ATP-induced endocytosis in human erythrocyte ghosts. Characterization of the process and isolation of the endocytosed vesicles.

ATP-induced endocytosis in human erythrocyte ghosts has been studied, and a procedure for the isolation of the endocytotic vesicles is described. Under isotonic conditions and 37 degrees C, optimal endocytosis occurs with concentrations of 4 to 10 mM MgATP. Within 30 min, up to 45% of the membrane is removed from the surface and converted into sealed inside-out vesicles. Local anesthetics, such as chlorpromazine, potentiate ATP-induced endocytosis in ghosts. Forcing cells containing endocytotic vesicles through a hypodermic needle leads to the exclusive fragmentation of the outermost plasma membrane. The endocytosed vesicles can then be separated from these fragments by centrifugation on a gradient of dextran T70. Biochemical analyses indicate that endocytotic vesicles contain full complements of the major membrane proteins (i.e. also spectrin and actin), common phospholipids, fatty acids, and cholesterol. Furthermore, they exhibit a fully intact spectrin component 2 phosphorylation machinery. In contrast, MgATPase activity is largely excluded from these vesicles. The novel inside-out vesicles described have properties different from those of previously analyzed fragments of the erythrocyte membrane. They will permit a detailed study of a native spectrin-actin network now exposed to the outside.


Structure of the major gangliosides from bovine thyroid.

After preparative isolation, the carbohydrate, long chain base, and fatty acid composition of the major gangliosides from bovine thyroid have been analyzed. The structures were elucidated by determining the molar ratio of the building blocks, permethylation analysis, and enzymatic degradation studies. The following structures are identified: N-Acetylneuraminyl(2,3)-galactosyl(1,4)glucosyl(1,1)ceramidie; N-glycolyneuraminyl(2,3)galactosyl(1,4)glucosyl(1,1)ceramide; galactosyl(1,3)N-acetylgalactosaminyl[(3,2)N-acetylneuraminyl](1,4)galactosyl(1,4)glucosyl(1,1)ceramide; fucosyl(1,2)galactosyl(1,3)N-acetylgalactosaminyl[(3,2)N-acetylneuraminyl](1,4)galactosyl(1,4)glucosyl(1,1)-ceramide. The structures were confirmed by direct inlet mass spectrometry of the permethylated gangliosides and the corresponding asialo derivatives. Structures are proposed for common ions in the different mass spectra.


Structural characterization of lactotetraosylceramide, a novel glycosphingolipid isolated from human meconium.

In the course of work on a systematic structural mapping of nonacid glycosphingolipids of human meconia, special attention was given to a major component preliminarily identified as an isomer of neolactotetraosylceramide (paragloboside). This component was isolated in its pure form from meconium of a blood group O individual and subjected to detailed structural analyses, using mass spectrometry and proton NMR spectroscopy on intact permethylated and permethylated-reduced (LiAlH4) derivatives, and gas liquid chromatography on degradational products of native, permethylated, and permethylated-reduced derivatives. The isolated compound was conclusively shown to have the structure Galp beta 1 yields 3GlcNAcp beta 1 yields 3Galp beta 1 yields 4Glcp beta 1 yields 1Cer, and is thus identified as lactotetraosylceramide. The major fatty acids were 2-hydroxy fatty acids with 16 and 20 to 24 carbon atoms, and the bases were sphingosine and phytosphingosine. This glycolipid, although not isolated and structurally characterized before, has long been thought of as a precursor substance of the Lewis active glycolipids and of ABH-active glycolipids with a type 1 saccharide chain.


Characteristics of tissue growth into Proplast and porous polyethylene implants in bone.

(1) Bone does not form within internal pores of undistorted Proplast implants because of the small interconnecting pore size of the material; (2) the nonosseous, fibrous tissue which exists in the pores of Proplast implants in bone is not attached to the surrounding bone (i.e., Sharpey's fibers are not present). The load-bearing support which can be afforded by Proplast implants is limited by the incomplete bone ingrowth along the margins of the material and the tensile strength of Proplast.


Behavior of three high-Cu amalgams.

Amalgams made from three single-composition high-Cu alloys of the same nominal composition were investigated for differences in creep behavior. The curves of creep vs. final Hg content for all three alloys showed a sudden creep increase (jump) at approximately 46% final Hg content. Previous studies have shown this creep jump to be associated with the initiation of the Sn--Hg phase (gamma 2). The anticipated final Hg content in clinical restorations for two of these alloys is below the creep jump and gamma 2 would not be expected. On the other hand, the clinical Hg content for the third alloy was above its creep jump and gamma 2 would be expected. The effect on clinical behavior of this finding require further investigation.


In vitro blood compatibility of glycosaminoglycan-precipitated collagens.

Precipitation of bovine hide collagen by chondroitin 6-sulfate at low pH and subsequent crosslinking enhances the blood compatibility of native collagen. Both dehydrothermal crosslinking and complexation with chrondroitin 6-sulfate separately decrease the platelet-aggregating activity of collagen. Crosslinking also decreases the number of free acidic and free basic residues on collagen, which suggests that crosslinking involves these residues in condensation reactions with formation of intrachain and interchain synthetic peptide bonds. Clotting times for collagen precipitated with chondroitin 6-sulfate indicate that this surface does not activate or interfere with coagulation via either the intrinsic or extrinsic pathway. These findings support further consideration of collagen modified by chondroitin 6-sulfate as a blood compatible material.


SEM fractography studies of porous vitreous carbon: a candidate biomaterial.

A new porous vitreous carbon material under development for use in orthopedic applications was investigated. Specimens were machined to appropriate sizes and fractured in one of the following modes: compression, cantilevered bending, or axial torsion. Scanning electron microscopy (SEM) was used to examine surface and internal features. Characteristics of a brittle, glassy material were noted. Findings included internal voids which appeared as craters, patches of whiskerlike fibrils, and edge impurities. Numerous microcracks caused by mechanical shaping and handling were the most remarkable structural defects. Pore channels which would allow bony ingrowth ranged in size from 50--500 micrometers with the majority between 200 and 300 micrometers. This study of porous vitreous carbon points to the need for stricter quality control in manufacturing, alternative methods for shaping and handling, and careful consideration in design and usage of a brittle material with marginal limits of safety for biomedical applications.


Microelectrode studies of stress-generated potentials in four-point bending of bone.

A microelectrode technique has been developed to enable the study of stress-generated potentials (SGP) in bone to a spatial resolution of 5 micrometers. The technique has been used to measure the electrical potentials as a function of bone micromorphology in four-point bending. Electric fields ranging from 30 to 10(3) times greater than is measured by conventional macroscopic methods have been discovered at the Haversian canals for human and bovine cortical bone. The amplitude and direction of the electric field in the osteons depend specifically upon the amplitude and the sign (i.e., compression or tension) of the stress. The implications of this finding with regard to the origin of SGP and their possible physiological significance are considered.


Microelectrode study of stress-generated potentials obtained from uniform and nonuniform compression of human bone.

By use of a previously developed microelectrode technique, the effect of nonuniform stresses on the stress-generated potentials (SGP) in bone were studied to a resolution of 5 micrometers. Comparison was made between uniformly and nonuniformly applied compression of human cortical bone. It was found that the radial electric fields for osteons in a specimen under uniform compression were equivalent, and such specimens possessed no macroscopic SGP; for nonuniform compression, the electric fields of osteons differed, and a macroscopic SGP was measured. The magnitude of the macroscopic SGP thus appears to be dependent upon local stress differences and, hence, on the SGP of local regions.


Platelet interaction with synthetic copolypeptide films.

Kinetic and equilibrium studies of blood platelet binding to copolypeptide films show that attachment and serotonin release are not dependent upon the composition of the copolypeptide. Data may be explained by postulating that platelets frequently collide elastically with the surface but leave behind material that modifies subsequent behavior. Similarly, material released from platelets adsorbs at the interface and the extent of attachment and serotonin release are modified and controlled by these adsorbed species. Basically, if the platelet is exposed to a clean surface, its collision with the surface leads to activation and release. In the presence of inert protein, the collision is cushioned by the protein and platelets do not attach or release to any extent. Finally, if protein (or other entities) released from the platelet provide attachment sites, then attachment occurs without release. It is postulated that the behavior of platelets at surfaces is controlled by these interrelated processes.


Degradation resistance of some candidate composite biomaterials.

The degradation resistance of matrix, fiber and composite systems which we have been studying as candidate orthopedic materials has been examined in two appropriate environments. Both resistance to steam sterilization in an autoclave environment and resistance to a simulated physiologic solution have been studied. In the autoclave study, samples were placed in a pressure cooker at 123 degrees C for differing amounts of time and tested for retention of mechanical properties. Results indicate that most of the materials tested could be autoclaved several times, as long as autoclave times did not exceed 1 hr. Longer autoclave times result in an accelerated degradation and loss of strength of all materials except the polypropylene. Polysulfone degrades after even the shortest autoclave duration. Resistance to the simulated physiologic environment was tested by measured retention of mechanical properties after immersion times in pseudo-extracellular fluid (PECF) at 37 degrees C for as long as three years. None of the materials showed any significant changes in properties after immersion in the PECF.


Release characteristics of dibucaine dispersed in konjac gels.

A possible use of konjac gel for sustained release of drugs was examined in a monolithic system containing dibucaine. Dibucaine was dispersed in the gel which was prepared by gelation of the konjac flour in a borax solution at 60 degrees. The cumulative amount of the drug released plotted against the square root of time was linear in the monolithic system. This relationship was in agreement with that expected from the theoretical equation for planar configuration. The mechanism of the release of the drug from the gel may be considered to be leaching of the drug by the permeating fluid. The release profile from dried konjac gel was similar to that from undried gel, but that from unwarmed gel showed a deviation from linearity although sustained release was similarly obtained.


Resistant congenital club foot--one-stage posteromedial release with internal fixation. A follow-up report of a fifteen-year experience.

I have reviewed my personal experience with 240 resistant congenital club feet (176 patients) treated surgically by one-stage posteromedial release with internal fixation. The end results in the 149 feet that had a follow-up of two to fifteen years were: excellent or good, 83.8 per cent; fair, 10.7 per cent; and failures, 5.3 per cent. The best results with the least complications were in children who were operated on between the ages of one and two years. A statistical analysis of fifty-three feet with a follow-up of less than two years is reported for comparison with the results after longer follow-up to allow analysis of all the known failures and complications that have been encountered with the operation. Possible causes and prevention of fair and poor results are discussed. Modifications of the technique, which originally was reported in 1971, are described.


Serratus anterior paralysis in the young athlete.

Ten cases of isolated, complete paralysis of the serratus anterior muscle were diagnosed in young athletes during a three-year period. One patient had recurrent partial paralysis of the serratus anterior muscle, the first such case reported. From studies on cadavera and clinical observations, we concluded that paralysis of the serratus anterior muscle results from a traction injury to the long thoracic nerve of Bell. Since full recovery usually occurs in an average of nine months, surgical methods of treatment should be reserved for patients in whom function fails to return after a two-year period. Non-strenuous use of the involved extremity with avoidance of the precipitating activity, followed by exercises designed to maintain the range of motion of the shoulder and to increase the strength of associated muscles, is advocated for treatment of acute or repetitive injuries to the long thoracic nerve of Bell.


Tennis elbow. The surgical treatment of lateral epicondylitis.

Of the 1,213 clinical cases of lateral tennis elbow seen during the time period from December 19, 1971, to October 31, 1977, eighty-eight elbows in eighty-two patients had operative treatment. The lesion that was consistently identified at surgery was immature fibroblastic and vascular infiltration of the origin of the extensor carpi radialis brevis. A specific surgical technique was employed, including exposure of the extensor carpi radialis brevis, excision of the identified lesion, and repair. The results at follow-up were rated as excellent in sixty-six elbows, good in nine, fair in eleven, and failed in two. There was an over-all improvement rate of 97.7 per cent, and 85.2 per cent of the patients returned to full activity including rigorous sports.


External pin fixation for unstable Colles' fractures.

During a five-year period, a double-pin Roger Anderson apparatus, with pins perpendicularly placed in the second and third metacarpals and in the distal part of the radius, was applied in 130 patients with an unstable Colles fracture. Sixty of the 130 were followed for two years. Shortening was limited to a median of two millimeters and dorsal angulation, to a median of 3 degrees. Wrist dorsiflexion averaged 58 degrees, and volar flexion averaged 50 degrees. Pronation and supination had an average loss of 5 degrees when compared with the uninjured side. Repeat reduction was required in only three patients. Patient assessment revealed that 85 per cent of the patients had good results; 12 per cent, fair; and 3 per cent, unsatisfactory. Objective analysis (McBride system) revealed that 90 per cent had good to excellent results; 8 per cent, fair; and 2 per cent, poor. Ninety-two per cent had no pain, 89 per cent had no deformity, and the mean grip strength was twenty kilograms. Sixteen patients had complications; seven of the sixteen had pin loosening, which occurred most frequently late during the course of treatment and without adverse sequelae.


A simple biplanar method of measuring femoral anteversion and neck-shaft angle.

A simple biplanar roentgenographic technique for measurement of femoral anteversion and neck-shaft angle is described in which no special equipment is required. An anteroposterior roentgenogram of the pelvis and the femoral neck is made with the hip in neutral rotation. The true lateral roentgenogram is made with the patient's limb in a lateral position with the hip and knee flexed and with the entire lateral aspect of the leg in contact with the top of the table. Projected cervicofemoral angulations are measured on the roentgenograms. The method is described to determine the true angles by dynamically illustrating the projected angles. Graphs have been prepared with which one can readily determine the anteversion and neck-shaft angles once the projected cervicofemoral angulations have been measured.


Patellar complications following total knee arthroplasty.

We studied the complications involving the patella following total knee arthroplasty in eighty-six knees in which thirty-four unconstrained and fifty-two offset hinge prostheses had been implanted. The abnormalities that we studied included: patellar dislocation, five knees; subluxation, eighteen knees; localized wear, three knees; and generalized wear, four knees. In twenty-three patients these complications were associated with patellar malalignment, and occurred predominantly when the offset hinge model of prosthesis was used. The complications were attributed partly to mechanical factors inherent in the prosthetic design and partly to anatomical abnormalities. Failure of the surgeon to compensate adequately for both of those factors at the time of operation was a factor in most of the complications.


Late reconstruction of the patellar tendon.

Late reconstruction of undiagnosed ruptures of the patellar tendon by transfer of the gracilis and semitendinosus tendons, supplemented by a heavy-gauge encircling wire to bridge the gap, was successful in four patients. These tendons are stronger than fascial strips which have previously been used for the purpose. The four patients treated by this technique returned to their preinjury functional levels.


Prosthetic replacement of the knee and a large segment of the femur or tibia.

Total replacement of the knee and of a long segment of the femur or tibia provided functional recovery of the knee joint in thirteen patients in whom the length of the bone replaced ranged from seventy-five to 150 millimeters. Nine patients had benign bone tumors, three had malignant lesions, and one patient had salvage of a failed total knee arthroplasty, associated with loss of bone structure. After follow-up of two years or more, all patients had a stable extremity, excellent relief of pain, and a useful range of motion, although one patient required revision for loosening.


Joint replacement in non-ambulatory patients.

We treated eighteen non-ambulatory patients by multiple operations consisting of total replacement of the hip or the knee, or both. Thirteen patients had rheumatoid arthritis, four had degenerative arthritis, and one had had bilateral resection of the femoral head and neck. The patients were followed for an average of forty months. We devised a classification based on the number of joints involved and a rating scale for function. Good to excellent results were achieved in the patients who had two or three joints operated on (with one exception). Fifteen patients became ambulatory and seven could climb stairs. Seven patients were pain-free. The factors responsible for poor results were significant neural problems and loss of motivation. The presence of severe upper-extremity involvement was not an obstacle preventing walking.


Tenosynovial chondromatosis in the hand.

Eight cases of tenosynovial chondromatosis in the hand were compared with twelve cases collected from the literature and available data on eleven cases showed that in three there were recurrences. No patients had metastatic lesions during a follow-up period averaging 7.8 years. Based on these data, we concluded that a moderately radical en bloc excision is adequate treatment for tenosynovial chondromatosis in the hand.


A simplified technique to correct hyperextension deformity of the metacarpophalangeal joint of the thumb.

A flexible hyperextension deformity of the metacarpophalangeal joint of the thumb in eleven patients was repaired by tendon reinforcement of the volar capsule of the joint using the tendon of the extensor pollicis brevis. This tendon, which was regarded as a deforming factor, was left attached at its insertion on the proximal phalanx and was divided at the musculotendinous junction proximally. The mobilized tendon was pulled out distally and then passed proximally and volarly, first superficial to the sheath of the long flexor tendon and then back through a hole in the neck of the metacarpal, and finally in a distal direction to form a criss-cross pattern and be sutured to the tendon of the adductor pollicis. The procedure gave reliable results after an average follow-up of 6.5 years in these eleven patients who had rheumatoid, paralytic, and traumatic lesions.


Multiple carpometacarpal dislocations. A review of four cases.

The cases of four patients with carpometacarpal fracture-dislocations of the ulnar four joints are reported, in all of whom there was extensive additional trauma. Open reduction was necessary in three patients, and only one was treated successfully by a closed reduction. He then had persistent but painless subluxation of the metacarpal bases. On end-result evaluation, two of the other three patients had occasional discomfort and diminished grip strength. One patient had a diminished range of motion of the wrist joint at one-year follow-up, and one patient had narrowing and sclerosis of the carpometacarpal joints.


Open reduction for congenital dislocation of the hip using the Ferguson procedure. A review of twenty-six cases.

Twenty-three patients with twenty-six congenital hip dislocations treated by the Ferguson procedure were reviewed. The study was undertaken to determine if this method of treatment has any effect on the incidence of avascular necrosis of the femoral head and if there are preoperative factors that can assist in predicting the success of treatment. Clinical and roentgenographic criteria were utilized and only dislocations of the hip unassociated with other disorders were included. Avascular necrosis did not occur in these patients during the period studied. It was also found that patients who were more than twelve months old at the time of operation and had a acetabular index of 40 degrees or more had a high incidence of redislocation.


Excisional arthroplasty of the elbow.

Thirty patients had excisional arthroplasty of the elbow, with interpositional Gelfoam and early mobilization. Most of the patients had residual disability from trauma. Most of the patients' results were evaluated four years or more after the operation: 80 per cent were pain-free and 90 per cent were able to return to their previous occupations. One patient had to be reoperated on and therefore was considered to have a poor result.


Stiff-legged gait in hemiplegia: surgical correction.

Selective tenotomy of one or two heads of the quadriceps based on electromyographic criteria improved knee flexion in hemiplegic patients who walked with a unilateral stiff-legged gait. The improvement was greatest in eight patients in whom the rectus femoris was released, either with or without release of the vastus intermedius, with activity in pre-swing stance and initial swing confined to those heads of the quadriceps; in these patients knee flexion improved an average of 20 degrees. Knee flexion improved an average of only 8 degrees in five patients in who activity was present in one head of the quadriceps that was not surgically released. No improvement in knee flexion occurred in seven of eight patients in whom activity was present in two or more of the quadriceps heads that were not surgically released.


Differential bone-scanning in the evaluation of a painful total joint replacement.

In an attempt to differentiate between loose and infected total joint replacements, seventy-nine patients were studied with both technetium-99 pyrophosphate and gallium-67 citrate bone scans. Seventy of these patients had positive technetium scans. Nineteen had positive gallium scans and all nineteen had positive cultures at the time of operation, while one patient had a false-negative gallium scan. Ten other patients who had negative technetium-99 scans and a painful total joint were followed without operative intervention. Of these, only two required operation after their technetium-99 scans became positive. Based on these studies, gallium-67 citrate scans combined with technetium-99 pyrophosphate scans are believed to be useful in differentiating between loose and infected prostheses. A negative technetium scan should weigh heavily against surgical exploration in an attempt to find the source of pain.


Volkmann's contracture in children: aetiology and prevention.

A review was conducted of the records of fifty-five children who were admitted to the Hospital for Sick Children in Toronto between 1955 and 1975 with a diagnosis of Volkmann's contracture in fifty-eight limbs. Ten patients had been transferred to this hospital with established ischaemia after Bryant's traction for a fractured femur; all had a very poor outcome. Thirteen other cases of Volkmann's contracture affecting the superficial posterior compartment had been treated with a fixed Thomas' splint and a Bradford frame after fractures of the femoral shaft. Supracondylar fractures of the elbow resulting in Volkmann's contracture frequently had both an arterial injury and a compartment syndrome. Most of the fifty-five children reviewed here had not had early appropriate treatment. For the past twenty-one years the frequency of Volkmann's contracture has not declined in spite of many published reports on the compartment syndrome, and the hazards of supracondylar fractures and of Bryant's traction.


The pathology and prevention of Volkmann's ischaemic contracture.

Ischaemia is a rare complication of injury to a limb but must be excluded in every case. Two distinct types occur: Type I, where a proximal arterial injury gives rise to ischaemia distally; and Type II, where a direct injury gives rise to ischaemia at the site of the injury. Whatever the nature of the insult, an ischaemic contracture only develops as a result of swelling of the soft tissues where these soft tissues are contained in un unyielding osteofascial compartment. This secondary ischaemia can only be relieved by a timely fasciotomy. The diagnosis of ischaemia in an injured limb and the indications to operate on it can usually be made on clinical grounds alone.


Factors in the development of the spinal stenosis syndrome.

The spinal stenosis syndrome is a potential hazard when congenital or developmental narrowing of the bony canal, particularly in its lateral recesses where it can be demonstrated by axial tomography, places the emergent nerve root and its blood supply at risk to further small compressive elements. When lumbar disc degeneration allows rotatory and lateral instability, posterolateral bulging of the annulus fibrosus into the root canal occurs when weight is taken on the ipsilateral lower limb. Symptoms of the spinal stenosis syndrome do not arise until the development of this instability. To relieve the symptoms, the nerve root must be freely mobilised and decompressed by full lateral decompression, with partial or total facetectomy if necessary, by enucleation of the intervertebral disc and by removal of the posterolateral portions of the annuli fibrosi.


Segmental neurophysiological mechanisms in scoliosis.

Segmental spinal reflexes (stretch reflexes) were studied in patients with scoliosis. The proprioceptive responses to the phasic stretch of the paraspinal muscles were asymmetric in all patients, and were increased on the convex side. The asymmetry was more pronounced when the patients were standing. The observed asymmetry of the reflex responses was taken to indicate asymmetry in the tone and postural activity of the superficial layer of the paraspinal muscles. A reciprocal relationship was found in the segmental reflex organisation between the superficial and deep layers of the paraspinal muscles. The increase in reflex response of the superficial muscles on the convex side can be due to diminished reciprocal inhibition from weak, deep muscles. Thus a segmental neurogenic disorder involving predominantly the deep paraspinal muscles of the convexity of the curve may be the primary lesion responsible for the development of scoliosis.


Two-stage corrective surgery for congenital deformities of the spine.

Sixty patients with congenital deformities of the spine were operated upon in the past fifteen years using a two-stage procedure. In the fifty patients with scoliosis half of the deformities were due to hemivertebrae and half to unilateral bars. The average correction of the deformity was 47 per cent. Early neurological signs observed in two patients with a diastematomyelia resolved. Of the ten patients with kyphosis nine had neurological signs of impending paraplegia and one was completely paraplegic before operation; all improved markedly. Posterior spinal fusion alone in the rapidly progressing congenital deformity may not prevent further progression, particularly in those cases iwth unilateral bars. Anterior resection of the vertebral body with later posterior fusion with Harrington instrumentation is safe and effective.


A prospective study of children with untreated Catterall group 1 Perthes' disease.

Twenty-four hips in twenty children affected by Group 1 Perthes' disease have been reviewed to assess the radiographic result after a minimum follow-up of four years. The children were allocated to Group 1 prospectively after examination of the early radiographs and no specific treatment of the affected hip was provided. The radiographic end results assessed by three methods were good even in those cases in which the additional stress of containment splintage of the contralateral hip was applied.


The African neonatal hip and its immunity from congenital dislocation.

The hips of twenty full-term African neonates have been examined in detail to determine any anatomical factors which might explain the difference in the incidence of congenital dislocation of the hip in the African and in the Caucasian. Measurements included the degree of anteversion of the femoral neck and the acetabulum and the diameter and depth of the acetabulum. The acetabulum tended to be deeper and to vary within a much narrower range than that reported for Caucasians, lending indirect support to the theoretical role of acetabular dysplasia in the aetiology of congenital dislocation of the hip. Measurements of the anteversion of the acetabulum and femoral neck were similar to those given for Caucasians.


Failure of sliding nail-plate fixation in subcapital fractures of the femoral neck.

A retrospective study of 200 cases of subcapital fracture of the neck of the femur treated by sliding nail-plate fixation has been carried out. Failure of fixation within three months occurred in forty-two cases (21 per cent). These failures could, in part, be attributed to a combination of the severity of the fracture and various imperfections in technique. The age of the patient and the angle of the nail-plate had no significant effect on the result.


Chip fractures of the os triquetrum: the mechanism of injury.

Studies derived from analyses of radiographs and dissections of cadaveric wrists have been directed at testing and disproving the commonly held theories that link chip fractures of the os triquetrum with avulsion. The authors found that the mechanism of such injuries involved a chisel action of the ulnar styloid upon the dorsum of the os triquetrum; furthermore, the impact of a fall on the outstretched arm with the hand rigidly held in strong dorsiflexion and ulnar deviation could be forceful enough to fracture the body of the os triquetrum as well. In all cases a striking prolongation of the ulnar styloid, beyond the surface of the ulnar head, was consistently noted.


A comparison of the Freeman-Swanson (ICLH) and Walldius prostheses in total knee replacement.

Seventy-five Freeman-Swanson (ICLH) Mark I total knee replacements, all performed in one orthopaedic unit between 1972 and 1975, were independently reviewed. The fifty-eight surviving patients, with sixty-eight arthroplasties, have been interviewed and examined and the clinical records of the deceased patients inspected. Sixty arthroplasties (80 per cent) were successful and fifteen failed (20 per cent). There were no disasters. Twenty (33.8 per cent) of the successful arthroplasties were excellent. It is expected that modification of the prosthesis and improved instrumentation will increase this percentage of excellent results and reduce the failure rate.


Free vascularised fibular transplant for replacement of the lower radius.

An operation is described in which a microvascular technique was used to transfer a living fibula, with its vascular pedicle intact, to replace the lower end of the radius after massive resection for giant-cell tumour. Angiography carried out six weeks later showed that the grafted bone was viable. Six months after operation the transplanted fibula showed no osteoporosis or bone resorption and bony union at the junction of host and graft.


Extra-abdominal desmoid tumours.

Desmoid tumours are not common but have a distinct resemblance to fibrosarcomata. Their clinical appearance and progress should be recognised since failure to distinguish them from sarcomata could result in extensive and unnecessarily mutilating operations. Three cases of extra-abdominal desmoid tumours are reported. Two of the patients had tumours arising from multicentric foci in the same limb. The disappearance of the tumours in two patients after the menopause, and the variation in the tumours during the menstrual cycle in the third patient, add weight to the theories about endocrine control.


[Neoplastic and preneoplastic lesions in rats after oral administration of a single dose of N-nitrosomorpholine (author's transl)].

The administration of a single oral dose of 320 mg/kg body wt. N-nitrosomorpholine (NNM) to 62 Sprague-dawley rats resulted in neoplastic and preneoplastic changes in different organs, especially in liver and kidney. After a lag period of 4 weeks, nearly all experimental animals developed preneoplastic (clear cell, acidophilic, basophilic, mixed cell) foci of the liver parenchyma. Sporadically, small neoplastic nodules were found in the liver as early as 4 weeks after the beginning of the experiment. After a lag period of 1--2 years, 4 of 13 rats showed multiple neoplastic hepatic nodules and one animal a hepatocellular carcinoma. The bile ductules of some animals responded to the carcinogen by forming mucous cholangiofibroses and cystic cholangiomas. After long lag periods, large cholangiofibromas were found in two experimental animals. One or two years after application of the carcinogen, many animals developed epithelial (clear cell, acidophilic, chromophobic, basophilic, oncocytic) kidney tumors, often cystic. Pathologically changed (clear cell, chromophobic, basophilic, oncocytic) tubules are regarded as precursors of the epithelial tumors. The latered tubules appear for the first time at about half a year after application of the carcinogen. Apart from multiple cysts of the liver and kidneys some pancreatic cysts developed in two animals. In addition, two mesenchymal kidney tumors, one malignant neurinoma, two subcutaneous fibromas, one fibroadenoma, and one squamous cell carcinoma of the skin were observed.


Tumors and hyperplastic lesions in Syrian hamsters following transplacental and neonatal treatment with cigarette smoke condensate.

Cigarette smoke condensate (CSC) in olive oil was injected into outbred Syrian hamsters: in adults i.p. on the 10th--14th days of gestation, total dose 1.5--2.5 mg/g b.w.; in 12 to 14-days-old animals s.c., total dose 0.5--1.5 mg/animal. Following 15--25 months of observation benign and malignant neoplasms of various location were found in 2/58 (3.4%) females, treated during pregnancy; in 17/51 (33.3%) of their transplacentally exposed offsprings; in 5/53 (9.4%) of neonatally treated hamsters. In the last two groups females were more affected than males. Most frequently occurred tumors of adrenal glands, pancreas, female sex organs, and liver. No tumors appeared in controls, either untreated or injected with olive oil. In addition, hyperplastic lesions, in particular multiple liver cysts and cholangiomatosis were also observed, mainly in animals exposed transplacentally and as neonates.


Cyclophosphamide-resistant Yoshida ascites tumor cells and their cross resistance to some alkylating agents.

A cyclophosphamide-resistant subline of a Yoshida ascites tumor was developed by giving increasing doses of the drug after transplantation. The effect of several alkylating agents on the cell proliferation of both the sensitive and resistance cell line was compared establishind dose response curves and D50 values. The developed subline revealed a 260 fold resistance to cyclophosphamide. It was completely cross-resistant to hydroperoxycyclophosphamide, whereas for triaziquone, N-oxide-mustard, and N-methyl-mustard only a partial cross resistance existed. These results give further evidence that cyclophosphamide and hydropeoxycyclophosphamide have the same mechanism of action. Regarding the other alkylating agents the results demonstrate differences concerning either the molecular mode of action or protecting effects. A decreased activation or uptake of substance is probably not the base for resistance.


The effects of 1-methyl-1-nitrosourea on the survival pattern of cycling and noncycling HeLa cells.

The biological behavior of HeLa cells exposed to 1-methyl-1-nitrosourea was examined by determining the survival fraction in asynchronous and synchronous cultures. Asynchronous cell population exposed to 1-methyl-1-nitrosourea for 1 h exhibited a shoulder type survival curve, indicating that damage must be accumulated before the lethal effect occurs. A fraction of 25% of cells survives the concentration of 100 micrograms/ml. The duration of treatment with the drug did not have a significant effect on the cell survival. The experiments with synchronized cells showed that MNU exhibits the killing in all phases of the cell age, but the most sensitive are these in S phase. However, they are still six times more resistant at the same concentration than the culture in plateau phase. At the concentration of 100 micrograms/ml nondividing plateau cells are about 35 times more sensitive than exponentially growing cells. We can conclude that MNU acts as the most acting killing agents on the cells which are in nondividing plateau phase.


Excretion patterns of alkylating metabolites in urine following cyclophosphamide treatment of tumor patients: influence of application route, dosage, liver and kidney function.

The excretion patterns of cyclophosphamide (CP) in urine were studied in 54 tumor patients aged between 21 and 61 years, using the nitrobenzyl-pyridine (NBP) reaction, with regard to the route of application (i.v., i.m. or oral), the CP dose and the functional state of the liver and kidney. The studies were carried out in nephrectomized patients and patients with liver affections caused by the basic disease, in particular with malignant lymphomas and mammary carcinomas. The following results were obtained: 1. There exists a direct relationship between the dose of CP applied and the quantitative excretion of alkylating metabolites in urine. According to these studies in which the patients received up to 2.8 g CP/m2 body surface, the upper CP dose was limited by the generally toxic side effects rather than by the metabolization rate. 2. At comparable CP doses the route of application (i.v., i.m. or oral) has no appreciable influence on the excreted NBP activity. 3. Disorders of the liver function without signs of icterus are not a contraindication to CP treatment. 4. The functional failure of one kidney has no statistically significant influence on the excretion of alkylating metabolites in urine.


Endodermal germ cell carcinoma (endodermal sinus tumor) of the vagina in infant girls.

An endodermal sinus tumor (endodermal germ cell carcinoma) was diagnosed in a 1-year-old girl in the vagina after hemorrhage; the tumor was completely removed by radical abdominal surgery. Postoperative polychemotherapy was performed for two years with Actinomycin D, Adriamycin, Vincristin, and Cyclophosphamide. The infant is now tumor-free for 26 months, showing almost normal somatic and psychic development. The characteristic histological patterns and clinical course of this strongly malignant tumor are demonstrated, based on 25 published case reports of endodermal sinus tumors in the vagina of little girls (aged 5-26 months). This neoplasm in early infancy has to be separated from the clear-cell adenocarcinoma of the vagina which occurs after puberty in adolescent girls and young women, and is induced by stilbestrol therapy to the mother during early pregnancy.


Hormone sensitivity of gynecological tumor cells in tissue culture.

Proliferating tumor cells obtained from ovarian, mammary, and endometrial tumors in tissue culture were tested for the influence of proteohormones and steroid hormones on cellular DNA synthesis and cell growth. The gonadotropic hormones stimulated DNA synthesis of ovarian tumor cells by single administration, or in combination with cortisol, up to the 11-fold of the comparable controls. The hormone sensitivity of the cell lines was variable, resulting in individual reaction patterns. There was no correlation to the histological diagnosis of the primary tumors with respect to the grade of differentiation. The results suggest that ovarian tumor cells in tissue culture can maintain sensitivity to organotropic hormones. Compared to the ovarian carcinoma lines, mammary or endometrial tumor cells did not respond to a similar extent. Progesterone decreased DNA synthesis of endometrial carcinoma cells.


[Malignant interstitial cell tumor of testis with a marker enzyme (author's transl)].

Metastatic interstitial cell tumor of the testis is one of the rarest human neoplasms. This is the nineteenth case to be reported. While most of these tumors are combined with hormonal dysfunction, the present tumor, apart from its uncommon hormonal profile, is remarkable because of its capacity of producing and secreting a marker enzyme, alkaline phosphatase. No response was seen after cytostatic therapy with new antineoplastic agents, such as a combination of adriamycin and cis-diamminedichloride-platinum (II), and ifosfamide. Considering the lack of radiosensitivity, surgery is the primary modality of treatment.


Vasospasm in the lower extremities during and following arterial reconstruction.

Foot temperatures of 36 patients were recorded continuously in the operating room and in the surgical intensive care unit (SICU) for 1-3 days. Operations included aortofemoral endarterectomy, arterioplasty, and bypass grafting. Regardless of severity of the operation, all had cold extremities on entering the SICU. The subsequent warming of the big toes, which started after 4-12 hours in the SICU, if at all, was classified according to a scheme of 6 deviations from a basic trend. This latter was a bilateral, continuous increase in 1-2 hours to 34 degrees C or higher where it remained with minor oscillations. Unilateral deviations were either diminished warming or no warming on one side. Bilateral deviations included stepwise increments to 34 degrees C, diminished increments, and no warming. Neurogenic vasospasm appeared to be the principal factor diminishing blood flow, with circulating vasoconstrictors and organic blocks as additional less important factors. Subsequent amputations of nine limbs were partially correlated with the categories of digital warming.


Arterial and venous blood viscosity in ischemic lower limbs in patients affected by peripheral obliterative arterial disease.

Blood, plasma and serum viscosity, packed red cell volume, and plasma fibrinogen concentration was measured in a group of 13 subjects, suffering from peripheral obliterative arterial disease of the lower limbs (stage III or IV), awaiting surgery, and in 9 control subjects, none of whom showed signs of circulatory disease of the lower limbs either by clinical examination or by instrumental measurements. The blood samples were taken from the: (a) antecubital vein; (b) femoral vein; (c) femoral artery. In the patients venous blood viscosity was significantly higher than arterial blood viscosity. No significant differences have been found between the femoral and the antecubital vein, but in the femoral vein blood viscosity was slightly higher. Packed red cell volume and plasma viscosity were slightly but significantly higher in venous blood, while no difference was seen in serum viscosity and in plasma fibrinogen concentration. In the control subjects blood viscosity values were decidedly lower than in patients and no statistically significant differences were to be found between artery and vein, even if viscosity values were lower in artery. These results support the hypothesis that an interrelation exists between hyperivscosity syndrome and vascular ischaemia-inducing diseases.


Vocal cord paralysis following carotid endarterectomy.

From 1964 to 1977, over 400 carotid endarterectomies were performed at the Rochester General Hospital. Two patients from this group experienced hoarseness secondary to vocal cord paralysis on the operated side, believed due to trauma to the vagus nerve. Although uncommon, it is important to stress the method of avoiding this complication.


Importance of bidirectional arterial flow in dialysis fistula construction.

The normal direction of blood flow in end of cephalic vein to side of radial artery arteriovenous dialysis fistulas in the wrist was antegrade in the proximal arterial limb and retrograde through the distal arterial limb. There were no instances of arterial insufficiency in the hand due to a steal phenomenon. These findings support the surgical construction of end of vein to side of artery fistulas.


Long-term results (from 5 to 7 years) with the Hancock S-G-P bioprosthesis.

This communication is concerned with the long-term results of the first 113 patients who underwent single or multiple heart valve replacement with the Hancock-SGP bioprosthesis at the Department of Cardiovascular Surgery, University of Padova Medical School, in a two years period (March 1970-March 1972). Hospital mortality was 18.5%; of the 92 survivors, whose follow-up is now 5 to more than 7 years, the authors report the clinical reevaluation, the complications and the causes of late death. Hemodynamic reevaluation has been performed so far in 26 patients and the prostheses have shown to function properly both in the mitral and the aortic position. The data available to date indicate that the glutaraldehyde preserved porcine valve is durable and its use is warranted in any valvular position.


Correction of tetralogy of Fallot after Waterston shunt.

The authors have reviewed their experience concerning twenty-seven patients who underwent intracardiac repair of Fallot's Tetralogy after a previous Waterston shunt. They are divided into 4 groups according to the classification of Shinebourne, Anderson and Bowyer. The risk factors are analyzed in group 1 patients in whom primary total correction is contra-indicated and in whom the mortality at repair is high in presence of kinking of right pulmonary artery. The type of surgery is still debatable. For the symptomatic patients of the other groups, primary total correction at any age seems, at this time, to be the treatment of choice. The contra-indications are an anterior descending coronary artery arising from the right coronary artery or associated complex anomalies which would make total correction difficult.


On the mechanism of 5-bromodeoxyuridine induction of prolactin synthesis in rat pituitary tumor cells.

GH12C1, a clonal strain of rat pituitary tumor cells in culture (GH cells), does not produce detectable amounts of prolactin. 5-Bromodeoxyuridine (BrdUrd), the thymidine analogue, at sublethal concentrations (3-5 microgram/ml) induces prolactin synthesis in these cells. BrdUrd also induces prolactin synthesis in F1BGH12C1 cells, a BrdUrd resistant (BrdUrdr) substrain isolated from GH12C1 cells. The F1BGH12C1 strain is not drug dependent, but its resistance to BrdUrd is a stable phenotype. The significant features of the induction of prolactin synthesis in the BrdUrdr strain are the increased net synthesis of prolactin and the shortening of the lag period of prolactin induction. As BrdUrd concentration in the growth medium is increased, the rise in prolactin synthesis parallels the increased incorporation of BrdUrd into DNA. Prolactin synthesis is first detected when BrdUrd replaces 20-25% of the thymidine in DNA. BrdUrd can replace up to 75-80% of the thymidine within 2 d of treatment. Partial starvation of these cells under specified growth conditions does not affect the general growth pattern of the cells, general protein synthesis, and thymidine uptake, but does affect DNA synthesis. When cells are cultured under conditions in which DNA synthesis is preferentially inhibited, BrdUrd does not induce prolactin synthesis, suggestive of a DNA-mediated mechanism of action for the drug.


Double outlet left ventricle: how should we repair it?

Two patients with D.O.L.V., V.S.D. and P.S. have been successfully corrected surgically. They represent the seventh and the eighth successful repairs of D.O.L.V. reported so far in the literature. Surgical correction was accomplished by an "extraventricular" type of repair, closing the V.S.D., interrupting the connection between left ventricle and pulmonary artery and creating a conduit between right ventricle and pulmonary artery. The conduit used was a fresh aortic homograft. An "intraventricular" type of repair of this rare congenital malformation has been also reported, but the authors believe that it is not applicable to all cases of D.O.L.V. and it is possibly more likely to incur complications, while the "extraventricular" one doesn't seem to have any contraindication. Preoperative diagnosis can be only achieved with careful angiographic study, including left ventriculography.


Hormonal modulation of ovarian interstitial cells with particular reference to gap junctions.

Thin-section and freeze-fracture studies on the rat ovarian interstitial cells revealed reductions in the size and the number of gap junctions after pituitary ablation. Small gap junctions, however, persist as long as 90 days after hypophysectomy even though regressive cytoplasmic changes are completed 75 d earlier. Administration of exogenous human chorionic gonadotrophin (HCG) results in the restoration of the normal interstitial cell morphology which is accompanied by amplification of junctional membrane. Within 24 h of hormone application, gap junction growth is characterized by the appearance of formation plaques. These studies suggest that the effect of hormone on interstitial cell gap junctions is to modulate the junctional surface area.


Clinical comparison between membrane and bubble oxygenators in cardiopulmonary bypass.

The clinical course of two similar patient groups was compared in whom, during cardiopulmonary bypass, a membrane or bubble oxygenator was employed. According to our results there is no significant functional difference between the two types of oxygenators as long as the perfusion time does not exceed 90 minutes. Beyond this time limit, the membrane oxygenator has distinct advantages, particularly with regard to hemolysis. We presently prefer the bubble oxygenator. The use of a membrane oxygenator is restricted to complex open heart procedures with suspected technical problems.


Myosin synthesis in embryonic chicken fibroblasts.

The rate of constitutive myosin synthesis was measured in cultures of replicating embryonic chicken skin fibroblasts by pulse labeling with [3H]leucine. These cells synthesized the 200,000-dalton heavy chain of myosin (MHC) at a rate of 3.2 x 10(3) molecules/cell/min. Additionally, an independent estimate of the MHC synthesis rate needed to maintain a constant level of constitutive MHC/cell was calculated from total protein content, percentage MHC, fibroblast doubling time, and MHC half-life. This calculated rate of approximately 2.9 x 10(3) molecules/cell/min was in close agreement with the measured rate. By comparison, the synthesis rate of myofibrillar MHC in fully activated muscle cell cultures was approximately 2.9 x 10(4) molecules/nucleus/min.


Early thoracotomy for stab wounds of the heart.

Fifteen consecutive patients with stab wounds of the heart are reviewed. The left ventricle was stabbed in six patients and right ventricle in four. Sinus tachycardia was present in 54% and hypotension in 67% of cases. Central venous pressure recorded in ten patients was elevated in all but one. Generalized ST segment elevation was the rule among the available preoperative recordings. One patient had right bundle branch block. Thirteen of the fifteen patients had a thoracotomy and two had pericardiocentesis only. Sixty nine percent of the thoracotomies were performed within two hours of the injury. There was only one death of a patient who arrived with no blood pressure or heart sounds. An average follow-up of 20 months revealed long-term sequela of an asymptomatic ventricular aneurysm in one patient. Our unusually gratifying results support the approach of emergency thoracotomy in patients with penetrating cardiac trauma.


Generation of asymmetry during development. Segregation of type-specific proteins in Caulobacter.

An essential event in developmental processes is the introduction of asymmetry into an otherwise undifferentiated cell population. Cell division in Caulobacter is asymmetric; the progeny cells are structurally different and follow different sequences of development, thus providing a useful model system for the study of differentiation. Because the progeny cells are different from one another, there must be a segregation of morphogenetic and informational components at some time in the cell cycle. We have examined the pattern of specific protein segregation between Caulobacter stalked and swarmer daughter cells, with the rationale that such a progeny analysis would identify both structurally and developmentally important proteins. To complement the study, we have also examined the pattern of protein synthesis during synchronous growth and in various cellular fractions. We show here, for the first time, that the association of proteins with a specific cell type may result not only from their periodicity of synthesis, but also from their pattern of distribution at the time of cell division. Several membrane-associated and soluble proteins are segregated asymmetrically between progeny stalked and swarmer cells. The data further show that a subclass of soluble proteins becomes associated with the membrane of the progeny stalked cells. Therefore, although the principal differentiated cell types possess different synthetic capabilities and characteristic proteins, the asymmetry between progeny stalked and swarmer cells is generated primarily by the preferential association of specific soluble proteins with the membrane of only one daughter cell. The majority of the proteins which exhibit this segregation behavior are synthesized during the entire cell cycle and exhibit relatively long, functional messenger RNA half-lives.


Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane.

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.


Local anesthetic-induced inhibition of collagen secretion in cultured cells under conditions where microtubules are not depolymerized by these agents.

Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.


Stereo electron microscopy of the 25-nm chromatin fibers in isolated nuclei.

Thin sections (0.1-0.25 micron) of isolated chicken erythrocyte nuclei were examined at various tilt angles. Stereo pairs of electron micrographs document the parallel alignment of 25-nm chromatin fibers adjacent to the nuclear envelope, and demonstrate a fiber substructure consistent with close-packed arrays of nucleosomes.


DNase-I-dependent dissociation of erythrocyte cytoskeletons.

The human erythrocyte contains a complex of peripheral membrane proteins which forms an extensive network or cytoskeleton on the cytoplasmic membrane surface. When I treat erythrocyte cytoskeletons with deoxyribonuclease I (DNase I), the cytoskeletons dissociate and erythrocyte actin is solubilized. The dissociation of the cytoskeletons by DNase I parallels the disruption of actin filaments in vitro by DNase I and is blocked by the addition of action to the DNase I. Large protein complexes remain after DNase I disrupts the cytoskeletons, but these complexes are no longer visible in the light microscope nor sedimentable and are selectively depleted with respect to actin. From these studies, I suggest that DNase I binds to and solubilizes actin, which serves as a structural link between protein complexes in the erythrocyte cytoskeleton.


Troponin in embryonic chick skeletal muscle cells in vitro. An immunoelectron microscope study.

The fine structurel distribution of troponin on thin filaments in developing myofibrils was investigated by the use of immunoelectron microscopy. Embryonic chick skeletal muscle cells grown in vitro were treated with antibodies against each of the troponin components (troponin T, I, and C) from adult chicken muscles. Each antibody was distributed along the thin filaments with a period of 38 nm. It is concluded that these newly synthesized regulatory proteins are assembled at their characteristic position from the initial phases of myofibrillogenesis.


Sperm binding and fertilization envelope formation in a cell surface complex isolated from sea urchin eggs.

An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.


Organization of tubulin in normal and transformed rat kidney cells.

We have carried out a quantitative biochemical and ultrastructural study of tubulin and microtubules in a normal rat kidney (NRK) cell line and its viral transformant (442) in culture. Under equivalent culture conditions, both cell lines contain the same amount of tubulin according to a colchicine-binding assay. The normal and transformed cells differ significantly, however, with respect to the state of organization of their tubulin. Counts of microtubules in sectioned cells indicate that NRK cells have almost twice as many microtubules per unit area of cytoplasm as the 442 cells. Centrifugation studies, on the other hand, show that 442 cells have almost twice as much pelletable tubulin as the NRK cells. We propose, therefore, that the transformed cells contain a large amount of tubulin which is in some alternative aggregate form that is not morphologically detectable as microtubles in the cytoplasm


Microtrabecular lattice of the cytoplasmic ground substance. Artifact or reality.

The cytoplasmic ground substance of cultured cells prepared for high voltage transmission electron microscopy (glutaraldehyde/osmium fixed, alcohol or acetone dehydrated, critical-point dried) consists of slender (3-6 nm Diam) strands--the microtrabeculae (55)--that form an irregular three-dimensional lattice (the microtrabecular lattice). The microtrabeculae interconnect the membranous and nonmembranous organelles and are confluent with the cortices of the cytoplast. The lattice is found in all portions of the cytoplast of all cultured cells examined. The possibility that the lattice structure is an artifact of specimen preparation has been tested by (a) subjecting whole cultured cells (WI-38, NRK, chick embryo fibroblasts) to various chemical (aldehydes, osmium tetroxide) and nonchemical (freezing) fixation schedules, (b) examination of model systems (erythrocytes, protein solutions), (c) substantiating the relaibility of critical-point drying, and (d) comparing images of whole cells with conventionally prepared (plastic-embedded) cells. The lattice structure is preserved by chemical and nonchemical fixation, though alterations in ultrastructure can occur especially after prolonged exposure to osmium tetroxide. The critical-point method for drying specimens appears to be reliable as is the freeze-drying method. The discrepancies between images of plastic-embedded and sectioned cells, and images of whole, critical-point dried cells appear to be related, in part, to the electron-scattering properties of the embedding resin. The described observations indicate that the microtrabecular lattice seen in electron micrographs closely represents the nonrandom structure of the cytoplasmic ground substance of living cultured cells.


Structural states of myelin observed by x-ray diffraction and freeze-fracture electron microscopy.

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.


Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane.

The receptor-rich postsynaptic membrane of the elasmobranch electric organ was fixed by quick-freezing and then viewed by freeze-fracture, deep-etching and rotary-replication. Traditional freeze-fracture revealed a distinct, geometrical pattern of shallow 8.5-nm bumps on the E fracture-face, similar to the lattice which has been seen before in chemically fixed material, but seen less clearly than after quick-freezing. Fracture plus deep-etching brought into view on the true outside of this membrane a similar geometrical pattern of 8.5-nm projections rising out of the membrane surface. The individual projections looked like structures that have been seen in negatively stained or deep-etched membrane fragments and have been identified as individual acetylcholine receptor molecules. The surface protrusions were twice as abundant as the large intramembrane particles that characterize the fracture faces of this membrane, which have also been considered to be receptor molecules. Particle counts have always been too low to match the estimates of postsynaptic receptor density derived from physiological and biochemical studies; counts of surface projections, however, more closely matched these estimates. Rotary-replication of quick-frozen, etched postsynaptic membranes enhanced the visibility of these surface protuberances and illustrated that they often occur in dimers, tetramers, and ordered rows. The variations in these surface patterns suggested that in vivo, receptors in the postsynaptic membrane may tend to pack into "liquid crystals" which constantly appear, flow, and disappear in the fluid environment of the membrane. Additionally, deep-etching revealed a distinct web of cytoplasmic filaments beneath the postsynaptic membrane, and revealed the basal lamina above it; and delineated possible points of contact between these structures and the membrane proper.


Localization of acetylcholine receptors by means of horseradish peroxidase-alpha-bungarotoxin during formation and development of the neuromuscular junction in the chick embryo.

The localization of acetylcholine receptors (AChR) in the surface of developing myogenic cells of the chick embryo anterior and posterior latissimus dorsi muscles in relation to the process of innervation has been studied at the ultrastructural level utilizing a horseradish peroxidase-alpha-bungarotoxin conjugate. Localized concentrations of AChR were found in small regions 0.1-0.4 micron in width on the surface of myogenic cells of 10- to 14-d-old muscles. Surface specializations consisting of an external coating of extraneous material and an internal accumulation of dense material are associated with the plasma membrane in the regions of AChR concentration. As the muscle fibers are innervated, reactive surface patches are found at the region of contact of the growing nerve fiber and the surface of myotubes or their fusing myoblasts. After the establishment of contact, the patches of reaction product become more numerous and coextensive within the region of the neuromuscular junction and its immediate surroundings forming a dense continuous deposit on the postsynaptic sarcolemma. Activity becomes increasingly restricted to the site of the neuromuscular junction as the embryos approach hatching. At all stages, specializations external and internal to the plasmalemma are found at regions of high density of AChR, suggesting that they play a role in the maintenance of a higher concentration of receptors at these sites. These specializations also occur at the region of initial synaptic contact, indicating that they might be recognized by the nerve and represent preferred sites of innervation. Innervation appears to exert a stabilizing influence on the area of high AChR concentration in contact with the nerve and to induce a further increase in the AChR density of this site while the number of AChR in the remaining portions of the muscle surface declines.


Nexuses between the smooth muscle cells of the guinea-pig ileum.

The circular musculature of the guinea-pig ileum has been studied by freeze-fracture to analyze quantitatively the gap junctions (nexuses) between its smooth muscle cells. The average cell surface area and cell volume are 5,074 micron 2 and 3,260 micron 3. The packing density of nexuses is 48/1,000 micron 2 of cell surface or approximately 244/muscle cell. Nexuses range in area from less than 0.1 to approximately 1.5 micron 2 and they occupy 0.212% of the cell surface. The average packing density of intramembrane particles or pits in nexuses is approximately 7,200/micron 2 of nexal surface, indicating that there may be approximately 77,000 intercellular channels in the full complement of nexuses of one muscle cell.


Characterization of the molecular components in kinetoplast-mitochondrial DNA of Trypanosoma equiperdum. Comparative study of the dyskinetoplastic and wild strains.

The structure of the kinetoplast DNA of Trypanosoma equiperdum has been studied and compared to the structure of the circular mitochondrial DNA extracted from a dyskinetoplastic strain of T. equiperdum. In T. equiperdum wild type, the kinetoplast DNA constitutes approximately 6% of the total cellular DNA and is composed of approximately 3,000 supercoiled minicircles of 6.4 x 10(5) daltons and approximately 50 circular supercoiled molecules of 15.4 x 10(6) daltons topologically interlocked; The buoyant density in CsCl of the minicircles is 1.691 g/cm 3. The large circles have a buoyant density of 1.684 g/cm 3, are homogeneous in size and are selectively cleaved by several restriction endonucleases which do not cleave the minicircles. The cleavage sites of six different restriction endonucleases have been mapped on the large circle. The minicircles are cleaved by two other restriction endonucleases, and their cleavage sites have been mapped. The mitochondrial DNA extracted from the dyskinetoplastic strain of T. equiperdum represents 7% of the total DNA of the cell and is composed of supercoiled circles, heterogeneous in size, and topologically associated in catenated oligomers. Its buoyant density in CsCl is 1.688 g/cm 3. These molecules are not cleaved by any of the eight restriction endonucleases tested. The reassociation kinetics of in vitro labeled kDNA minicircles and large circles has been studied. The results indicate that the minicircles as well as the large circles are homogeneous in sequence and that the circular DNA of the dyskinetoplastic strain has no sequence in common with the kDNA of the wild strain.


Organization of neuronal microtubules in the nematode Caenorhabditis elegans.

We have studied the organization of microtubules in neurons of the nematode Caenorhabditis elegans. Six neurons, which we call the microtubule cells, contain bundles of darkly staining microtubules which can be followed easily in serial-section electron micrographs. Reconstruction of individual microtubules in these cells indicate that most, if not all, microtubules are short compared with the length of the cell process. Average microtubule length varies characteristically with cell type. The arrangement of microtubules gives an overall polarity to each bundle: the distal ends of the microtubles are on the outside of the bundle, whereas the proximal ends are preferentially inside. The distal and proximal ends each have a characteristic appearance indicating that these microtubules may have a polarity of their own. Short microtubules in processes of other neurons in C. elegans have also been observed.


Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins.

To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS-polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface.


Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. II. Internalization of proteins.

The distribution of surface proteins during phagocytosis by rabbit peritoneal polymorphonuclear leukocytes was studied to determine whether the proteins of the phagocytic vesicles of these differentiated cells were representative of the entire set of plasma membrane proteins. Phagocytosis of bovine serum albumin-diisodecylphthalate emulsion by lactoperoxidase-iodinated rabbit neutrophils was linear over 15-20 min at a rate of 96 microgram oil/min/mg cell protein. This rate was similar to that of unlabeled cells. Incorporation of cell-associated free iodine by endogenous myeloperoxidase during phagocytosis was inhibited by 1 mM cyanide, which had no effect on the rate of particle uptake. The surface of intact neutrophils contained at least 13 iodinated proteins distinguishable by polyacrylamide gel electrophoresis followed by autoradiography. Isolated phagosomes were deficient in six of these proteins. The plasma membrane fraction of these cells was missing five of these same proteins which, however, were enriched in a dense surface fraction (Willinger, M., and F. R. Frankel. J. Cell Biol. 82: 32-44). When experimental conditions were reversed, and the PMNs were labeled after phagocytosis, these five proteins remained on the cell surface, while at least three of the major proteins found on resting cells were depleted. Incubating the cells with colchicine, which has been shown to affect the distribution of some plasma membrane constituents during phagocytosis, had no effect on the distribution of surface proteins in our system. These results indicate that a nonrandom interiorization of lactoperoxidase-labeled surface proteins of polymorphonuclear leukocytes occurs during phagocytosis.


Cardiac gap junction configuration after an uncoupling treatment as a function of time.

Rabbit ventricle either was fixed in glutaraldehyde without injury (control) or was injured before fixation, presumably causing electrical uncoupling of the gap junctions. All tissue was then processed for freeze-fracture. Replicas of control gap junctions exhibited irregular packing of the P-face particles and E-face pits. Average center-to-center spacing of the particles was 10.5 nm. Tissue fixed 1-5 min after injury showed clumping of gap junctional particles and pits. Within the clumps, the particles and pits were hexagonally packed and the center-to-center spacing of the particles averaged 9.5 nm. In tissue fixed 15-30 min after injury, the clumps of gap junctional particles had coalesced into a homogeneous structure in most junctions. The packing of the particles and pits was hexagonal and the spacing of the particles averaged 9.5 nm. A few pieces of rabbit atrium were frozen without prior fixation or cryoprotection to try to assess the effect of glutarldehyde fixation on gap junction structure. In this tissue the gap junctional particles were irregularly packed and their spacing averaged 10.0 nm.


Helical substructure of neurofilaments isolated from Myxicola and squid giant axons.

Neurofilaments purified from invertebrate giant axons have been analyzed with the electron microscope. The neurofilaments have a helical substructure which is most easily observed when the neurofilaments are partially denatured with 0.5 M KCl or 2 M urea. When the ropelike structure comprising the neurofilaments untwists, two strands 4--5.5nm in diameter can be resolved. Upon further denaturation these strands break up into rod-shaped segments and subsequently these segments roll up into amorphous globular structures. Stained, filled densities can be resolved within the strand segments, and these resemble similar structures observed within the intact neurofilaments. The strands appear to consist of protofilaments 2--2.5 nm in diameter. These observations suggest that the neurofilament is a ropelike, helical structure composed of two strands twisted tightly around each other, and they su-port the filamentous rather than the golbular model of intermediate filament structure.


Identification of the subunit proteins of 10-nm neurofilaments isolated from axoplasm of squid and Myxicola giant axons.

Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and squid. The axoplasm was fractionated by discontinuous sucrose gradient centrifugation and gel filtration on Sepharose 4B. The fractions were monitored for neurofilaments by electron microscopy. When isolated in the presence of chelating agents, the neurofilaments of Myxicola are composed almost entirely of protein subunits with mol wt of 150,000 and 160,000. Squid neurofilaments contain two major proteins with mol wt of 200,000 and 60,000. These proteins are compared with other intermediate filament proteins which have been reported in the literature.


Localization of submembranous cations to the leading end of human neutrophils during chemotaxis.

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.


Calcium-induced asymmetrical beating of triton-demembranated sea urchin sperm flagella.

Asymmetrical bending waves can be obtained by reactivating demembranated sea urchin spermatozoa at high Ca2+ concentrations. Moving-film flash photography shows that asymmetrical flagellar bending waves are associated with premature termination of the growth of the bends in one direction (the reverse bends) while the bends in the opposite direction (the principal bends) grow for one full beat cycle, and with unequal rates of growth of principal and reverse bends. The relative proportions of these two components of asymmetry are highly variable. The increased angle in the principal bend is compensated by a decreased angle in the reverse bend, so that there is no change in mean bend angle; the wavelength and beat frequency are also independent of the degree of asymmetry. This new information is still insufficient to identify a particular mechanism for Ca2+-induced asymmetry. When a developing bend stops growing before initiation of growth of a new bend in the same direction, a modification of the sliding between tubules in the distal portion of the flagellum is required. This modification can be described as a superposition of synchronous sliding on the metachronous sliding associated with propagating bending waves. Synchronous sliding is particularly evident in highly asymmetrical flagella, but is probably not the cause of asymmetry. The control of metachronous sliding appears to be unaffected by the superposition of synchronous sliding.


Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve.

We examined the role of nerve terminals in organizing acetylcholine receptors on regenerating skeletal-muscle fibers. When muscle fibers are damaged, they degenerate and are phagocytized, but their basal lamina sheaths survive. New myofibers form within the original basal lamina sheaths, and they become innervated precisely at the original synaptic sites on the sheaths. After denervating and damaging muscle, we allowed myofibers to regenerate but deliberately prevented reinnervation. The distribution of acetylcholine receptors on regenerating myofibers was determined by histological methods, using [125I] alpha-bungarotoxin or horseradish peroxidase-alpha-bungarotoxin; original synaptic sites on the basal lamina sheaths were marked by cholinesterase stain. By one month after damage to the muscle, the new myofibers have accumulations of acetylcholine receptors that are selectively localized to the original synaptic sites. The density of the receptors at these sites is the same as at normal neuromuscular junctions. Folds in the myofiber surface resembling junctional folds at normal neuromuscular junctions also occur at original synaptic sites in the absence of nerve terminals. Our results demonstrate that the biochemical and structural organization of the subsynaptic membrane in regenerating muscle is directed by structures that remain at synaptic sites after removal of the nerve.


Fate of plasma membrane during endocytosis. I. Uptake and processing of anti-plasma membrane and control immunoglobulins by cultured fibroblasts.

The uptake and processing by cultured rat embryo fibroblasts of control rabbit immunoglobulins (C IgG) or IgG directed against plasma membrane constituents (anti-PM IgG), and labeled with fluorescein (F) or with radioactive acetate (A), have been investigated by cell fractionation and immunological techniques. Both F and A anti-PM IgGs become bound to the cell surface, by a process that is slow, but largely temperature-independent. In the presence of an excess of high-affinity antibodies, binding reaches an absolute limit which corresponds to extensive coating of the plasma membrane. The anti-PM IgGs remain attached to the membrane for at least several days, even at 37 degrees C, with no significant transfer to lysosomes or degradation. In contrast, C IgGs are handled very differently by the fibroblasts, and their fate is strikingly affected by the type of labeling used. AC IgG is taken up slowly, at a rate proportional to its concentration, and is subsequently broken down in what appears to be lysosomes. Part of the AC IgG also binds to the plasma membrane. FC IgG is taken up many times faster than AC IgG, though with the same strict linearity as a function of concentration. Most of the FC IgG taken up is stored in cytoplasmic granules which behave like lysosomes. For reasons that are not understood, only about half of the stored FC IgG can be broken down. Cells exposed simulatnaously to AC IgG and FC IgG, or to A anti-PM IgG and FC IgG, handle each type of IgG in its characteristic fashion. Kinetic analysis of these results indicates that Ac IgG could be taken up by fluid endocytosis, but that FC IgG must be interiorized by a selective mechanism, presumably adsorptive in nature. That anti-PM antibodies remain stably bound to the plasma membrane and do not interfere with the uptake of FC IgG is interpreted to indicate either that two distinct membrane domains are involved in the two phenomena, or that membrane patches coated with anti-PM IgG participate in endocytosis, and are recycled back to the cell surface after delivering their contents intracellularly.


Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents.

Cultured rat embryo fibroblasts were first allowed to store for 24 h fluorescein-labeled goat immunoglobulins directed against rabbit immunoglobulins (F anti-R IgG), and were subsequently exposed for 24 h to [(3)H]acetylated rabbit immunoglobulins known to bind to the cell membrane either specifically (anti-plasma membrane IgG: A anti-PM IgG) or unspecifically (contol IgG: AC IgG). As a result of immunological interaction between the two antibodies (no effect was found if the cells had been preloaded with control goat FC IgG), a substantial portion of the stored F anti-R IgG was unloaded from its intracellular storage site, appearing in the medium in the form of soluble immune complexes with rabbit A IgG. Part of the unloaded F anti-R IgG also was recovered in association with the plasma membrane, but only when A anti-PM IgG was used. In addition, significant reverse translocation of AC IgG from plasma membrane to lysosomes or some related intracellular storage compartment was also observed. With A anti-PM IgG, this translocation was less marked and affecte at the same time the plasma membrane marker 5'- nucleotidase. Cells that had stored horseradish peroxidase (HRP) simultaneously with F anti-R IgG did not unload HRP when exposed to A anti-PM IgG. These results support strongly, though not unequivocally, the concept that plasma membrane patches interiorized by endocytosis are recycled, or shuttled, back to the cell surface. In the framework of this concept, recycling antibody-coated membrane is taken to serve as vehicle for the selective intracellular capture and extracellular discharge of immunologically bound F anti-R IgG. The alternative explanation of regurgitation triggered off by immune complexes is considered less likely in view of the lack of HRP unloading.


Binding of hyaluronate to the surface of cultured cells.

The binding of hyaluronate to SV-3T3 cells was measured by incubating a suspension of cells (released from the substratum with EDTA) with 3H-labeled hyaluronate and then applying the suspension to glass fiber filters which retained the cells and the bound hyaluronate. The extent of binding was a function of both the concentration of labeled hyaluronate and the cell number. Most of the binding took place within the first 2 min of the incubation and was not influenced by the presence or absence of divalent cations. The binding of labeled hyaluronate to SV-3T3 cells could be prevented by the addition of an excess of unlabeled hyaluronate. High molecular weight preparations of hyaluronate were more effective in preventing binding than low molecular weight preparations. The binding of [3H]hyaluronate was inhibited by high concentrations of oligosaccharide fragments of hyaluronate consisting of six sugars or more, and by chondroitin. The sulfated glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparin, and heparan sulfate) had little or no effect on the binding. The labeled hyaluronate bound to the cells could be totally removed by incubating the cells with testicular hyaluronidase, streptomyces hyaluronidase, or trypsin, indicating that the hyaluronate-binding sites are located on the cell surface.


Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography.

We have used fiber autoradiography to examine the DNA product made in vitro in a lysed cell system. CHO cells were treated with 0.01% Brij-58 and the lysates were incubated at 30 degrees C in a complete reaction mixture for in vitro DNA synthesis with [3H]thymidine triphosphate ([3H]TTP) as the radioactive tracer. Fiber autoradiograms prepared from the DNA showed that it was synthesized on tandemly arranged replication units that were of average size of 20 micrometers, very similar to the size of units found in vivo. The rate of replication fork movement was 25--50% of the in vivo rate. More than 80% of forks stopped functioning by 15 min, and 95% stopped by 60 min. This suggests that synthesis is halted by premature terminations. Evidence for new initiations was provided by replication units with labeled origins in DNA synthesized in an in vitro reaction in which radioactivity was omitted for the first 10 min of incubation. This, plus the observations that the distance between initiation points (replication unit size) is not increased and that premature termination accounts largely for the cessation of synthesis, suggest that significant initiation takes place in this in vitro replication system.


Clusters of intramembrane particles associated with binding sites for alpha-bungarotoxin in cultured chick myotubes.

Developing chick myotubes in tissue culture were freeze-fractured to yield complementary replicas of large areas of membrane. Regions of muscle fibers with high concentrations of acetylcholine receptors were identified by binding of fluorescent-labeled alpha-bungarotoxin. Membranes in such regions contained clusters of large (100 A Diam) angular particles, similar in appearance to particles found in postsynaptic membranes of cholinergic synapses. Particles appeared in apposing areas of cytoplasmic and external leaflets but were more prevalent in the cytoplasmic leaflet. The areas of high particle concentration were coextensive with the fluorescence due to bound toxin. Treatment of cultures with tetrodotoxin increased the size of fluorescent spots and areas of high concentration of particles relative to those found in control cultures. In muscle cultures grown in the presence of spinal cord explants, some neurites contacted and innervated nearby myotubes. Intense fluorescence due to binding or alpha-bungarotoxin was present at portions of such neurite-myotube contacts. At these same portions, a high concentration of large angular particles was present in the sarcolemma adjacent to the neurite. In addition, an ordered arrangement of large particles was seen in the cytoplasmic leaflet of the neuronal plasmalemma directly apposing the muscle. The possible significance of these arrangements is discussed. Clusters on myotubes tended to be larger (contain more particles) when they occurred in groups, defined as three or more clusters with an intercluster distance of less than 0.5 micrometers. Clusters were also larger in myotubes treated with tetrodotoxin and in myotubes adjacent to some neurites in nerve-muscle cocultures. Several depressions containing particles similar to those in the clusters were found in the sarcolemma. The implications of these depressions are discussed in light of current theories of incorporation of proteins into cell membranes.


Sensory adaptation of leukocytes to chemotactic peptides.

The morphology and behavior of polymorphonuclear leukocytes (PMNs) were studied after rapid changes in the concentration of a chemotactic factor N-formylnorleucylleucylphenylalanine (f-NorleuLeuPhe) (Schiffmann et al., 1975, Proc. Natl. Acad. Sci. U. S. A. 72:1059--1062). After an increase in peptide concentration, the cells round, form lamellipodia or ruffles over most of their surface, and stop locomotion. These changes are transient. After a delay, the cells, still in the presence of peptide, withdraw most of the ruffles and resume locomotion, forming ruffles only at their front. Cells repeat the transient generalized ruffling upon further increase in peptide concentration. The behavioral changes occur over the same dose range as binding to a saturable receptor. The duration of the transient response after a concentration increase is roughly proportional to the increase in the number of cell receptors occupied as a result of the concentration change. Decreasing the concentration of peptide causes the cells to round transiently and form blebs before they recommence locomotion. The transient nature of these aspects of the cell's responsiveness to chemotactic factors appears to be due to adaptation by the cells. The ability to adapt to the concentration of a chemotactic factor may be important in leukocyte chemotaxis.


Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers.

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.


Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle.

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.