spine]. A case of 40 years old male with left sided avulsion injury of L5, S1 nerve roots due to severe trauma. Diagnosis was confirmed with CT and radiculography. The presence of prolapsed sheath of the avulsed root in radiological examination combined with clinical picture establishes correct diagnosis.


[Sacrum fracture associated with neurologic complications]. A care of the patient, aged 39, who sustained a fracture of the sacrum complicated by compression of the roots L5 and S1 is reported. Surgical treatment on root decompression was performed. Following 9 months neurological signs subsided, but some intermittent low back pain persisted.


[Long-term results of treating histiocytosis X of the spine]. A series of 38 children and juveniles with eosinophilic granuloma located in 44 vertebral bodies was treated. The diagnosis was based on typical radiographic appearance, bone marrow examination and histology in cases where the lesion was surgically resected. Management consisted mainly of immobilization and unloading of the spine for 2-3 years. Since 1972 in order to shorten this period posterior arthrodesis of 3 vertebral bodies in thoracic spine and 2 vertebral bodies in lumbar spine by Albee-Gruca method is performed. Radiotherapy was applied in 1 case. In most of the cases full or partial regeneration of the vertebral body was achieved; the height was restored in 76% on average. The extent of regeneration was higher in younger children and in lumbar and cervical spine possibly because of lordosis in these segments.


[Surgical procedures for bone neoplasms in children]. The treatment of 40 patients with bone tumors have been presented. The primary tumors were located in the following sites: femur (14), tibia (8), fibula (4), humerus (4), scapula (1), clavicle (2), pelvis (5), hand (1). Investigated group were: osteosarcoma (18), Ewing's sarcoma (14), chondrosarcoma (2), fibrosarcoma (1), synovial sarcoma (1), chondroblastoma (4). In the most frequent malignant bone tumors, osteosarcoma and Ewing's sarcoma, unified management was adapted. The treatment was initiated with multidrug chemotherapy and followed by surgery or radiotherapy (Ewing's sarcoma) of the primary site. Surgery was performed in 30 cases: 19 mutilating operations because of the broad local invasion, 11 conservative surgical procedures (limb -- salvage operations). Satisfactory oncological and functional effect can be achieved after limb-salvage surgical procedures in the cases of localized, especially semimalignant bone tumors.


[Remarks concerning diagnostic principles and early treatment of congenital hip dislocation]. Principles of early diagnosis and treatment of congenital dislocation of the hip are presented. The clinical course, complications and results of treatment by Frejka pillow are reported. Indications for surgical treatment in cases of failure by conservative management, as well as in cases of deficient rebuilding of the hip are discussed.


[Ultrasound diagnostics of the spine].

[Ultrasound diagnostics of the spine]. A short delineation of contemporary ultrasound applications, its possibilities and limitations in diagnostics of the spine and related structures is presented. Ultrasound has an established position in diagnosing fetal spinal defects and dysraphic syndromes especially in children. It is also an important method of intraoperative imaging. Ultrasound can be used in diagnosing sciatica and in identification of pathological spondylosis cisterns.


[Traumatic hip dislocation in children]. Three children aged 7, 9 and 11 were treated because of traumatic dislocation of the hip. There was one case of anterior and two cases of posterior iliac dislocation. The reduction was performed under general anaesthesia directly after admission. In two children derotational shoe for 6 weeks and a plaster for 3 weeks in third case were used in after treatment. The unweighing of the limb was maintained for 6 weeks more. Subsequently, the gradual weight bearing was allowed. Radiographic examination of two children 1.5 and 3.75 years after injury revealed normal hip joints. There was no restriction of physical activity of these children.


[Nonspecific osteogenic arthritis of the hip]. Osteogenic arthritis of the hip in children depending on the localization of the primary focus of infection can be classified into three types of femoral, acetabular and pelvic origin. These types differ as to the pathology as well as clinical course of the disease. Diagnostics and treatment is discussed. It was found that improper treatment in the early stage led to the chronic form of the disease. Clinical and radiological picture of osteogenic hip arthritis as well as its treatment is discussed on the basis of 125 cases reviewed. Different sequel to osteogenic hip arthritis depending on pathological origin, stage of the disease and method of treatment were pointed out.


[Evaluation of knee joint function after fracture of the patella]. Clinical assessment of the function of the knee joint after surgically and conservatively treated fracture of the patella was carried out. On the basis of C. Ranawat criteria it was found that regardless the method of treatment applied restriction of the joint function was expressed mainly by limited range of motion and by weakening of the knee extensors. The results of surgical and conservative management are similar. The best results of treatment for fracture of the patella were achieved in patients operated with the use of wire loop-cerclage.


[Radiologic analysis of the patello-femoral joint after treatment of patella fracture]. Anatomical condition and congruence of the patello-femoral joint after differently treated fracture of the patella was assessed on the basis of roentgenometric analysis of radiographs of 39 patients. The best situation was found in surgically treated cases with the use of the wire loop (cerclage), less favorable in patients managed conservatively and the worst in patients operated after Weber.


[Surgical treatment of spastic flatfoot using Gorynski's method]. After failure analysis in so far applied surgical treatment of spastic flatfoot the technique of synthesis of already existing procedures have been offered. Foot alignment is achieved by the reduction of the talus in relation to the calcaneus that is secured by bony graft after Grice and front-rearfoot alignment with Kirschner's wire fixation through calcaneus to talus and secondly cuboid to calcaneus. The muscular balance is provided by elongation of the calcaneal tendon and transposition of peroneal brevis tendon into tendon of tibialis posterior muscle. In the after treatment long cast immobilization is after 3 weeks replaced by walking cast for next 9 weeks. This management proved to be clinically successful for 15 years.


[Evaluation of surgical treatment for spastic flatfood using Gorynski's method]. The assessment of surgical treatment of 100 spastic flatfeet by Goryński method was presented. After average follow up of 7 years restoration of correct shape of the foot was found in 86 cases.


[A trial of ultrasonographic use for evaluating leg lengthening]. Modified de Bastiani method of callotasis with Wagner apparatus was used as a lengthening procedure of 4 femurs and 1 tibia. The ultrasonography was applied to measure the degree of distraction, to determine the eventual axial deviation of the bone ends and to follow up the callus formation in the gap. The obtained results were compared with radiography.


[Apert Syndrome--orthopedic aspects and treatment outcome]. Several years follow up of 3 patients aged from 4 to 14 years with acrocephalosyndactyly of Apert Syndrome type is described. Functional evaluation of the hands was performed before the onset and after completion of treatment. Psychological tests run since the beginning of treatment were analyzed. In all cases remarkable improvement of function and shape was achieved, but progressive bony changes within phalanges negatively influence all over results.


[Torsion and rotation of apical vertebrae and thoracic deformity in idiopathic thoracic scoliosis in computer tomography]. CT examination of 9 patients with primary thoracic right sided scoliosis aged from 13 to 19 years (15 years and 4 months on an average) was carried out. The angle of curvature (Cobb method) ranged from 38 degrees to 99 degrees (68 degrees on an average). Rotation of the apical vertebra was measured and torsion evaluated. Rotation ranged from 5 degrees to 28 degrees (17 degrees on an average). No relation among rotation, angle of curvature and size of the hump was found. The directions of rotation and torsion were opposite. At the apex of curvature due to torsion the end of spinous process was deviated towards the hump but the vertebrae into opposite direction.


[Fracture of both humeral bones after electrocution]. A case of 58-years-old male electrocuted with 220 V/50 Hz voltage is presented. The electrocution resulted in fracture within proximal end of both humeral bones.


[A case of carpal tunnel syndrome caused by an abnormality of the palmaris longus muscle]. The authors present a rare case of carpal tunnel syndrome caused by accessory head of palmaris longus muscle of a soft tissue tumor appearance. Clinical examination was typical for carpal tunnel syndrome. X-ray revealed no bony abnormalities. An ultrasound aided diagnosis, that was finally confirmed intra operatively and by histopathology.


[Principles of forming a diagnosis in orthopedics and orthopedic-trauma]. The rules of writing accurate and profound-diagnoses in orthopedics and traumatology were presented. The most common mistakes were pointed out. An attention was paid to the eponyms. Latin language and correct vocabulary.


[What is research?].

[What is research?]. The issue of research is presented in this paper. It is classified and characterized. The factors that motivate physicians to undertake research are pointed out as well as danger of superficial or even dishonest attitude towards this problem.


[Electrostimulation in treatment of scoliosis]. The efficiency of electrical stimulation in the treatment of idiopathic scoliosis was assessed on the basis of 95 patients reviewed. In 63% of cases an improvement or inhibition of progression of the curvature was achieved. The infantile and juvenile positive therapeutic effect was found in 70% patients, providing the onset of the therapy took place before the tenth year of life. Adolescent scoliosis were affected by this kind of therapy only to the very little extent.


[The value of Sowinski coracoid-plasty for recurrent dislocation of the shoulder]. The value of Sowiński coracoplasty for recurrent, trauma related dislocation of the shoulder was evaluated on the basis of multidirectional clinical, Xray, CT and ultrasound examinations in 15 patients operated on between 18 and 40 years of age. Good results were achieved in 14 cases after 2-14 years follow up. In one case of epileptic patient 4.5 years after surgery traumatic dislocation of the shoulder occurred with a fracture of incorporated bony graft. The authors have proved the coracoplasty performed to be efficient in preventing recurrent dislocation of the shoulder by means of creating new biomedical conditions around the joint. The bony graft by elongation of the coracoid process of ca 35 mm and change in its alignment imposes strong mechanical support for the head of the humerus and indirectly makes the acetabulum deeper. The graft located above anterior rim of the acetabulum increases the tension of subscapular muscle and stabilizes the capsule thus aiding to the stability of the joint. In 4 cases some adhesions between the graft and subscapularis muscle have slightly restricted external sotation of the shoulder.


[Use of arthroscopy for knee joint diagnosis in children]. Diagnostic arthroscopy in 25 patients below 18 years of age was performed. Clinical diagnosis was confirmed in 48% of cases. The differences occurred mainly in diagnoses of menisceal injuries. In 10 cases (40%) arthroscopy indicated no need for further surgical treatment. In the author's view more frequent use of arthroscopy in diagnosing knee problems in children is justified.


[Evaluation of long term results of treating patellar fractures with polyester sutures]. The late results of patellar fractures treatment with polyester suture in 24 patients with an average follow up of 8.1 year was presented. The method proved to be useful in transverse fractures of the central and distal part od the patella as well as in multi-fragmented ones. The evaluation of patello-femoral arthritic changes has remarkably influenced the overall clinical result. The displacement of 2 mm or more within articular surface existing postoperatively implicates bad prognosis, polyester suture is mechanically strong and requires no additional operation to remove the material used for fixation.


[Principles of assessing the treatment of knee instability]. Multifactorial system of assessment of the results of treatment of the injured knee is presented. It comprises of clinical evaluation, modified Lysholm scale, set of fitness tests and patient's opinion.


[Results of treatment for anterolateral knee instability using Ellison's operation]. In 7 patients with anterolateral knee instability Ellison procedure was performed. After an average follow up of 56.7 months excellent and good results were found in 3 cases using functional assessment scale, in 6 cases according to patients opinions and in all cases submitted to fitness tests. The deterioration of function in operated knees observed on average 3 years after surgery was due to distraction of transplanted structures.


[Results of suturing peripheral meniscal tears]. Since numerous complications after meniscectomy, in cases of peripheral tear of the meniscus an average follow up of 32.2 months after meniscal suture was presented. Excellent and good results in 90 percent of cases reviewed prove this procedure to be efficient.


[Modification of the surgical technique for one stage peri-talar correction of congenital clubfoot]. The surgical technique for one-stage peri-talar correction of congenital clubfoot was modified by the author who introduced an accessory lateral approach to the tarsus. It enables true elimination of the soft tissue contracture maintaining deformity of the foot.


[Technique of surgical treatment for congenital clubfoot using complete subtalar release by the Cincinnati approach]. The technique of congenital clubfoot surgical treatment based on independently described method by Mc Kay and Simons is delineated. We have modified skin incision making it ca 2 cm above the calcanean tuber in order to facilitate an access for elongation of the calcaneal tendon. In some cases the procedure was supplemented with cuneonavicular and first tarsal-metatarsal capsulotomy and tibialis anterior muscle tendon transfer to the dorsum of the first metatarsal bone.


[Coexistent paralytic drop foot and gluteal fibrosis after intramuscular infections--therapeutic implications]. Six children with paralytic drop foot, which developed after intramuscular injections and who had co-existing gluteal fibrosis are presented in this study. Paralytic drop foot was diagnosed on an average of 5.5 months after intra-gluteal injections. This was the major therapeutic problem. The diagnosis of gluteal fibrosis was made on an average only 3 years and 7 months later. In 3 cases the external rotation and abduction contracture of the extremity in the hip joint, caused by gluteal fibrosis, with active plantar flexors and supinators of the foot could contribute to the recurrence of the equinovarus deformity of the surgically corrected foot.


[Morphological evaluation of fascia lata tissue implantation for chondral defects in the knee joint of sheep under active motion and load]. Experiments carried out on 64 knees of 32 Lein sheep with superficial and deep chondral defects of the patella and femoral condyles covered by autogenic fascia lata grafts indicate that under active motion and load the grafted tissue after 6 weeks is thinner, fragile and lustreless. Quantitative and qualitative deterioration of collagen fibers, almost complete atrophy of elastic fibers and increase in number of cells and vessels is observed under microscope. After 9 weeks remodeling of the fascia occurs which thickens and increases in number of fascicular collagen fibres, after 12 weeks the foci of neochondrogenesis can be found. After 26 weeks a new hyaline like cartilage is generated surrounded by connective tissue containing fascicular collagen and elastic fibers. At this point remodeling is not yet completed.


[Densitometric bone mineral analysis in the radial metaphysis and its use in assessing fluoride changes]. Theoretical foundations and practical application of densitometric bone mineral analysis is presented. The radiographs of forearm and aluminum wedge were compared. Bone mineral content (BMC) was calculated on the basis of densitometric measurements as well as bone and soft tissue thickness evaluation. The method error determined on the basis of tests is 4.89%. The measurements were taken in 754 fluoride exposed workers and in the control group of 60. A distinctive decrease of BMC with age was found in both groups. In older fluoride exposed workers this process was slowed down. Minor increase of BMC was found in workers exposed to fluoride to the higher degree.


[Intraoperative diagnostic isotopic bone scanning]. Advantages of intraoperative bone scintigraphy are presented on the basis of surgical treatment of 50 osteomyelitis patients. Tc99m was given either intravenously or into the fistula and the radiation was measured by a set of scintillating probe and radiometer. Small size, simple handling and quick measurement make this set extremely useful for intraoperative diagnostics contributing to complete removal of infected tissue from the operating site. This method requires only one tenth of usual dose of radioisotope what must be recognized as an additional advantage.


[Long term treatment results of open malleolar fractures]. Twenty four cases of open malleolar fractures with minimal follow up of 8 years after surgery were reviewed. Neglecting to repair ligament injury and in 2 cases infection gave bad results of treatment in 6 cases.


[Correction stabilizing unit for closed reduction of calf bones before their union with external fixators]. The author presents construction and function principles of the correction stabilizing unit serving as a device reducing fractured tibial bone before its fixation. This unit ensures as well the stability of bone fragments throughout surgical fixation.


[Fracture of the acetabular cup as an early complication of total hip replacement]. The authors have presented 12 patients with non traumatic fracture of the acetabular cup of the Weller hip prosthesis. All the fractures have occurred 10 to 40 months following hip arthroplasty 9 of the broken sockets have been removed at the time of revision arthroplasty. They showed marked destruction with multiple fragmentation of the polethylen. The area of the destruction was surrounded by active tissue reaction described as resorbtion granulation. The remowed fragments of the polethylen have been examined physicochemically and showed marked increase in the degree of crystalisation. The authors have come to the conclusion that the main reason for the failure of the presented series of the polietylene cups was defect of the material used in their production.


[Long term treatment results for acute hematogenous osteomyelitis of the radius]. The 37 years follow up of hematogenous osteomyelitis of the radius with the whole shaft sequestrum is presented. The treatment consisted of surgical removal of the sequestrum and administration of both local and systemic antibiotics. One year after surgery and subsiding of the flare the fragments were united with an autogenic bone graft. After trauma related fracture the incorporated graft has united in usual period of time. Clinical and radiological examination carried out after 37 years revealed full remodeling of the shaft with features of growth disturbances of the radius.


[Surgical treatment of neglected trauma related dislocations of the cervical vertebrae]. The series of 67 patients operated on for inveterate (more than 3 weeks since injury) trauma related dislocation of the cervical vertebrae between 1979 and 1990 is presented. Up to 3 months after injury an attempt to reduce the dislocation by means of the cranial traction has been undertaken. In cases of failure surgical reduction of the dislocation was performed. If this was unsuccessful in some cases the vertebral body narrowing spinal canal was removed. In 60% patients presenting neurological deficits a remarkable improvement was -- achieved what proves the surgical treatment to be useful even several months after injury.


[Late results of surgical treatment of infantile idiopathic scoliosis by Harrington's method]. The late results of surgical treatment of infantile scoliosis by the Harrington method have been presented in 43 patients. The patients age at surgical treatment ranged from 11 to 21 years (14.1 years in the average). The patients age at final follow up examination ranged from 18 to 34 years (25.4 years on the average). The curvature angle at operation was 102.1 degrees according to Cobb on the average. 43.7 per cent of curvature was corrected. Although an improvement of posture was present in all the patients, only 51 per cent of the patients, had good posture. After the treatment 81 per cent of the patients worked.


[Remarks on treatment of unstable fractures of the thoracolumbar spine]. An evaluation of treatment results of unstable spine fractures using the Harrington instruments in 37 patients has been performed. The described method can result in anatomical restoration of the vertebral canal. If vertebral canal lumen is not restored, anterior decompression should be performed making possible removal of posteriorly dislocated bone fragments. The earlier anterior decompression, the greater chance of improvement of neurological conditions. The described procedure is especially useful in fractures with partial palsy. After treatment 70 per cent of the patients with partial palsy resumed their normal lives.


[Attempts at hierarchic classification of radio-morphologic changes of the lumbar spine in screening examinations]. An objective evaluation of condition of the spine in candidates for study and employment in continuous and excessive loading of the lumbar spine is difficult because of the lack of suitable criteria classifying pathomorphological changes found on X-ray examination. In order to fill this gap, the authors analyzed 85 X-ray pictures of the lumbar spine of candidates within the preliminary screening examinations. A 5-points-scale considering the clinical value of individual deviations in lumbar spine structure was accepted. On the basis of that research the groups of the examined were determined: able to physical work (0-3 points), admitted on condition (4 points) and disqualified (5 points and over). On the basis of this model 57.6 per cent of the candidates were disqualified and only 25.9 per cent were admitted to study and work with no conditions.


[Spinal fracture in the course of spondylarthritis ankylopoietica (Bechterew disease)]. In 1965-1986 fifty-six patients were treated for fracture of the spine in the course of spondyloarthritis ankylopoetica. In 47 patients the cervical spine was injured. In 53 patients concomitant neurological symptoms were observed. Total or partial palsy were dominant in injuries of the cervical spine (72 per cent). A marked neurological improvement was achieved in the course of treatment in most of the patients. The authors recommend non-surgical treatment.


[Current state of knowledge about the application of cryotherapy for treatment of musculoskeletal diseases]. On the basis of literature data and his own investigations, the author discusses differential reactions of the human organism to cooling, the varieties of cryotherapeutic procedures, their therapeutic efficacy and current views on their mechanisms of action, and contraindications to their application. The present data indicate that the reflexotherapeutic mechanism plays a fundamental role in the analgetic action of cryotherapeutic procedures. The transient antiinflammatory influence of a single cryotherapeutic procedure does not undergo any additive effect during repetitive application of cooling. Regarding the facilitation of the kinesitherapy, cryotherapy appears a valuable means in the symptomatic management of inflammatory and noninflammatory diseases of the locomotor system.


[Surgical treatment of residual congenital dysplasia with dislocation of the hip joint after treatment with the Frejka pillow]. The characteristics of patients and the radiological condition of hip joints that required surgery because of residual dysplasia in the course of growth after treatment by the Frejka pillow have been presented. That treatment was employed in 4 per cent of the treated hips. The operations consisted in inspection of the joint, transiliac osteotomy after Dega with subtrochanteric osteotomy with detorsion of the femur, of the same type of operation without opening of the joint, performance of only transiliac osteotomy or only subtrochanteric osteotomy. Because late evaluation at over 10 years of life revealed 5 per cent of hips with poor rebuilding that should be treated surgically, it has been determined that the total number of cases requiring surgery after treatment by the Frejka pillow is 9 per cent.


[Aseptic necrosis of ischiopubic synchondrosis (osteochondrosis ischiopubica) as the cause of diagnostic difficulties in pathology if the infantile hip]. Two cases of aseptic osteochondrosis ischiopubica in a 6-years-old girl and a 10-years-old boy have been presented. In the described cases the radiological and clinical picture was the reason of diagnostic difficulties suggesting diagnosis of tumor and hematogenic osteitis.


[Early results of leg bone lengthening using epiphysis-metaphysis distraction]. The methods and results of elongation of the lower extremity by epiphysis-metaphysis distraction obtained at treatment of 13 children aged from 4 to 15 years, with abbreviation of 30-80 mm have been presented. In 11 children full elongation of the abbreviation of 30-57 mm was obtained. In two children full elongation was not obtained.


[Injury of the acromial end of the clavicle imitating dislocation of the acromio-clavicular joint]. Two patients have been presented: a 15-years-old boy with rupture of the clavicular-coracoid ligaments as in total acromioclavicular dislocation, in whom dislocation of that joint did not occur but acromial epiphysiolysis of the clavicle (this patient, was successfully treated surgically) and 5-years-old boy with strain of the clavicular-coracoid ligaments and fracture of the acromial end of the clavicle; he was treated conservatively with remaining of angular dislocation of the distal fragment. The hope for rebuilding of the axis was fulfilled in that child. The anatomical and functional result in both patients is good.


[A self-constructed device for reduction and stabilization of multi-fragmented fractures of the patella. Technical data and analysis of clinical material]. The author has presented the construction and use of his own device for reduction and stabilisation of multi-fragment fractures of the patella ("patella-clamp"). The device was examined in action first on cadavers and then introduced to clinical use. The use of the "patella-clamp" made possible to obtain better results in surgical treatment of multi-fragment fractures of the patella.


[Examination the extent of flap rotation from the medial head of the gastrocnemius muscle]. The authors evaluated various methods of range lengthening of muscular and cutaneous--muscular flaps from the medial head of the gastrocnemius muscle on 18 amputated lower extremities and section specimens. It has been found that lengthening of flaps size by Cheng and transformation of the muscular and cutaneous--muscular flap into the island flap produced marked lengthening of flap arch. The authors believe that incisions of the perimysium can not be recommended as a method of lengthening of flaps range.


[Free cutaneous-muscle flap from the latissimus dorsi muscle]. Free cutaneous--muscular flap from the latissimus dorsi muscle was used to cover extensive posttraumatic skin defect of the distal tibia in a patient with lesion of the anterior tibial artery. The technique of flap transfer and vascular anastomosis has been presented. Full healing was achieved.


[Arthrodesis of the hip joint as a treatment of degenerative changes--anachronism or an alternative for endoprosthetic arthroplasty?]. On the basis of the observation of 22 patients with arthrodesis of the hip (the average age -- 34 years), the value of the method in the treatment of osteoarthritis that joint has been discussed. The comparison of intraarticular arthrodesis with stable compression with pelvis osteotomy and stabilization by the "cobra" plate has proved that the second method is better because of elimination of immobilization by a plaster cast and low percentage of complications of union. The clinical analysis of the majority of the patients has not revealed negative influence of arthrodesis on the other hip, the lumbar spine and the knee. Arthrodesis of the hip has improved walking efficiency and ability to work in 17 from 22 patients. Very good and good results have been obtained in 16 patients, fair in 3, and poor in 3.


[Disorders of lipid metabolism in aseptic necrosis of the femoral head in adults]. 14 cases of aseptic necrosis of the femoral head, in which disorders of lipid metabolism were found, have been presented. Literature on aseptic necrosis qualities lipid disorders as agents of risk. In the presented cases, lipid disorders have indicated occurrence of the IV type of hyperlipoproteinemia according to Fredrickson.


[Clinical evaluation of the usefulness of knee arthroscopy]. The authors have analyzed the results of 570 cases of knee arthroscopy performed in 1974-1987, which included 441 diagnostic and 129 operative arthroscopies. The greatest diagnostic group consisted from 163 cases of suspicion of meniscus injury. Chondromalacia of the patella was the reason for arthroscopy in 53 cases. The importance of joint clinical and arthroscopic examination has been stressed.


[Radiologic examination for diagnosis of intraarticular knee injuries]. A set of 4 projections of radiological examination performed on patients with intraarticular injuries of the knee has been presented, namely: 1) the a-p view of the knee in the standing position, 2) the 1-1 view with 30 degrees of flexion in the lying position, 3) the axial view of patellae, 30 degrees of flexion of the knee, standing position, 4) the tunnel view of the knee joint. The significant role of radiological examination, beside clinical examination and arthroscopy in diagnosis of these injuries has been stressed. Clinical practice has proved great usefulness of the performed projections.


[Results of treating recurrent dislocation of the patella by the Garlick-Salamon method]. The Garlicki-Salamon method consists in isolation of the patella ligaments, loosening of the lateral pulleys, medial and downward transposition of the patella ligament insertion, wrapping of the medial joint capsule, and transfer of the m. vastus medialis insertion to the medial-upper quadrant of the patella. The late follow-up (at least 5 years of observation) was carried on in 26 knees. 4 results were excellent, 10 -- good, 8 -- fair, 4 -- poor. In nine cases (35 per cent) development osteoarthritis of the patello-femoral joint was found. Recurrence of dislocation was found in 4 cases.


[The possibility of using carbon material in surgery of joints]. The experiments consisting in replacement of a dissected anterior cruciate ligament of the knee in rabbits with a bundle of parallel carbon fibers and a braided carbon covered with animal collagen lyophilized dura mater have been performed. Similar experiments have been performed on dogs. The lateral ligaments of the knee were also restored, longitudinally cut menisci and the Achilles tendon were sutured. The produced carbon plate and metal screws were used to fix bone fracture. The positive results of the experiments on animals with the use of carbon fibers of Polish make have proved the possibility of their employment for periarticular ligament reconstruction, suturing, Achilles tendon injuries and juxta capsular meniscus rupture. The union of bone fracture with a composite carbon plate has shown the superiority of "carbon" osteosynthesis over "metal" one. It is believed that carbon materials will be used clinically more widely.


[Osteitis and reconstruction of shaft defects of long bones]. The results of treatment of 18 patients with post-inflammatory shaft defects of the long bones within the femur, the tibia, and the humerus have been presented. The defect ranged from 2 to 4 cm in most of the cases; it was 8 cm in one case, 10 cm in two cases and over 20 cm in two cases. The treatment method consisted in active, multidirectional surgical procedure overcoming the inflammatory process and changing the "infected" case into a "clean" one making possible to use the generally accepted treatment methods. Healing of grafts and bone union was obtained in all 18 cases, marked restriction of movement in the knee joint was found in 4 patients, and recurrence of the inflammatory process of mild clinical course was found in 4 cases.


["Taurolin-Gel" and Taurolin-Trockengel" in treatment of osseous tissue inflammation]. The activity and principles of administration of Taurolin have been presented. That drug was used in 15 patients with chronic osteitis. Healing by first intention was obtained in 10 patients, necrosis of the skin occurred in 3 patients, recurrence of inflammation was found in one patient in whom accessory sequestroctomy was performed in the last case of osteitis and arteriosclerosis healing was obtained by second intention. The clinical observation of 15 patients has revealed: a) irritating influence of Taurolin on the soft tissue, b) increased phase of secretion of exudate since the 5 day after surgery, c) bactericidal influence of the drug. Sterilization of the focus occurred at 2 to 3 weeks after surgery. The use of Taurolin requires experience, complete abiding by the principles of use of this drug and equipment with a set of Charrier's drains. The authors are carrying on further observation of the use of Taurolin in the treatment of osteitis.


[A remark on diagnosis of osteogenic sarcoma in a rare location]. Two cases of osteogenic sarcoma localized in the femoral neck have been presented. As it has resulted from literature, such a localization is very rare and it matches great diagnostic difficulties, mainly caused by absence of typical periarticular reactions and ambiguous X-ray pictures. Angiography and computerized tomography are helpful in diagnosis of the type of tumor. Histopathological examination is decisive.


[Composite antebrachial island flap].

[Composite antebrachial island flap]. Two cases of antebrachial island flap including the radial bone have been presented. In the first patient it was used to fill infected defect of skin and the 2nd metacarpal bone. In the second patient, the operation was indicated by shortening (2 cm) of the 3rd metacarpal bone after its intraarticular fracture. In both patients, the bone and skin grafts healed.


[Activities of the podiatric group in the Lublin orthopedic departments under the direction of Professor Stanisław Piatkowski]. The development of podology in the Lublin society as well as the personal achievements of prof. Stanisław Piatkowski have been presented. The originated in the 60-ies activities concerning podology have included scientific research, cooperation with shoe industry institutions, and wide prevention of the foot health in the Lublin region.


[Radiologic and clinical evaluation of the wrist in some movement disorders of children]. The wrist angle in X-ray pictures of the hand in 47 children, aged 6-16 years, treated for bone growth disorders, was evaluated. The obtained results were compared with the wrist angle in normal children. The increase of the wrist angle was observed in deformations: dwarfism, gargoylism and dysplasia multiplex, and the decrease of this angle in the Turner syndrome. The range of passive and active movements of the wrist was evaluated. Passive and active mobility of the wrist (especially the radial and the ulnar deviations) was smaller in the cases of increased wrist angle.


[Surgical treatment of Smith and Barton type fractures]. The method and the results of surgical treatment of Smith and Barton fracture of the distal radial epiphysis has been presented in 10 patients. Dislocation of fragments is reduced after exposure of the palmar side of the distal radial bone. Stabilization is secured by a metal plate, which supports the epiphysis and is fixed to the proximal fragment by screws. It prevents from redislocation of fragments and helps to recover full activity of the hand, which was obtained in 8 patients. Delayed reduction or stabilization of fragments by Kirschner wires in 2 persons has failed.


[Specific character of movement disorders in children studying string instruments]. Orthopaedic examinations were carried out in 242 children studying string instruments. Various indispositions and deformations of the motor organs were found in 31 children. It has been demonstrated that they were mainly the result of technical errors in holding of the instrument, which caused overloading of the upper extremities and the spine.


[The role of static-dynamic factors in genesis of femoral head and neck deformation in dysplastic hips. A physical-mathematical interpretation ]. The clinical material for a physical-mathematical analysis of femoral head and neck deformations in subluxated and luxated hips consisted of 1.574 case histories of children operated on in 1954-1988. In that material 21.9 per cent of the children had preoperative growth disorders of the head, and in the half of the cases also the femoral neck was involved. In 10.79 per cent of the children pre- and postoperative trophic disorders were also observed. On the basis of the physical-mathematical analysis of static-dynamic factors of the hip, it has been determined that the disordered shape of the femoral head and neck is most of all the result of defective formation of the roof. The oblique roof changes the static and dynamic forces acting on the femoral head and neck in such a way that large overloading of the growth zones of the medial side occurs and is followed by development of proximal femur deformation.


[The protease-antiprotease system in pathogenesis of transient arthritis of a single joint in children]. In 35 patients with transient arthritis of a single joint, levels of alpha-1-antitrypsin were determined in the serum in the acute stage of the disease, the recovery period, and the control group. The results were compared. Determination was that the results of examination have indicated a markedly increased level of carried out by the use of M-Partigen, immunodiffusive plates by the Mancini me-alpha-1-antitrypsin in the serum in children in the acute period of this disease and a significant decrease of its level in the period of subsidence of the clinical symptoms in comparison with the control group. The changes in the level of this enzyme indicate disturbance of protease-antiprotease balance in pathogenesis of this disease.


[Ultrasonography in transient hip synovitis in children]. Ultrasonography was performed in 10 children with the symptoms of transient hip synovitis. In all the patients inflation of the capsule was found. At the following examinations subsidence of intraarticular exudate was observed.


[The influence of geometry of the total hip endoprosthesis on late functional results]. The influence of some parameters of incorrect position of elements of an endoprosthesis of the hip on the late functional result of 61 endoprosthetic arthroplasties have been analyzed. The authors believe that the group of several factors of incorrect mounting of elements of an endoprosthesis is usually the cause of deterioration of treatment results due to occurrence of clinical and radiological symptoms of loosening of the endoprosthesis.


[Functional result and telemetric evaluation of gait after total endoprosthetic arthroplasty of the hip]. The late functional results of one-side endoprosthetic arthroplasty in 35 patients, supplemented with telemetric examination of temporal parameters of gait, have been presented. The marked correlation of the obtained results of telemetric examination with clinical and radiological state of the operated on hip has been found. The authors stress the favorable and rational effect of the cane in unloading of the inefficient lower extremity.


[Partial patellectomy in treatment of fresh fractures of the patella]. The late results of partial patellectomy in treatment of fracture of the apex or the 1/3 of the peripheral patella have been presented. Fourteen patients were evaluated. Attention was paid to the dependence of the result on the correct surgical technique. Full painless range of movements in the operated on knee was achieved in 7 persons. In the other 7 patients, extension of the knee was full but flexion was moderately limited. Periodical pain occurs in 3 persons.


[Resection of the patella and plasty of the knee by the de la Caffiniere method]. The surgical technique of patellectomy with preservation of continuity of the extension apparatus of the knee by the de la Caffinière method and the early results of treatment in 8 operated on patients have been presented. The indications for surgery were: posttraumatic stiffness of the knee in extension in 4 patients, patello-femoral arthrosis with preserved wide range of movement in 3 patients, fresh multi-fragmented fracture of the patella without rupture of the extension apparatus in one patient. The good treatment result was obtained in 6 patients (3 treated for patello-femoral arthrosis, 1 for fresh fracture of the patella, and 2 for stiffness of the knee in extension). The poor results in the other 2 patients operated on or stiffness of the knee in extension were due to large damage of the cartilaginous surface of the condyles and extensive intra- and periarticular adhesions.


[Characteristics and diagnosis of neoplastic metastasis to bones]. On the basis of the analysis of 156 hospitalized patients, the most important traits differentiating metastasis of various organs to the bones have been presented. It has been found that the bones are most frequently invaded by kidney cancer, somewhat less frequently by breast cancer and the bronchus cancer and markedly more rarely by cancer of other organs. The types of metastasis expansion in the bones were determined radiologically: the most frequent--osteolytic, less frequent--mixed, and the osteoplastic type (prostate cancer, gall-bladder cancer, and pancreas cancer). Metastasis is situated most often in the spine and the femur. The authors have also presented the tactics of diagnosis of metastasis by using data from anamnesis, clinical and radiological examination and directed specialist examinations, for instance arteriography of the kidneys at suspicion of kidney cancer. In spite of complex diagnostics the source of metastasis was not found in over a dozen of patients.


[Outcome of treating solitary osseous cyst with acetate methylprednisolone injections]. Among 24 children with solitary osseous cyst of various localization treated by local methyl prednisolone injections, 20 children had excellent and good results. The radiological criteria of evaluation were as follows: full rebuilding of the bone in the site of cyst--excellent result, irregular trabecular structure or widening of the bone remain--good result, small radiological translucency with simultaneous full rebuilding of width of the cortical bone, securing proper mechanical resistance--fair result, absence of radiological traits of rebuilding--poor result. In each patient, 4-6 instillations were performed every 3 months. Subsidence of local compression and auscultative pain, deliberation of balloon inflation of the metaphysis, normalization of width of the cortical bone, rebuilding of the spongy bone and "going away" of the epiphyseal cartilage from the cyst have been observed.


[History of functional treatment of fractures]. A concise history of fracture treatment from ancient times to XIX century, when the old rules of management started to be modified are presented at the beginning. An isometric traction methods of treatment as well as isotonic ones both indirect and direct have been discussed. The shortcomings of fraction treatment induced application of functional treatment that was a combination of the traction and joint movements assisted by the mechanical devices. The author indicates that functional treatment methods were not known to polish orthopaedic surgeons up to the seventies. This empty space was filled only in the last decade. From historical analysis author concludes that external fixation is the future of fractures treatment.


[Axial spondylometer--a device for measurement of the configuration and mobility of the spine in three planes]. A method of triplanar assessment of the configuration and mobility of the spine with the use of the original axial spondylometer is described. The device allows to present mobility in the plane of two perpendicular axes X and Y in new categories: elasticity, susceptibility and deshaping of the spine.


[Spondylometric assessment of the configuration and flexion-extension mobility of the spine in students]. An original method of assessment of the configuration and mobility of the spine in the sagittal plane with the use of axial spondylometer allowed reproducible and more objective evaluation of the spinal movement then previously employed methods. A qualitatively new assessment of the spinal mobility was achieved due to the introduction of mathematical indicators of elasticity, susceptibility and reshaping of the spine. The evaluation of the spinal range of motion in 167 students indicated prominent sexual dimorphism. The elasticity and flexion reshaping indicators in males exceeded of 17.6% and 21.6% respectively the females ones. The circular mobility of the spine greater then its distensibility is characteristic for males. In both sexes flexion surpasses extension of the spine from 157% to 215%.


[Trauma related subtalar dislocation of the foot]. Eight cases of subtalar foot dislocation (5 cases of medial displacement, 2 lateral and 1 very rare anterior one) has been presented. On the basis of authors experience and literature the mechanism and the principles of diagnostics and treatment of this injury has been discussed.


[Early results of surgical treatment for congenital dislocation of the hip by the Degi method in young children]. The early results of surgical treatment of congenital dislocation of the hip by complex Dega method in children aged from 18 to 30 months has been presented. The reduction of the hip after all the obstacles and the elements of dysplasia has been removed was very stable. Derotation osteotomy of the femur in the hips of less than 50 degrees anteversion was not necessary. Avascular necrosis after surgical treatment alone was not observed, however its presence after previous conservative management worsened the results after the operation.


[Treatment of the deformities associated with hypoplasia or agenesis of the sacral bone]. On the basis of follow-up of 5 children treated because of the deformities associated with sacral agenesis or hypoplasia as well as on the literature the authors came to the conclusion that congenital dislocation of the hip, clubfoot and flatfoot are the most common. The aim of the treatment is to prepare the child for walking before the end of the first year of life. Since the deformities mentioned are resistant to conservative treatment they require a very sophisticated surgery with the constant adjustment of the operating plan in every stage of the management. The type of hip surgery should be selected individually.


[A case of sacral agenesis coexisting with Goldenhar syndrome]. A case of two coexisting embryopathies has been described. The deformities within the sacral bone and systemic anomalies led to the diagnosis of sacral agenesis; Goldenhar syndrome has been differentiated with other disturbances of branchial arches formation.


[Chronic rotational dislocation in the lateral atlanto-axial joint in a seven year old girl]. A case of inveterate, rotational dislocation in the atlanto-axial joint type III in 7 year old girl hit by a car was presented. Direct skull traction was applied resulting in complete reduction and regaining of the full range of motion in the cervical spine.


[Mechanical properties of tissue generated as a result of fascia lata grafted into an articular cartilage defect of the knee joint (experimental observations)]. In 64 sheep knees crushing strength and elastic recovery of the tissue generated on the basis of fascia lata grafted into articular cartilage defect in the knee after active motion and load has been assessed. The newly generated tissue is characterized by increasing in time thickness, deformability and elastic recovery the greater the deeper the defect was. Primarily, the new tissue is able to carry smaller loads but after 26 weeks is capable of carrying much greater loads then the cartilage; it has also better plastic and elastic properties.


[Angiosarcoma of the femur following chronic osteomyelitis]. Available literature concerning neoplastic metaplasia of the bone undergoing chronic osteomyelitis has been cited. The treatment of 62 years old patient with lasting 47 years chronic osteomyelitis of the femur after shotgun wound was presented in details. The diagnosis of angiosarcoma following chronic osteomyelitis has been established histologically. The extremity has been amputated with good result--no metastasis or recurrence was found during follow-up.


[Mineral changes occurring with age in the intervertebral disks of L4-L5 vertebrae]. The results of mineralogical examination of 26 L4-L5 intervertebral discs taken from cadavers has been presented. Radiology did not reveal marked osteoarthritic changes at this level. The age of the individuals at the fatal accident ranged from 4 to 87 years (mean 41.2). -- Scanning microscopy, atom absorbtion spectroscopy and roentgen microscopy was used in the examination. Two age related phenomena were discovered; demineralisation of the nucleus pulposus and mineralisation of the anulus fibrosus. Two types of mineralisation were found; the microscopic one visible as granules of different shape and size consisting of crystalline hydroxyapatite aggregates and the one detectable exclusively by sensitive chemical methods as an increase of Ca and P content and disturbance of the remaining elements content in biological structures.


[Mechanism of articular fracture of the calcaneus in experimental, pathological, roentgenographic and computerized tomography examination]. The experiments on amputated extremities, pathological examination as well as plain and computerized radiology confirmed existence of two basic types of calcaneal articular fractures: tongue type and crush type. An accurate anatomy of the experimentally fractured os calcis could be restored only after Westhues. An articular surface in crush fracture could be restored only in open arthrotomy. Computerized tomography revealed the type of the fracture and the degree of calcaneal destruction very precisely but was of no practical value in the treatment of the os calcis fractures.


[Assessment of the value of stimulation from a preparation of Tołpa peat for treatment of trauma related osteomyelitis]. Tołpa peat preparation at the dose of 5 mg per day was administered to 10 patients with chronic posttraumatic osteomyelitis for 6 weeks period of time. Good clinical result (ceasing of the inflammatory process) was found in 4 patients but 3 of them were concurrently treated surgically. Immunological examination (angiogenesis test and chemiluminescence test) performed before the onset and after completion of the treatment with Tołpa preparation did not reveal meaningful changes indicating immunomodulating action of the preparation on the course of posttraumatic osteomyelitis.


[Use of the Weber method in treatment of acromioclavicular dislocation]. The results of treatment of 10 patients with acromioclavicular dislocation has been presented. In all cases acromioclavicular fixation was done with Weber tension band (Zuggurtung). Additional immobilisation was not used so an early rehabilitation was possible. Full range of motion of the shoulder joint was found in 9 patients.


[Use of Dynasplint in prevention of muscular contractures in limbs undergoing lengthening]. Dynamic splinting with Dynasplint for prevention of muscular contractures and subluxations in the joints during lower limb lengthening in 10 children has been used. In all cases the splint successfully prevented subluxation in the knee and the foot deformity. Ability to move splinted joints allowed effective rehabilitation. The splint was well tolerated by all the patients during whole period of distraction.


[Use of magnetotherapy for treatment of bone malunion in limb lengthening. Preliminary report]. Two children with bone malunion after lengthening of congenitally shortened lower leg have been presented. The crus has been elongation 8 cm by Ilizarov method in 9 years old boy and 5 cm elongation of the tibia has been achieved with the use of Bastiani method in 8 years old girl. In both cases malunion has occurred. The radiographs revealed pseudoarthrosis (the girl, 6 month after operation) and centrally located bony defect (the boy, 2 months postoperatively). In both cases pulse sinusoidal magnetic field was applied successfully with cortical bone and marrow cavity restored in 2 months.


[The value of ultrasonography in monitoring limb lengthening]. The authors attempted to assess the value of ultrasound for monitoring of the new bone formation at the site of distraction in patients undergoing limb lengthening after Bastiani. Twenty patients were assessed; 12 cases of femur lengthening and 8 cases of tibia lengthening. It was found, that ultrasound allowed to detect the new bone formation visible as hyperechogenic foci as soon as 4 to 14 days postoperatively, while regular radiology showed this only 4 to 8 weeks after operation. Detection of the new bone formation at the site of lengthening permits, in authors opinion, to choose an optimal onset of distraction and its pace according to the speed of bone formation. Regular ultrasound monitoring prevents premature bone union or disintegration of the newly generated bone in case of to slow or to fast distraction respectively. In two cases sonography early demonstrated cystic changes at the location of distraction. It also permits detection of an axial displacement of the fragments of bone in every case.


[Universal hip fixator "Zespol". Construction of the fixator, principles of function, instrumentation, surgical technique, indications and results of treatment]. The construction and principles of function of hip fixator based on Zespol method, with compression of the fragments provided by elastic deformation of a plate is presented. Original instrumentation, operative technique and indications for this type of osteosynthesis is described. Results analysis revealed that out of 64 patients 10 died within 6 month from surgery, all of them older than 80 years of age. In 46 from remaining 56 patients a union of fractured bone was found. There were 82.2% of good results in patients with femoral neck fracture and 87.5% in the group with trochanteric fractures. Few complications produces hope for Zespol hip fixator to become valuable device for treatment both trochanteric and femoral neck fractures.


Effect of luminal acidification on guinea pig gastric mucosa. H+ secretion was studied in guinea pig fundic mucosa incubated in (A) bicarbonate-Ringer's gassed with 95% O2-5% CO2, or (B) HEPES gassed with 100% O2 before and after luminal pH was lowered to 2.0 for periods up to 90 min. At pH 2.0 for 60 min, H+ secretion in group A tissues fell by 35 +/- 4% (P = 0.02) from a control rate of 1.35 +/- 0.09 mu eq/cm2/hr and in group B tissues by 50 +/- 11% (P = 0.01) from a control rate of 1.59 +/- 0.08 mu eq/cm2/hr. After 90 min at pH 2.0, H+ secretion in group A fell by 53 +/- 8% (P = 0.02) from a control rate of 1.47 +/- 0.07 mu eq/cm2/hr and in group B fell by 44 +/- 6% (P = 0.01) from a control rate of 1.38 +/- 0.07 mu eq/cm2/hr. Histamine 1 x 10(-4) M stimulation following exposure to pH 2.0 for 90 min increased secretion in group A tissues from 0.80 +/- 0.14 to 1.06 +/- 0.13 mu eq/cm2/hr (P < 0.05), compared with an increase in nonacidified controls from 1.15 +/- 0.22 to 1.80 +/- 0.20 mu eq/cm2/hr (P < 0.05) and in group B tissues from 1.27 +/- 0.10 to 1.56 +/- 0.19 mu eq/cm2/hr (P < 0.05) compared with nonacidified controls from 1.43 +/- 0.22 to 2.23 +/- 0.41 mu eq/cm2/hr (P < 0.05). Secretory function and electrical characteristics were adversely affected by luminal acidification to pH 2.0 and suggested a breach in the mucosal barrier with damage to parietal cells.


L-lysine repression of penicillin biosynthesis and the expression of penicillin biosynthesis genes acvA and ipnA in Aspergillus nidulans. The addition of 0.1 M L-lysine to the fermentation medium reduced the production of penicillin by about 50% in Aspergillus nidulans. To analyse this effect at the molecular level, the expression of the penicillin biosynthesis genes acvA and ipnA, encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, was studied by using translational fusions with different reporter genes (strain AXB4A, acvA-uidA, ipnA-lacZ fusions; AXB4B, acvA-lacZ, ipnA-uidA fusions) integrated in single copy at the chromosomal argB locus of Aspergillus nidulans. Irrespective of the reporter genes used the expression of acvA and ipnA fusion genes was repressed in L-lysine grown cultures. The expression of a fusion gene of an A. nidulans primary metabolism gene (oliC-lacZ) was not affected by L-lysine.


Use of a modified cupric acetate method for the detection and quantitation of xylanolytic activities: a comparative study with the congo red method. A modified cupric acetate method for the screening and quantitation of xylanolytic activities was comparable with the more widely used congo red method with respect to sensitivity and ease of use and was shown to have points of merit over the latter. The use of a non-linear correction, in comparison to the conventional linear one, for the effect of dilution on the quantitative plate assay was evaluated.


A general method to generate DNA probes for microorganisms. We present a method that permits the rapid generation of DNA probes for eubacteria. In the procedure the variable regions for the 16s rRNA genes are amplified using polymerase chain reaction (PCR) technology and primers based on the conserved regions of these genes. Following sequencing of the variable regions, a choice is possible for a probe specific for that organism. No knowledge of the molecular biology of the microorganism is required prior to the application of this approach. The generality of the method is shown using Salmonella typhimurium, Staphylococcus aureus, Clostridium perfringens, Klebsiella pneumoniae, Pseudomonas fluorescens, Aeromonas salmonicida and Mycobacterium bovis. A. salmonicida was examined in detail and a DNA probe was prepared that distinguishes it from other Aeromonas species.


Fat-body-specific expression of the Drosophila Lsp-2 gene. The larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is expressed at a very high level in the fat body of third-instar larvae. Here we report that Lsp-2 transcription in adult flies produces a unique mRNA localized in the adult adipose tissue of the head in both sexes. To identify regulatory regions of this Drosophila gene, Lsp-2 5'-flanking DNA sequences were fused to the E. coli beta-galactosidase gene (lacZ). Transient expression of the hybrid gene in third-instar larvae indicates that 230 bp just upstream from the 'TATA box' of the Lsp-2 gene are sufficient for larval fat body-specific expression.


Synthesis and characterization of soluble dextran-adenosine phosphate complexes: kinetic effects of coenzyme loading. Soluble dextran-ATP complexes have been synthesized using a bifunctional oxirane as the coupling agent. The degree of coupling is time-dependent, allowing materials of varying coenzyme loadings to be produced very simply. Characterization studies have shown that at the maximum coenzyme loading obtained (34 molecules per complex) all coenzyme moieties were coenzymically active with hexokinase. The extent of coenzyme loading was shown to have a considerable influence on the values of Km and Vmax of the complex as a substrate for hexokinase. Enzyme activity was also found with acetate kinase and myokinase, and coenzyme recycling (ATP, ADP) was demonstrated in an ultrafiltration reactor.


Discrimination of fetal bovine serum from other sera by silver staining or lectin blotting of SDS/PAGE-separated serum proteins. In order to develop a methodology to discriminate fetal bovine serum from other sera with which it might be contaminated, proteins from fetal bovine, newborn calf, colostrum-free newborn, and calf sera were separated by SDS/PAGE and analyzed by silver staining of the gels or lectin blotting, using concanavalin A (Con A), following transfer of the proteins to nitrocellulose. A high molecular weight Con A-reactive glycoprotein of an apparent molecular mass greater than 200,000 daltons was present in newborn, colostrum-free, and calf sera, but absent or at a significantly lower concentration in fetal serum. These two methods accurately and reliably identified contamination of fetal serum with other sera in a series of blind studies on unknown, coded samples. As little as 1% colostrum-free serum in a batch of fetal serum can be detected by either procedure.


Functional protein-polysaccharide conjugate prepared by controlled dry-heating of ovalbumin-dextran mixtures. A functional ovalbumin-dextran conjugate was prepared by dry-heated storage at 60 degrees C and 65% relative humidity for 3 weeks. The emulsifying properties of the ovalbumin-dextran conjugate were about three times higher than those of an ovalbumin-glucose conjugate. SDS-electrophoresis patterns showed that the ovalbumin-dextran conjugate obtained by dry-heating was not as polydispersed as that obtained by cyanogen bromide-activated dextran. The average molecular weight of the ovalbumin-dextran conjugate was about 200,000. The excellent emulsifying properties of ovalbumin-dextran conjugate were maintained even at pH 3 and were further improved at pH 10. In addition, the emulsifying properties of the ovalbumin-dextran conjugate were greatly enhanced by preheating the conjugate at 100 degrees C. Thus, it is suggested that an ovalbumin-dextran conjugate prepared by controlled dry-heating can be used as a macromolecular emulsifier for food applications.


Comparison between the antigenicity of native and unfolded beta-lactoglobulin. The antibody binding sites (B-cell epitopes) on beta-lactoglobulin (beta-LG) were surveyed by assaying the reactivity of the tryptic fragments of beta-LG to mouse anti-beta-LG antiserum with ELISA. Four peptide fragments (the residues 21Ser-40Arg, 41Val-60Lys, 102Tyr-124 Arg, and 149Leu-162Ile) bound the antibodies. We considered that B-cell epitopes of beta-LG were included in these fragments. Furthermore, these four tryptic fragments were also reactive with the antiserum to RCM beta-LG. Therefore, the unfolding of the beta-LG molecule is considered not to influence the localization of the antibody binding sites on beta-LG.


Bovine alpha S1-casein gene sequences direct high level expression of active human urokinase in mouse milk. We have produced a line of transgenic mice carrying a hybrid bovine alpha S1 casein/human urokinase gene. Bovine alpha S1-casein gene regulatory sequences specifically direct expression of the human urokinase gene in lactating mammary tissue from these mice. Urokinase is a 54 kD protein with 9 disulfide bonds that is normally synthesized in the kidney; however, the casein/urokinase transgenic mice secrete active human urokinase into their milk at concentrations of 1-2 mg/ml. The mice show no other abnormalities. They give birth to, and nurse, normal sized healthy litters. Thus it is possible to produce high concentrations of a large, cysteine rich, non-milk protein in the milk of transgenic animals. This line of transgenic mice provides a model for the eventual production of transgenic farm animals producing high levels of recombinant proteins in their milk.


Monoclonal antibodies specific for human IgE-producing B cells: a potential therapeutic for IgE-mediated allergic diseases. Monoclonal antibodies (MAbs) specific for surface antigens of lymphocytes are being used to target and deplete tumorous or normal lymphocytes in vivo. Here, we report evidence for the existence of antigenic epitopes on IgE that are accessible on IgE-secreting B cells but not on other cells bearing IgE. Among 42 murine MAbs specific for human IgE, two were shown by fluorescence flow cytometric analyses to bind to IgE-secreting cell lines but not to IgE bound to high-affinity IgE.Fc receptors (Fc epsilon RI) on basophils or low-affinity IgE receptors (Fc epsilon RII) on other cell types. Neither could they induce histamine release from basophils of various donors even under very permissive conditions. These antibodies may be useful for targeting IgE-secreting B cells in patients suffering from IgE-mediated allergies.


Expression in yeast of amino-terminal peptide fusions to hepatitis B core antigen and their immunological properties. Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell-dependent and a T-cell-independent antibody response. It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting particle integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope. Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively. These cores could not be produced in E. coli in soluble form but were expressed to high levels in yeast. We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes. Both FMDV and hCG-cores were shown to present the epitopes at the surface of the particles. The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose.


High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. We have used a glutamine synthetase (GS) gene as an amplifiable marker in Chinese hamster ovary (CHO) cells. GS was combined with an efficient transcription unit to produce tissue inhibitor of metalloproteinases (TIMP). Initial transfectant cell-lines selected using a GS gene secreted up to 9 micrograms TIMP/10(6) cells/24h. After one round of GS gene amplification expression levels of 110 micrograms TIMP/10(6) cells/24h were achieved. These GS gene amplified CHO cells, when adapted to grow in suspension, accumulated 180mg/l in shake flask culture. This system therefore provides a rapid method of achieving high level gene expression in mammalian cells.


Methods for the estimation of the number and quality of animal cells immobilized in carbohydrate gels. Rapid and reliable methods for the determination of survival, proliferation, and metabolic activity of immobilized cells in gels are described. The first method is based on an MTT assay that measures qualitatively and quantitatively the metabolic activity of the cells. The second method determines cell number by measuring the amount of DNA available for Feulgen staining. In the third method, two fluorescent dyes are used to differentially stain viable and dead cells. The fourth method involves the use of glutaraldehyde to protect the cells when melting the gel to facilitate hemocytometric count. The presented techniques should help to test the efficiency of the immobilization procedures and to monitor the growth and survival of immobilized cells.


Long-term culture of human esophageal explants and cells. Human esophageal epithelium obtained from intermediate autopsies (less than 12 h) was maintained as cell and explant cultures. In order to develop a serum-free, defined media culture model, several medias and additives were evaluated. The viability and differentiation of the epithelial cells cultured with serum-free, Keratinocyte Growth Media (KGM, Clonetics Co., San Diego, CA) was improved over that of esophageal cells and explants cultured in either serum-supplemented CMRL 1066 (OCM), serum-free additive-supplemented CMRL 1066, or cimetidine-supplemented CMRL 1066. The KGM component EGF was determined to be trophic for esophagus cells on the basis of findings of increased 3H-TdR labelling in KGM cultures when compared to control cells grown in KGM without EGF (KBM). The morphologic pattern of the cytoskeletal proteins actin, keratin, and vimentin were characterized in isolated cell populations. The intermediate filaments, keratin, and vimentin were co-expressed in these epithelial cells. Esophageal explant viability, differentiation, and outgrowth from 15 cases were also evaluated in dishes coated with basement membrane associated proteins. Explants cultured in these dishes were equally well-preserved and differentiated. There were no significant differences in the explant histology when there was protein coating of the culture dishes, although one case showed improved outgrowth with laminin coating. A main advantage for using this culture system is that the same medium (KGM) can be used for both the culture of explants and isolated epithelial cells. Future applications of this model include determining: (1) the effect different concentrations of EGF and calcium in the media will have on esophageal proliferation and differentiation, and (2) the role of different basement membrane associated proteins on the plating efficiency of either isolated or outgrowth epithelial esophageal cells.


Vaxstrate: an anti-reproductive vaccine for cattle. This paper describes the development of a vaccine for the prevention of pregnancy in female cattle. The vaccine is based on the established principle that antibodies to the hypothalamic releasing hormone, gonadotrophin releasing hormone (GnRH) block the action of GnRH on pituitary secretion of luteinizing hormone and follicle stimulating hormone, leading to gonadal atrophy in mammals. The vaccine comprises an immunogenic GnRH:ovalbumin conjugate formulated in a novel double adjuvant system and is administered in a two-dose treatment regimen. Field trials have confirmed efficacy and the product, Vaxstrate, has now been registered and commercialized.


Target amplification systems in nucleic acid-based diagnostic approaches. Currently, PCR is the standard method for target amplification because it is the oldest and most developed procedure. However, several new alternative approaches for target amplification have recently been developed. Although these new methods are at a relatively early stage of development, each has some advantages over PCR, such as greater amplification per cycle (TAS, 3SR, Q beta), isothermal reaction (3SR), or coupled amplification-mutation detection (LAR/LAS). As a result, each may eventually gain widespread use after further development.


Removal of a proteolytic activity associated with aggregates formed from expression of creatine kinase in Escherichia coli leads to improved recovery of active enzyme. Expression of creatine kinase (CK) from a Torpedo californica electric organ cDNA in Escherichia coli results in an insoluble protein product with no detectable CK activity. Although this is a stable aggregate that can be isolated in an enriched form by centrifugation, initial attempts to generate enzyme activity by denaturing and refolding yielded only minute amounts of active protein. We find that these low recoveries are due to proteolysis of the CK during denaturation and refolding. While this proteolytic activity is not inhibited by either phenylmethanesulfonyl fluoride (PMSF) or EDTA, it can be largely removed from the CK aggregate by extraction with a detergent-containing buffer prior to denaturation. This treatment improves the recovery of active CK approximately 100-fold. We have also found similar proteolytic activity associated with the aggregate formed when a mutant of bovine pancreatic trypsin inhibitor (BPTI) is expressed in E. coli. Discovery of this proteolytic activity in two different expression systems suggests that it should be considered as a potential problem for recovery of active protein from other inclusion bodies as well.


Indole alkaloid formation by Catharanthus roseus cells in a biofilm reactor. Catharanthus roseus cells producing indole alkaloids were grown in the form of a biofilm. Production medium was circulated through the reactor parallel to the upper surface of the horizontal biofilm. Sugar consumption and indole alkaloid formation were followed to compare the performance of cultures with different biofilm thicknesses. Dissolved oxygen concentrations gradients within the biofilms were determined at the end of each run. RNA and protein content of the cells in the upper and lower layers of the the biofilms were compared. Results obtained in the biofilm experiments were compared to those obtained with suspension cultures. At optimized biofilm thicknesses, the biofilm reactor was more effective than suspension cultures in maximizing indole alkaloid titers. This is thought to be due to better cell-cell contact within the biofilm and nutrient concentration gradients, which resulted in low growth rates.


Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable alpha-amylases and pullulanases. For the production of cell-free thermostable alpha-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full as well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60 degrees C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10(12) cells up to 700 U/10(12) cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450,000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes.


N-laurylbiotinamide as affinity surfactant. N-laurylbiotinamide (NLB), which retains strong affinity for the protein avidin, was synthesized from biotin and N-laurylamine via the biotin ester of N-hydroxysuccinimide and characterized by NMR. When the synthesized NLB was used as a cosurfactant with AOT to form a reverse micellar system in isooctane, it was found to extend the pH range over which avidin can be transferred from a continuous aqueous solution to the reverse-micellar phase. This behavior is similar to that already reported for a different affinity surfactant, n-octyl beta-D-glucopyranoside.


Characterization of an alkaline protease from Bacillus sp. no. AH-101. The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0-12.0 respectively. The specific activity toward elastin and keratin was 10,600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes.


Enhancement of gene expression by somatic hybridization with primary cells: high-level synthesis of the hepatitis B surface antigen in monkey Vero cells by fusion with primary hepatocytes. Vero cells transfected with the S gene encoding the surface antigen (HBsAg) of the hepatitis B virus (HBV) synthesize HBsAg at low levels. We have obtained a large increase in S gene expression by somatic hybridization of Vero cells with primary hepatocytes, which are the natural target cells for HBV infection. Fusion with cells other than hepatocytes did not enhance expression of the S gene. The Vero/hepatocyte hybrid clones analyzed are stable and have maintained a high level of HBsAg synthesis over prolonged periods. Hybrid cell lines may be of general interest for the high-level synthesis of proteins using cloned genes.


The development and application of a new affinity partitioning system for enzyme isolation and purification. A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.


A family of expression vectors based on the rrnB P2 promoter of Escherichia coli. We describe here the construction of a family of expression vectors, based on the P2 promoter of the Escherichia coli rrnB gene by removing regulatory sequences downstream of the Pribnow-box and replacing them with the lac operator. These vectors allow cloning of foreign genes in such a way that their products are synthesized either in the form of fusion proteins of different length, or without fusion partners, with or without the original translational initiation signals. One of the vectors contains a synthetic oligothreonine-coding sequence that helps to stabilize the product of the cloned gene. These vectors allow high-level regulated expression of foreign genes, even if their products are relatively short peptides.


Potent and specific inhibitors of protein kinase C of microbial origin. Potent and specific inhibitors of protein kinase C have been found in streptomyces and fungi: Staurosporine, an alkaloid from Streptomyces sp., is the most potent inhibitor of protein kinases with an IC50 in the nanomolar range. UCN-01 (7-hydroxy staurosporine), isolated from Streptomyces sp., is a selective inhibitor of protein kinase C with antitumor activity. Calphostin, isolated from the fungus Cladosporium cladosporioides, specifically inhibits protein kinase C (IC50 = 0.05 microM) without inhibiting other protein kinases. Microbial metabolites appear to be a promising source of inhibitors that target signal transduction pathways of eukaryotes.


Characterization of the swelling of a size-exclusion gel. The swelling of a dextran gel, Sephadex G-75, was observed in an aqueous environment at room temperature by a noninvasive technique that uses light microscopy coupled to an image analysis system via a video camera. The rate of swelling was found to follow the Tanaka and Fillmore theory, from which the overall gel diffusion coefficient was estimated as 6.3 x 10(-7) cm2/s. In addition to giving a quantitative measure of gel swelling that could be useful in the mechanical design of liquid chromatography columns, this approach provides data on wet particle size and particle size range, which is needed for the modeling of diffusional and mass transfer effects in size-exclusion chromatography. In this context, key observations are that the gel particles are nearly spherical with an elliptical shape factor of 0.98 (perfect sphere = 1) and that there is little difference between sizes of particles obtained in water, 50 mM Tris-glycine buffer (pH 10.2), and buffer containing 1 mg/mL protein. The diameter of the dry material ranged from 20 to 100 microns, while the hydrated particles had diameters of 40-350 microns. The rate of swelling is rapid, with 50% swelling occurring in about 10 s and swelling to 99% of the final wet particle size being obtained in less than 90 s.


Cystic fibrosis--the way forward from the gene. Cloning of the cystic fibrosis (CF) gene and elucidation of the physiological functions of the encoded protein is a triumph, not only for molecular biology, but also for people affected by CF. For them, not only is there now the possibility of screening for the commonest mutations, but they may also look forward to the prospect of improved therapies being developed.


Protective effect of methylcellulose and other polymers on insect cells subjected to laminar shear stress. The relative sensitivity of two insect cell lines to laminar shear stress was determined, and the protective effect of polymers added to the growth media of two insect cell lines, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), was evaluated. TN-368 and SF-9 cells were found to be equally sensitive to laminar shear stress. Methylcellulose [0.5% (w/v) Dow E4M Methocel] and dextran [4.5% (w/v)] increased the resistance of suspended cells to lysis due to laminar shear stress by factors of up to 76 and 28, respectively, compared to cells in media without additives. It was observed that the protective effect of Pluronic F-68 was concentration-dependent: 0.2% and 0.3% (w/v) F-68 increased the resistance of SF-9 cells to shear stress by factors of 15 and 42, respectively. However, increasing the concentration to 0.5% did not significantly increase the cells' resistance compared to 0.3% (w/v). F-68 at 0.2% only increased the resistance of TN-368 cells by a factor of 6. It is believed that the protection is a result of the polymer adsorbing to the cell membrane. None of the polymer additives tested had a significant effect on SF-9 or TN-368 growth rate.


Alkaline serine protease produced from citric acid by Bacillus alcalophilus subsp. halodurans KP 1239. Maximum production of alkaline serine protease by Bacillus alcalophilus subsp. halodurans KP 1239 was achieved after 24 h cultivation, at an initial pH of 7.6, on a medium containing 1.0% sodium citrate, 0.3% yeast extract, and 0.3% KH2PO4. The enzyme was purified to crystalline form from culture broth. The enzyme was most active at 60 degrees C and at pH 11.5. The molecular weight, isoelectric point and sedimentation coefficient in water at 20 degrees C were estimated as 29,000, 8.8 and 3.3S, respectively. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala- His-Asn-Arg-Gly-. The enzyme shared its antigenic determinants with B. alcalophilus ATCC 21522 serine protease, but not with the subtilisins Carlsberg and BPN'.


DNA fingerprinting of Streptococcus uberis based on polymorphism of DNA encoding rRNA. The rRNA gene restriction patterns of strains of Streptococcus uberis and Strep. parauberis were determined. Chromosomal DNA was digested with endonucleases and probed with radiolabelled DNA complementary to rRNA synthesized by random oligonucleotide priming using reverse transcriptase. Reproducible restriction patterns were obtained which readily distinguished Strep. uberis and Strep. parauberis. In addition, considerable variations in the patterns at the intra-specific level were observed indicating that rRNA gene restriction profiles are of epidemiological, as well as taxonomic value.


Enterococcus dispar sp. nov. a new Enterococcus species from human sources. The partial 16S rRNA sequences of two unknown human enterococcal isolates were determined by reverse transcription in an attempt to clarify their taxonomic position. The sequence data indicate that they belong to a hitherto unknown species of Enterococcus, for which the name Enterococcus dispar sp. nov. is proposed. The type strain is NCIMB 13000.


Automated surface area measurement of cultured cardiac myocytes. An automated method for rapidly measuring surface area of individual cardiac myocytes was used as an index of myocyte growth. Hearts from 2- to 4-day-old rats were digested by overnight incubation in cold trypsin solution. Enriched suspensions of myocytes were plated at 2 x 10(5) cells/well in 12-well-culture plates. Cells were grown in M199 supplemented with 1%, 10% serum or 10% serum plus 10(-7) M norepinephrine. On days 1-4 after plating, cells were fixed in Bouin's Solution and stained with Weigert's Iron Hematoxylin and Biebrich Scarlet-Acid Fuchsin. An inverted microscope, video camera and monitor were coupled to a video image processor (Image Technology Corp.). The enhanced image of stained heart cells was digitized, and perimeter, length, width and area of each selected cell were calculated. One hundred randomly selected cells were measured in each of eight wells from each treatment-day group. Areas of individual myocytes varied widely in culture dishes and the distribution was skewed toward larger cells. The standard deviation increased in proportion to an increase in mean cell area. A logarithmic transformation of the data normalized the data and yielded a more homogeneous variance. The geometric mean area of heart cells supplemented with 1% serum increased only slightly, but significantly, during four days in culture. Geometric mean area of cells supplemented with 10% serum increased nearly four-fold. Supplementing cells with norepinephrine (10(-7) M) in addition to 10% serum did not induce a further increase in cell size. This technique has the potential to rapidly and objectively monitor heart cell growth following pharmacological or toxicological treatments.


Protein recovery from reversed micellar solutions through contact with a pressurized gas phase. We describe a new process for the recovery of encapsulated protein from reversed micellar solution in concentrated form. The method involves desolubilization of the protein by decreasing solvent density through gas dissolution. Under appropriate thermodynamic conditions, the micellar water pool can be converted to clathrate hydrates. Protein recovery is facilitated by clathrate hydrate formation, which causes the desolubilized protein to exist in a solid phase, distinct from the micellar supernatant. The process is carried out without any ionic strength or pH modification.


Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers. Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (10(7) - 2 x 10(7) cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors.


Continuous cell substrate considerations. The debate over the potential risk of tumorigenicity attributable to the use of CCL substrates for biologicals production has continued for over 30 years and may continue for some time to come. Manufacturers and regulatory agencies are developing scientifically based guidelines for such products. It is currently possible to follow these guidelines to prepare recombinant biologicals and monoclonal antibodies in CCLs which do not pose unreasonable risks. This chapter has attempted to describe the scientific tools available to evaluate the putative risk of tumorigenicity due to potential virus DNA and protein contaminants. No theoretical or experimental basis exists to hypothesize that residual cellular protein might present a significant risk of tumorigenicity. The tools are certainly adequate for characterization of putative risks due to viruses and DNA but are not sufficiently powerful by themselves to assure product safety. The subsequent chapter on process validation describes how adequate assurances of safety ultimately can be obtained for products of CCLs against theoretical risks of tumorigenicity due to putative viruses and DNA. In addition to these safeguards, no evidence of tumorigenicity has been found in human or livestock animal recipients of the products prepared in CCL substrates. Many patients have received inoculations of tissue plasminogen activator, erythropoeitin, factor VIII, soluble CD4, GM-CSF, hepatitis B surface antigen vaccine, and various monoclonal antibodies and other recombinant products of continuous cell lines in clinical trials. For tissue plasminogen activator, large doses of 100 mg per patient or more have been used. At the time of writing over 10 kg of CHO-derived tissue plasminogen activator has been sold since late 1987 for administration to over 100,000 human patients. For recombinant factor VIII, erythropoeitin, and soluble CD4 proteins, chronic administration has been employed. Millions have received polio and rabies vaccines prepared in continuous Vero cells. In addition to this human experience, livestock animals have received annual inoculations of foot-and-mouth virus vaccine prepared in BHK-21 (a highly tumorigenic CCL) for up to 14 years without effect (69). No effects have been reported which might be attributed to oncogenic factors. Thus, scientific tools of characterization and principles of process validation are available to protect patients from putative risks of tumorigenicity associated with products prepared in CCLs. Increasing clinical experience also supports this conclusion.


Single cell assay with an automated capillary microinjection system. An automated capillary microinjection system with computer controlled positioning of the cells and of the capillary, and its applications and advantages are described. About 1500 injections are possible in one hour, with high reproducibility. In cytoplasmic and nuclear injections more than 90% and 85% of the cells are successfully injected. Using FITC-Dextran at a concentration of 0.5% as a fluorescently labelled coinjection marker, 99% of the cells can be retrieved in culture medium even 48 hours after injection. The coordinates of the cells are stored in the computer and accuracy in statistical evaluation of experiments is improved in comparison to the manual techniques. Methods for preparation and handling of glass capillaries were developed resulting in reproducible form and significantly reduced clogging rate. The improved characteristics offered by this system are demonstrated in studies leading to the confirmation of existence of mRNA(s) inhibiting cell proliferation. Functional screening by cell injections of cDNA libraries and of size fractionated mRNA molecules can be performed efficiently with the automated microinjection system.


High-level secretion of biologically active aprotinin from the yeast Pichia pastoris. A synthetic gene encoding aprotinin (bovine pancreatic trypsin inhibitor) was fused to the Saccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site. Pichia pastoris strains were developed to express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion. P. pastoris containing a single copy of the vector secreted approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipotent with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.


Flow cytometric study of cultured mammalian cells. Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating "stationary" phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.