Isolation and incorporation of rabbit liver epoxide hydrase into phospholipid vesicles. Methods are described for the incorporation into phospholipid vesicles of epoxide hydrase isolated from liver microsomes of phenobarbital-treated rabbits. Chromatography on a Sephadex G-50 column of epoxide hydrase and egg yolk phosphatidylcholine treated with sodium cholate yielded homogeneous vesicles with a diameter of about 25 nm and containing 80 to 85% of the protein applied. At high substrate concentrations, the vesicles catalyzed the hydration of benzo(a)pyrene-4,5-oxide and styrene-7,8-epoxide at a rate similar to that obtained with the enzyme in a soluble form. However, the kinetics of styrene glycol formation catalyzed by the vesicular or microsomal preparations were complex. Convex Lineweaver-Burk plots and concave Hill plots were obtained, whereas normal Michaelis-Menten kinetics characterized the hydration catalyzed by the enzyme in a soluble form. The results could be explained if reconstitution of the enzyme into the vesicles gives rise to low affinity high capacity sites for the substrate on the enzyme, or alternatively facilitates the interaction of the substrate with such sites already present. It is suggested that reconstituted liposomes containing both the liver microsomal hydroxylase system and epoxide hydrase may prove to be a good model system for evaluating substrate specificity and factors of importance in the formation of toxic and carcinogenic metabolites by these enzymes.


Effect of implant surface chemistry upon arterial thrombosis. A series of poly(alpha-amino acid)s with controlled chemical variations were investigated in order to assess the effect of different chemical moieties upon arterial thrombosis. The gross implant surface properties ranged from hydrophobic to hydrophilic ionic and nonionic. The materials were tested by implantation within canine femoral and carotid arteries. Results were compared with the response to the polyurethane Biomer. The changes in implant surface chemistry elicited a range of response that varied from intense thrombosis and rapid vessel occlusion to minimal thrombosis and endothelialization. The results showed that no simple relationship exists between a gross surface property, such as hydrophobicity, and the degree of thrombosis resistance. Some hydrophobic and hydrophilic materials were found to have good thrombosis resistance, while others were found to have poor thrombosis resistance. Leukocytes were shown to play an important role in both initial thrombosis and endothelialization. The major difference between materials that progressed to rapid vessel occlusion and materials that remained patent was the degree of direct leukocyte adherence and spreading on the implant surface prior to extensive platelet aggregation (less than 30 min). It was consistent for both hydrophobic and hydrophilic materials that the lack of direct leukocyte adherence to the implant surface was associated with intense thrombosis and rapid vessel occlusion. Conversely, the presence of numerous leukocytes directly adherent to either hydrophobic or hydrophilic surfaces appeared to have a moderating effect upon thrombosis and vessels with these implants remained patent. In instances when thrombosis was nonocclusive, the surfaces of the thrombi became endothelialized, primarily through the transformation of mononuclear leukocytes into endothelial cells. This article includes a hypothetical model representing the sequence of events and alternative pathways occurring at the blood-material interface, with special attention given to the involvement of leukocytes in arterial thrombosis.


Durability of the bond between bone and various 2-cyanoacrylates in an aqueous environment. The durability of the bond strength developed between 2-cyanoacrylate esters and bone has been determined by aging specimens in water. One-day bond strength of the isobutyl and isomeric amyl 2-cyanoacrylates varied from 6.2 to 7.2 MPa. The strength of the bond decreased on storage or on thermocycling in water. Hydrolytic stability increased with increasing length of the alkyl ester group. After a six-month storage in water the various amyl 2-cyanoacrylates retained from 70% to 73% of their one-day bond strength. Pretreatment of the bone surface prior to application of the adhesive did not prove beneficial. The cured 2-cyanoacrylate can be removed from the substrate surface by appropriate solvents. Thus, it is not bonded covalently to bone. The bond strength, especially of the isobutyl and amyl 2-cyanoacrylates to bone in an aqueous environment, appears to be superior to other adhesives. Provided these monomers are biocompatible, they may be useful clinically where an intermediate-term adhesion is desired.


Mechanical properties of antibacterial silicone rubber for hydrocephalus shunts. In an effort to find a solution to the serious problem of bacterial colonization of cerebrospinal-fluid shunting devices, room-temperature and heat-vulcanizing silicone rubbers were impregnated with gentamicin sulphate. The effects on the mechanical properties of the two rubbers were studied. Results show that the tensile strength and extensibility of the room-temperature-vulcanizing silicone rubber reduced with increasing concentration of the drug. For the heat-vulcanizing silicone rubber, the tensile strength was also found to decrease with increasing drug content. The extensibility after an initial reduction at low concentrations was found to increase at drug concentrations in excess of 10 mg/g. Nevertheless, the changes in mechanical properties measured are considered not to be so great as to preclude the application of drug-impregnated silicone rubbers to cerebrospinal-fluid shunting devices.


Influence of cold working on fatigue behavior of stainless steels used for prothesis: application to the study of wires with small sections. Orthodontists mainly use stainless austenitic wires with a wide range of ultimate tensile strength. To characterize the dynamical behavior of these wires, the authors have perfected machines to test materials with their original surface roughness without any prior machining of surfaces. Fatigue limits are discussed through mechanical and structural properties of the austenitic steels, with special attention given to cold working. Typical features of fracture, and particularly of the first cracks, are shown by means of scanning electron microscopy.


Reconstruction of the hip. A mathematical approach to determine optimum geometric relationships. The normal mechanical function of the hip is substantially altered by a variety of disorders. The surgical treatment of such conditions, particularly total hip replacement, offers the opportunity not only to replace the articular surfaces of the joint, but also to improve long-term mechanical function by reducing the loads on the joint. A mathematical model of the hip was developed to evaluate the effects of such surgically achievable mechanical alterations as acetabular placement, femoral shaft-prosthetic neck angle, neck length of the femoral prosthesis, and transfer of the greater trochanter. The loads on the hip were lowered significantly by placing the center of the acetabulum as far medially, inferiorly, and anteriorly as was anatomically feasible. Minimum joint contact forces occurred when the femoral shaft-prosthetic neck angles were small, while the minimum moments about the prosthesis stem-neck junction were found when the angles were 130 to 140 degrees. A neck length of the femoral prosthesis of thirty-five millimeters resulted in moments that were lower than those for a neck length of forty-five millimeters. Lateral transfer of the greater trochanter reduced hip-joint forces and moments but distal transfer had little mechanical effect.


The effect of total hip replacement and general surgery on antithrombin III in relation to venous thrombosis. This investigation was undertaken to identify and correlate one factor that makes patients undergoing total hip replacement more susceptible to venous thrombosis and pulmonary embolism than those who have almost any other elective orthopaedic procedure, and to determine why the operation of total hip replacement has proved to be relatively resistant to antithrombotic prophylaxis compared with general surgical procedures. Using the depletion of antithrombin III as a marker of activation of the coagulation system, two groups of patients were compared: twenty-one who were subjected to hip arthroplasty and fourteen who underwent general surgical procedures. Both during and after operation the decrease in the quantity of antithrombin III in hip-arthroplasty patients was significantly greater (p less than 0.05) than the decrease in general surgical patients. Seventy-three per cent of hip-replacement patients had venographic evidence of recent thrombosis, 60 per cent of which were discontinuous femoral-vein thrombi. Femoral-vein thrombosis occurs frequently in hip-arthroplasty patients and is relatively resistant to current antithrombotic prophylaxis. The data presented suggest that during hip surgery there is a strong systemic activation of the clotting cascade that is associated with local vessel injury and local stasis in the femoral vein, an association not found in most general surgical procedures.


A method for the postmortem evaluation of an in situ total hip replacement. A method was developed for the post-mortem evaluation of a total hip replacement retrieved in situ. The hip replacement had been implanted for six years. The evaluation procedure employed existing techniques in a logical sequence such that earlier tests would not compromise results from subsequent ones. These techniques included the measurement of range of motion, aspiration of the joint for analysis of debris, radionuclide arthrograms for determination of looseness of the components, gross and histological inspection of the capsule and synovial tissue, serial sectioning of the femoral component, determination of some mechanical properties of the bone and femoral stem, and scanning electron microscopy of the articulating surfaces of the prosthetic components. Positive findings in the specimen studied were: excessive wear of the articulating surface of the acetabular component, and associated polyethylene debris in the aspirate and surrounding synovial and granulation tissue.


Glenohumeral arthrodesis. Operative and long-term functional results. Seventy-one shoulders of seventy patients were fused for treatment of various conditions, and the results were analyzed after an average follow up of nine years and six months. The operative technique always included the use of internal fixation. The average position of arthrodesis was 45 degrees of abduction and 25 degrees of flexion of the arm, with the flexed forearm rotated 21 degrees above the horizontal plane, measured with the arm abducted and flexed. In sixty-eight shoulders, one operation achieved a solid fusion; in the other three, a second arthrodesis was required. Complications included tenderness over the outer ends of the internal fixation device, which required its removal from seventeen shoulders; a fracture in the fused extremity in ten patients; and a post-operative infection in one. Relief of pain was adequate in three-quarters of the patients. Three-quarters of the patients could perform activities involving reaching the trunk, one-half could do activities requiring reaching the head, and one-quarter were able to do light work with the arm at shoulder level or higher. The position of fusion had little effect on the result. Eighty-two per cent of the entire group believed that they had benefited from the operation, and none of the results deteriorated with time.


Vertebral excision for kyphosis in children with myelomeningocele. For twenty-three children with myelomeningocele, three types of operations, all including vertebral-body excision, were done to decrease and stabilize the kyphosis: (1) excision of the apical vertebra and a portion of the neighboring vertebra was done in five patients; (2) excision of the apical vertebra and of one or more vertebrae on each side of it, in eight patients; and (3) partial resection of the apical vertebra and of the proximal lordotic curve, in twelve patients. Loss of correction and increase of the kyphotic prominence equal to or greater than preoperative levels were encountered after the first two operations. The third operation provided persistent correction and the remaining lumbar vertebrae then continued to grow, increasing the capacity of the abdominal cavity.


Lateral hamstring transfer and gait improvement in the cerebral palsy patient. A retrospective analysis of twenty-three spastic patients who underwent forty-three transfers of the semitendinosus muscle to the lateral intramuscular septum and of the semimembranosus muscle to the biceps is presented. Decreased knee-flexion deformity as well as improved walking function were achieved in 91 per cent. An unsatisfactory result was associated with complications of the procedure. Only one knee of the forty-three that were operated on showed late genu recurvatum. This procedure appears to be both effective and relatively free of late comlications.


An innovative removable rigid dressing technique for below-the-knee amputation. The below-the-knee removable rigid dressing is a below-the-knee plaster cast held by a suspension stockinette to a supracondylar plastic cuff. It has proved to be an effective method for postoperative, pre-prosthetic, and prosthetic care of below-the-knee amputees. Being removable, it permits frequent observation and progressive shrinkage of the stump (by adding socks), and eliminates the need of elastic stump bandaging. In addition, it still maintains the advantages of immobilization of soft tissue (to reduce pain and facilitate wound healing) and prevention of trauma to the stump as does the conventional rigid dressing. It has significantly reduced the incidence of pre-tibial skin breakdown and distal edema, produced fast stump shrinkage, and shortened time to ambulatory discharge with a temporary prosthesis by ninety days.


A fluted intramedullary rod for subtrochanteric fractures. A new intramedullary rod has been developed for the treatment of subtrochanteric fractures. The stepped fluted rod is designed as a single unit and has exceptional bending strength and rigidity as well as excellent torsional load-carrying capacity. It has been used successfully in eighteen patients with a variety of subtrochanteric fractures. Union was achieved in all instances and no failure of the implant occurred. The simplified technique of insertion, the strength of the device, and the results of this study indicate that the fluted subtrochanteric rod has several advantages over other available devices.


Congenital radio-ulnar synostosis: surgical treatment. The results of an operative approach to the problem of radio-ulnar synostosis were assessed in thirteen patients, ten to twenty-five and one-half years after the procedure was performed. We concluded that in a patient with bilateral synostosis one hand, the one not used in writing, should be shifted to a position of 20 to 35 degrees of supination. With one hand in this position, the other may be left in considerable pronation. Often after such a shift it is not necessary to rotate the second arm. However, if the pronation is marked in the second forearm, and if function is impaired unduly by this position, surgical correction is indicated. The arm should be placed in a position of 30 to 45 degrees of pronation. In unilateral radio-unlar synostosis, the ordinarily ideal position of the radius is between 10 and 20 degrees of supination. In an adult, the patient's occupation should be considered in deciding on the rotatory positions of the forearms. We usually prefer a method of transverse osteotomy through the conjoined mass of the radius and ulna. Careful observation of the effect on the vascular status of the limb during and immediately after surgery is important.


In vitro measurement of articular cartilage deformations in the intact human hip joint under load. Using a new roentgenographic technique for measuring cartilage deformation in intact joint specimens, twenty-eight normal human hip joints from subjects twenty-five to eighty-five years old were loaded with a force of five times body weight in a testing machine. The initial unloaded thickness of the articular cartilage of the femoral head and the changes in thickness of this cartilage under load were measured roentgenographically at seven to twelve sites on each femoral head. These measurements showed that the deformations of femiral-head articular cartilage under load in the intact joint are non-uniform and increase greatly with age. In twelve specimens measurements were also made of the increase in cartilage deformation with time when the load of five times body weight was maintained on the joint. A single osteoarthrotic joint was also studied. The experimental findings imply changes in the fundamental mechanical properties of the cartilage with age, which probably result from age-related alterations in cartilage microstructure and chemical composition.


Normal range of motion of joints in male subjects. With a clinical goniometer, we measured the arcs of active motion of the shoulder, elbow, forearm, wrist, hip, knee, ankle, and foot in 109 normal male subjects ranging in age from eighteen months to fifty-four years old. The normal limits were determined for subjects who were one to nineteen years old and for those who were twenty to fifty-four years old. Significant differences were found between the two age groups for most motions. The data were compared with estimations in the handbook of The American Academy of Orthopaedic Surgeons as the standard reference. The data constitute a more detailed set of measurements, based on a sample described according to height and age, than has been available hitherto.


Congenital intrathoracic bronchopulmonary foregut malformations in childhood. Bronchopulmonary foregut malformations (BPFM) are a group of related disorders. They include tracheoesophageal fistula with or without atresia, bronchopulmonary sequestration, intestinal duplication cyst, bronchogenic cyst, aberrant systemic arterial supply to the lung, and bronchial mucosal rests in the esophagus. This paper describes 12 cases which illustrate the spectrum of clinical and radiographic features of these anomalies. The embryogenesis of these malformations is reviewed and appropriate radiographic investigation is discussed.


Hill-Sachs deformity with an unusually large defect. Anterior shoulder dislocation may cause a compression fracture in the posterolateral aspect of the humerus, known as the Hill-Sachs deformity. We wish to present a patient with an unusually large defect.


Intrathoracic desmoid at previous thoracotomy site. Desmoid tumor (aggressive fibromatosis) of the chest wall is rare and usually extrathoracic. A large extrapleural intrathoracic desmoid occurred at the site of previous thoracotomy, and recurred five years later.


Cerebral aneurysms and cardiac myxoma.

Cerebral aneurysms and cardiac myxoma. Cerebral aneurysms with a characteristic pattern may arise as a complication of cardiac myxomas. In our patient, hemiparesis resulted 12 years after the myxoma was resected.


Primary pulmonary sporotrichosis.

Primary pulmonary sporotrichosis. The diagnosis of primary pulmonary sporotrichosis was made in a 53-year-old male alcoholic four years after initial presentation with cavitary lung disease. Clinical and radiologic awareness of this extremely rare entity will allow optimal study of bronchial secretions in the bacteriology laboratory. Important clues include: (1) exposure to thorny plants, (2) cavitary pulmonary disease resistant to both diagnosis and treatment, and (3) a history of alcoholism.


False negative mammograms causing delay in breast cancer diagnosis. Eighty patients with negative mammograms and proven breast cancer are described. The incidence of negative mammograms is 5 to 7% of all breast cancer in Northern Alberta. In 30 patients, immediate biopsy because of clinical abnormality, and mastectomy, revealed only 3.3% axillary node involvement. The remaining 50 patients gave a history of finding a lump and then had a negative mammogram. Biopsy was delayed for a mean of 45 weeks. Their incidence of axillary node involvement was 42%. The mean age of both groups of patients was seven to eight years younger than the mean for all breast cancer patients. The implications of such delay on prognosis and survival are obvious. More public and medical education is needed.


Brain stem tumors in children (a review of 26 cases 1960--1976). We reviewed 26 patients with brain stem tumors. None had a pre-treatment tissue diagnosis. Four of the 20 treated patients are alive from 22 months to 12 years later, representing no improvement from a preceding series in the same centre.


An improved radiographic view of the glenohumeral joint. In addition to the standard views of the shoulder (external rotation, internal rotation, and axillary), a 40-degree posterior oblique and a 60-degree anterior oblique projection were used to evaluate 105 consecutive shoulder examinations. Of the 32 abnormal shoulders evaluated, the diagnosis was established 97% of the time with a single 40-degree posterior oblique view, as opposed to a 78% rate with the three standard views. The 40-degree posterior oblique view of the shoulder is especially useful in evaluating the glenohumeral joint.


Radiographic gradation of lumbosacral dysgenesis. The caudal dysgenesis syndrome comprises partial or complete agenesis of the lumbosacral vertebrae, as well as associated musculoskeletal, neurologic and visceral abnormalities. Maternal diabetes mellitus appears to be the most important etiologic factor, but for most patients the etiology is unknown. In the majority of cases, radiologic diagnosis is established readily, but minor degrees often coinciding with a serious urologic disease or a lower limb abnormality may be overlooked.


Carpal involvement in inflammatory (erosive) osteoarthritis. Inflammatory or erosive osteoarthritis, in addition to the classical digital distribution, may involve the carpus. Characteristic carpal changes are restricted to the radial aspect of the wrist and consist of narrowing, eburnation, osteophytosis, and mild subluxation of the first carpometacarpal and trapezio-scaphoid joints. Radiographic findings and differential diagnosis are discussed.


Lymphangiomas in children: report of 121 cases. We reviewed 121 children with lymphangiomas. The commonest sites of involvement are the extremities and trunk followed by the head and neck. Four patients with unusual lymphangiomas are presented in some detail. Clinical and radiographic features may strongly suggest the presence of a lymphangioma.


The periodic association of MAP2 with brain microtubules in vitro. Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP1 (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/ml. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately 1 mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.


Mechanisms of transcription in nucleoli of amphibian oocytes as visualized by high-resolution autoradiography. In oocytes of Pleurodeles waltlii, the method of Miller and Beatty has been combined with a method of high-resolution autoradiography especially suitable for the study of isolated molecules. In vitro labeling of RNA by tritiated precursors was carried out with increasing incubation times (1, 4, 15, 24, 48, and 72 h). Silver grains were present over ribonucleoprotein fibrils in amounts sufficient for quantitative analysis of nucleolar DNA transcription. Statistical analysis of the data revealed that: (a) The units of any one nucleolus exhibited a large degree of heterogeneity in their number of grains. (b) There was a parallelism between the increasing grain number and the ribonucleoprotein-fibril lengthening as observed along the transcription unit.


Morphological studies of stimulated adrenergic axon varicosities in the mouse vas deferens. The postganglionic axons of sympathetic neurons innervating the mouse vas deferens were stimulated transmurally in vitro by passing square pulses between two platinum electrodes. The ultrastructural appearance of the adrenergic nerve terminals was compared to samples fixed immediately after 30 min of stimulation and in samples allowed to recover for 2 h before fixation. The contralateral vasa deferentia served as controls, and these were incubated in Krebs solution for the same period as stimulated muscles. For each of four experiments, the mean number of large and small dense-core vesicles per square micrometer was calculated, as were the mean area and perimeter of the axon varicosities in each group. It was found that the number of small vesicles per square micrometer decreased by 60% during the stimulation period, but returned almost to control levels 2 h later. Large vesicles did not change in number during the stimulation or recovery periods. The proportion of vesicles containing cores was also determined for each group and found to decline just after stimulation in the small vesicle population, but to remain constant in the large vesicle population. The core depletion was partly reversed after 2 h. The vesicle recovery process was studied by use of the extracellular tracer horseradish peroxidase (HRP). When HRP was present in the extracellular space during stimulation, large numbers of vesicles contained the marker after recovery from stimulation. Thus, it is proposed that adrenergic axon varicosities recycle vesicle membrane through the plasma membrane in a manner similar to that already described for cholinergic nerve terminals.


Studies on the function of cell surface glycoproteins. I. Use of antisera to surface membranes in the identification of membrane components relevant to cell-substrate adhesion. An antiserum prepared against purified surface membranes of transformed BHK21/C13 cells (C13/B4) reversibly rounded and detached hamster cells from plastic tissue culture plates but did not affect cells of other species. Antiserum treatment did not alter the growth rate of C13/B4 or BHK21/C13 cells; however, NIL-8 cells exposed to the antiserum detached from the substrate and stopped growing, but remained viable for up to 72 h in the presence of the antiserum. Rounding and detachment were not inhibited by DNP or cycloheximide. Antiserum-detached cells did not reattach in the presence of these inhibitors. F(ab)' fragments also induced rounding, thus ruling out the involvement of complement and ligand-induced rearrangement of surface antigens in rounding and detachment. Three different surface-reactive immunoglobulin preparations were used in indirect immunoprecipitation studies in an attempt to identify cell surface antigens involved in regulating adhesion and morphology. Antiserum against surface membranes (anti-M) and against material shed by the cells into serum-free medium (anti-SFM) caused rounding and detachment, but a third antiserum (anti-LIS) prepared against a partially purified glycoprotein did not. All three immunoglobulin preparations precipitated glycoproteins with an apparent mol wt of 120,000 daltons from a crude membrane preparation solubilized by Nonidet NP-40. The two immunoglobulin preparations that caused rounding precipitated an additional glycoprotein peak of 140,000 daltons. Extensive preabsorption of the extract with anti-LIS immunoglobulin enriched the anti-membrane and antiserum-free medium precipitates for the 140,000-dalton peak. Anti-M immunoglobulin eluted from intact cells and subsequently used to precipitate NP-40 solubilized membrane constituents also reacted with a group of glycoproteins of approximately 140,000 mol wt. Therefore, this group of glycoproteins was considered most likely to be the glycoproteins involved in substrate adhesion and maintenance of cellular morphology.


Studies on the function of cell surface glycoproteins. II. Possible role of surface glycoproteins in the control of cytoskeletal organization and surface morphology. Immunoglobulin from goat antiserum directed against purified surface membranes from transformed BHK21/C13 cells (anti-M) has been shown to cause both control and transformed hamster cells to round and detach from the substrate (see accompanying paper). This paper documents the effects of the antiserum on the cytoskeletal organization and cell surface morphology of control BHK21/C13 cells examined by scanning and transmission electron microscopy. As a result of antiserum-induced rounding, the normally smooth cell surface becomes covered with filopodia and blebs, and the organization of all three components of the filamentous cytoskeleton is altered. In terms of cell surface morphology and cytoskeletal organization, the cells resemble rounded, postmitotic or trypsinized BHK cells rather than cells treated with either anticytoskeletal drugs or lectins. Immunocytochemical and radioimmune assay experiments support the suggestion that the rounding reaction induced by anti-M serum results from the specific interaction of antibodies with molecules on the cell surface. It is suggested that anti-M serum induces alterations in cytoskeletal organization via a transmembrane signal and that cytoskeletal reorganization is a fundamental part of the rounding and detachment process.


Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study. Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.


Element concentration changes in mitotically active and postmitotic enterocytes. An x-ray microanalysis study. Unfixed freeze-dried and uncoated tissue sections of the mouse duodenum were suspended across a hole in a carbon planchet and analyzed in a scanning electron microscope fitted with energy-dispersive x-ray analytical equipment. Computer analysis of the x-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and late anaphase-early telophase chromatin regions in the cryptal and villus enterocytes. Elemental concentrations (mmol/kg dry wt) were measured for Na, Mg, P, S, Cl, K, and Ca. None of the elements were compartmentalized preferentially in either the nucleus or the cytoplasm of interphase enterocytes of crypts or in postmitotic enterocytes of villi. In contrast, Ca, S, and Cl are detectable in significantly higher concentrations in mitotic chromatin of dividing enterocytes of the crypt as compared to surrounding mitotic cytoplasm, but Na, Mg, and P are in lower concentrations in the mitotic chromatin as compared to mitotic cytoplasm. Interphase enterocytes of crypts have higher concentrations of Mg, P, and K, and lower concentrations of Na than do postmitotic enterocytes of villi.


Quantitative conservation of chromatin-bound RNA polymerases I and II in mitosis. Implications for chromosome structure. RNA synthesis almost ceases in mitosis. It is ambiguous whether this temporal, negative control of RNA synthesis is solely because of the nature of chromosomes per se, (i.e., their condensed state), or to a physical loss of RNA polymerases along with other nuclear proteins which have been shown to pass into the cytoplasm in mitosis, or to their combined feature. Aside from such regulatory considerations, a question has also been raised as to whether RNA polymerases are constituents of metaphase chromosomes. To clarify these aspects of RNA polymerase-chromatin interaction in mitosis, the enzymes in chromosomes were quantitated and their levels compared to those in interphase nuclei and cells at various phases of the cell cycle. The results show that the amounts of form I, form II, and probably form III enzymes bound to a genome-equivalent of chromatin stay constant during the cell cycle. Thus, the mechanism for the negative control of RNA synthesis in mitosis appears to exist in the chromosomes per se, but not to be directly related to the RNA polymerase levels. This quantitative conservation of chromatin-bound RNA polymerases implies that they may persist as structural components of the chromosomes in mitosis.


The role of actin in temperature-dependent gel-sol transformation of extracts of Ehrlich ascites tumor cells. Ehrlich ascites tumor cell extracts form a gel when warmed to 25 degrees C at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0 degrees C. This gel-sol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg2+, KCl, and a reducing agent. Gelation was inhibited reversibly by 0.5 microM free Ca2+, and 25--50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt greater than 300,000, 270,000, 89,000, 51,000, 48,000, and 42,000 daltons. The last component was identified as cell actin because it had the same molecular weight as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Gelation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter, and it also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, the extracts showed temperature-dependent, cytochalasin-sensitive, and Ca2+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation.


Domains of receptor mobility and endocytosis in the membranes of neonatal human erythrocytes and reticulocytes are deficient in spectrin. It has previously shown (Schekman, R., and S.J. Singer, Proc. Natl. Acad. Sci. U.S.A. 73:4075-4079) that receptors in the membranes of neonatal human erythrocytes show a restricted degree of lateral mobility, whereas in adult human erythrocytes the receptors are essentially immobile. This restricted mobility is exhibited, for example, when concanavalin A (Con A) induces a limited clustering of its receptors in the neonatal erythrocyte membrane, resulting in the formation of invaginations and endocytic vesicles. This does not happen with adult cells. By the use of indirect immunoferritin labeling of ultrathin frozen sections of Con A-treated neonatal blood cells, we now show that the invaginations and endocytotic vesicles do not stain for spectrin, whereas the adjacent unperturbed membrane is heavily stained. The reticulocytes in the neonatal cell population undergo substantially more Con A-induced invagination and endocytosis than do the erythrocytes. These results lend strong support to the hypothesis that specialized discrete domains exist, or are induced, in the membranes of these neonatal cells, in which receptors are laterally mobile, whereas in the remaining (and predominant) part of the membrane the receptors are immobile. Such mobile domains are characterized by an absence of spectrin. During the maturation of the neonatal reticulocyte to erythrocyte, it is proposed that these domains are in large part, but not completely, eliminated.


Concanavalin A-induced endocytosis in rabbit reticulocytes, and its decrease with reticulocyte maturation. Concanavalin A (Con A) was taken up to a limited extent by endocytosis in rabbit reticulocytes but not in rabbit erythrocytes. This process was observed by the use of ferritin-labeled Con A and transmission electron microscopy of thin sections of plastic-embedded cells. Furthermore, the extent of endocytosis among the reticulocytes decreased with the extent of their maturation, reticulocyte age being measured by ribosome configurations. These results are consistent with the proposal that there are domains in the membranes of reticulocytes in which the Con A receptors are laterally mobile, and can be clustered and endocytosed. These mobile domains exist, or are formed, within a larger framework of immobile membrane. During reticulocyte maturation, these domains are gradually eliminated, eventually disappearing upon formation of the mature erythrocyte. Possible molecular mechanisms for this proposed elimination process are discussed.


Fibroblast cellular and plasma fibronectins are similar but not identical. Fibronectins are multimeric, adhesive glycoproteins present on cell surfaces and circulating in blood. Cellular fibronectin produced by fibroblasts in vitro and fibronectin isolated from plasma are known to be very similar immunologically and biochemically. We investigated whether or not they are identifical. Purified chicken and human cell-surface fibronectins are 150-fold more active in hemagglutination of fixed erythrocytes than plasma fibronectins. Cell-surface fibronectin is also 50-fold more active in restoring a more normal morphology to transformed cells originally missing the protein. However, in two other assays that measure cell attachment to collagen and cell spreading, cell-surface and plasma fibronectins have identical specific activities. In sodium dodecyl sulfate polyacrylamide gels, the subunits of human and chicken plasma fibronectins have significantly smaller apparent subunit molecular weights than cellular fibronectins present on cell surfaces or secreted into culture media. These differences are also present in a characteristic large subfragment of both forms of fibronectin after limited proteolysis by trypsin. We conclude that by both biological and biochemical criteria, cellular and plasma fibronectins are similar but not identical.


Motility of the microtubular axostyle in Pyrsonympha. The rhythmic movement of the microtubular axostyle in the termite flagellate, Pyrsonympha vertens, was analyzed with polarization and electron microscopy. The protozoan axostyle is birefringent as a result of the semi-crystalline alignment of approximately 2,000 microtubules. The birefringence of the organelle permits analysis of the beat pattern in vivo. Modifications of the beat pattern were achieved with visible and UV microbeam irradiation. The beating axostyle is helically twisted and has two principal movements, one resembling ciliary and the other flagellar beating. The anterior portion of the beating axostyle has effective and recovery phases with each beat thereby simulating the flexural motion of a beating cilium. Undulations develop from the flexural flipping motion of the anterior segment and travel along the axostyle like flagellar waves. The shape of the waves differs from that of flagellar waves, however, and are described as sawtooth waves. The propagating sawtooth waves contain a sharp bend, approximately 3 micron in length, made up of two opposing flexures followed by a straight helical segment approximately 23 micron long. The average wavelength is approximately 25 micron, and three to four sawtooth waves travel along the axostyle at one time. The bends are nearly planar and can travel in either direction along the axostyle with equal velocity. At temperatures between 5 degrees and 30 degrees C, one sees a proportionate increase or decrease in wave propagation velocity as the temperature is raised or lowered. Beating stops below 5 degrees C but will resume if the preparation is warmed. A microbeam of visible light shone on a small segment of the axostyle causes the typical sawtooth waves to transform into short sine-like waves that accumulate in the area irradiated. Waves entering the affected region appear to stimulate waves already accumulated there to move, and waves that emerge take on the normal sawtooth wave pattern. The effective wavelengths of visible light capable of modifying the wave pattern is in the blue region of the spectrum. The axostyle is severed when irradiated with an intense microbeam of UV light. Short segments of axostyle produced by severing it at two places with a UV microbeam can curl upon themselves into shapes resembling lockwashers. We propose that the sawtooth waves in the axostyle of P. vertens are generated by interrow cross-bridges which are active in the straight regions.


Electrophysiological recordings from spontaneously contracting reaggregates of cultured smooth muscle cells from guinea pig vas deferens. Smooth muscle cells were enzymatically dispersed from vasa deferentia of adult male guinea pigs (250-400 g). These cells reassociated in vitro to form monolayers and small spherical reaggregates (0.05-0.3 mm in Diam). Within 48 h of being placed in culture, cells in both types of preparation began to contract spontaneously. The contractions were rhythmic and slow. Cells in the monolayers stopped contracting after approximately 1 wk in vitro, but the reaggregates continued to contract spontaneously for at least 3 wk. Electron microscopy of the reaggregates revealed the presence of thick and thin myofilaments. Overshooting action potentials were recorded in many of the cells penetrated (primarily in reaggregates), and were accompanied by visible contractions of the aggregate or monolayer. Quiescent cells could often be excited by intracellularly applied depolarizing and hyperpolarizing (anodal-break) current pulses. The resting potentials had a mean value of -58 +/- 2 mV. The action potentials were usually preceded by a spontaneous depolarization. The action potentials had slow rates of rise (1--4 V/s) which were unaffected by tetrodotoxin (TTX, 1 microgram/ml), a known blocker of fast Na+ -channels. Verapamil (1 microgram/ml) blocked the action potentials. The mean value of input resistance was 6.9 +/- 0.5 M omega (n = 12). These electrophysiological properties are similar to those of intact adult vas deferens smooth muscle cells. Thus, the cultured adult vas deferens smooth muscle cells retain their functional properties in vitro even after long periods.


Microtubule protein preparations from C6 glial cells and their spontaneous polymer formation. C6 cell tubulin is indistinguishable from hog brain tubulin with respect to its molecular weight, amino acid composition, and colchicine-binding activity. Moreover, microtubule assembly systems from both sources form the same structures: rings, ribbons, tubules, and drug-induced polymers. There is, nevertheless, a difference between the cultured cell and brain systems which lies in the nature of their microtubule-associated accessory proteins. C6 microtubule preparations exhibit few rings at 0 degrees C, have low polymerization yield, and have a low content of accessory proteins. The addition of brain accessory proteins enhances the numbers of rings, and the yield of microtubules, to levels comparable with those of brain preparations. The polymerizing ability of C6 microtubule protein decays much faster than that of brain, but it can be restored by the addition of brain accessory protein. The results suggest that C6 accessory proteins are more labile than their brain counterparts.


Selective neutrophil desensitization to chemotactic factors. In the presence of extracellular calcium and magnesium, a series of chemotactic oligopeptides and C5a caused aggregation of human polymorphonuclear neutrophils (PMNs). This cellular response developed rapidly and began to reverse 2 min after exposure to the chemotactin. In the absence of the bivalent cations, none of the chemotactins stimulated the aggregation response. If cells were first exposed to a chemotactin and then treated with calcium and magnesium, aggregation was detected only after addition of the cations, and the magnitude of the response fell sharply as the interval between the addition of chemotactin and addition of cations was lengthened: when this interval exceeded 2 min, aggregation was barely detectable. This loss of reactivity persisted even when cells were re-exposed to fresh chemotactic factor and washed between the first and second exposures. In all instances, however, loss of cellular reactivity was highly selective: cells preincubated with any chemotactic oligopeptide were hyporesponsive to subsequent stimulation with an oligopeptide but remained fully responsive to C5a; cells preincubated with C5A were hyporesponsive to C5a but retained their responsitivity to the oligopeptides. Because this selectivity parallels the known specificities of these chemotactic factors for their receptors in or on the neutrophil, desensitization may reflect functional loss of receptors after stimulation. Alternatively, this selectivity may indicate that morphologically identical neutrophils contain subpopulations of cells with varying reactivities to receptor-bound chemotactic factors. In either event, desensitization may be useful in functionally defining chemotactic factors and their respective receptors. The rapidity of development of desensitization suggests that it may operate to limit or moderate various in vitro and in vivo neutrophil responses to chemotactic factors.


Active movements of the chromatoid body. A possible transport mechanism for haploid gene products. Recent data indicate that the chromatoid body typical of rat spermatogenesis may contain RNA synthesized in early spermatids by the haploid genome. Analyses of living step-1 and step-3 spermatids by time-lapse cinephotomicrography have shown that the chromatoid body moves in relation to the nuclear envelope in two different ways. Predominantly in step 1, the chromatoid body moves along the nuclear envelope on a wide area surrounding the Golgi complex and has frequent transient contacts with the latter organelle. In step 3, the chromatoid body was shown to move perpendicular to the nuclear envelope. It was seen located very transiently at the top of prominent outpocketings of the nuclear envelope with apparent material continuities through nuclear pore complexes to intranuclear particles. The rapid movements of the chromatoid body are suggested to play a role in the transport of haploid gene products in the early spermatids, including probably nucleocytoplasmic RNA transport.


Ligatin from embryonic chick neural retina. Ligatin, a filamentous protein previously found in suckling rat ileum, has been purified from plasma membranes of embryonic chick neural retina. The isolated plasma membranes are covered in part by 4.5-nm filaments that can be released from the membranes by treatment with Ca++. Subsequent dialysis against EGTA followed by sieve chromatography results in purification of the 10,000-dalton ligatin monomer. When labeled either with radioisotopes or with fluorescamine, the monomer is shown to electrophorese as a single discrete band in polyacrylamide gels. However, during standard fixing and staining procedures it diffuses from the gels and thus is not visualized. Ligatin's amino acid composition is distinguished by its high content of polar residues, especially Glx and Asx, and by the presence of phosphorylated serine. Upon re-addition of Ca++, purified ligatin monomers polymerize to form filaments 3 nm in Diam, identical to those formed by purified ileal ligatin. However, in both retina and ileum, the filaments observed on plasma membranes are greater than 3 nm in Diam. In ileum, this enlargement results from ligatin's function as a baseplate for the attachment of another protein, a beta-N-acetylhexosaminidase, to the cell surface. In retina, a corresponding difference in diameter between filaments seen in vivo and those formed from repolymerized ligatin alone and the co-solubilization of other proteins with ligatin suggest that ligatin may also function there as a baseplate for other cell surface proteins. The proteins associated with ligatin in retina differ morphologically from beta-N-acetylhexosaminidase and do not possess this enzymatic activity.


Uptake and release of [3H]gamma-aminobutyric acid by embryonic spinal cord neurons in dissociated cell culture. We have investigated the uptake and release of [3H]gamma-aminobutyric acid (GABA) by embryonic chick spinal cord cells maintained in culture. Cells dissociated from 4- or 7-d-old embryos were studied between 1 and 3 wk after plating. At 3 degrees C, [3H]GABA was accumulated by a high affinity (Km approximately equal to 4 microM) and a low affinity (Km approximately equal to 100 microM) mechanism. The high affinity transport was markedly inhibited in low Na+ media, by ouabain, at 0 degrees C, and by 2,4-diaminobutyric acid. Autoradiography, after incubation in 0.1 microM [3H]GABA, showed that approximately 50% (range = 30-70%) of the multipolar cells were labeled. These cells were neurons rather than glia; action potentials and/or synaptic potentials were recorded in cells subsequently found to be labeled. Non-neuronal, fibroblast-like cells and co-cultured myotubes were not labeled under the same conditions. The fact that not all of the neurons were labeled is consistent with the suggestion, based on studies of intact adult tissue, that high affinity transport of [3H]GABA may be unique to neurons that use GABA as a neurotransmitter. Our finding that none of fifteen physiologically identified cholinergic neurons, i.e., cells that innervated nearby myotubes, were heavily labeled after incubation in 0.1 microM [3H]GABA is significant in this regard. The newly taken up [3H]GABA was not metabolized in the short run. It was stored in a form that could be released when the neurons were depolarized in a high K+ (100 mM) medium. As expected for a neurotransmitter, the K+-evoked release was reversibly inhibited by reducing the extracellular Ca++/Mg++ ratio.


Tight junctions in the choroid plexus epithelium. A freeze-fracture study including complementary replicas. The tight junctions of the choroid plexus epithelium of rats were studied by freeze-fracture. In glutaraldehyde-fixed material, the junctions exhibited rows of aligned particles and short bars on P-faces, the E-faces showing grooves bearing relatively many particles. A particulate nature of the junctional strands could be established by using unfixed material. The mean values of junctional strands from the lateral, third, and fourth ventricles of Lewis rats were 7.5 +/- 2.6, 7.4 +/- 2.2, and 7.5 +/- 2.4; and of Sprague-Dawley rats 7.7 +/- 3.4, 7.4 +/- 2.3, and 7.3 +/- 1.6. Examination of complementary replicas (of fixed tissue) showed that discomtinuities are present in the junctional strands: 42.2 +/- 4.6% of the length of measured P-face ridges were discontinuities, and the total amount of complementary particles in E-face grooves constituted 17.8 +/- 4.4% of the total length of the grooves, thus approximately 25% of the junctional strands can be considered to be discontinuous. The average width of the discontinuities, when corrected for complementary particles in E-face grooves, was 7.7 +/- 4.5 nm. In control experiments with a "tighter" tight junction (small intestine), complementary replicas revealed that the junctional fibrils are rather continuous and that the very few particles in E-face grooves mostly filled out discontinuities in the P-face ridges. Approximately 5% of the strands were found to be discontinuous. These data support the notion that the presence of pores in the junctional strands of the choroid plexus epithelium may explain the high transepithelial conductance in a "leaky" epithelium having a high number of junctional strands. However, loss of junctional material during fracturing is also considered as an alternative explanation of the present results.


Parathyroid hormone biosynthesis. Correlation of conversion of biosynthetic precursors with intracellular protein migration as determined by electron microscope autoradiography. The formation of parathyroid hormone (PTH) in the parathyroid gland occurs via two successive proteolytic cleavages from larger biosynthetic precursors. The initial product coded for by PTH mRNA is pre-proparathyroid hormone (PreProPTH), a polypeptide of 115 amino acids. Within 1 min of synthesis, the polypeptide, proparathyroid hormone (ProPTH), is formed as a result of the proteolytic removal of the NH2-terminal 25 amino acids from Pre-ProPTH. After a delay of 15-20 min, the NH2-terminal six-amino acid sequence of ProPTH is removed to give PTH of 84 amino acids. To investigate the subcellular sites in the parathyroid cell where the biosynthetic precursors undergo specific proteolytic cleavages, we examined, by electron microscopy autoradiography, the spatiotemporal migration of autoradiographic grains and, by electrophoresis, the kinetics of the disappearance of labeled Pre-ProPTH and the conversion of labeled ProPTH to PTH in bovine parathyroid gland slices incubated with [3H]leucine for 5 min (pulse incubation) followed by incubations with unlabeled leucine for periods up to 85 min (chase incubations). By 5 min, 85% of the autoradiographic grains were confined to the rough endoplasmic reticulum (RER). Autoradiographic grains increased rapidly in number in the Golgi region after 15 min of incubation; from 15 to 30 min they migrated within secretory vesicles still in the Golgi region and then migrated to mature secretory granules outside the Golgi area. Electrophoretic analyses showed that Pre-ProPTH disappeared rapidly (by 5 min) and that conversion of ProPTH to PTH was first detectable at 15 min and was completed by 30 min. At later times of incubation (30-90 min), autoradiographic grains within the secretion glanules migrated to the periphery of the cell and to the plasma membrane, in correlation with the release of PTH first detected by 30 min. We conclude that proteolytic conversion of Pre-ProPTH to ProPTH takes place in the RER and that subsequent conversion of ProPTH to PTH occurs in the Golgi complex.


Transverse sarcomere splitting. A possible means of longitudinal growth in crab muscles. Transversely split sarcomeres are seen in mouthpart muscles of the blue crab in the electron microscope. Sarcomeres split only at the H zone. Two new sarcomeres are formed by a Z disk which appears in the H zone of the splitting sarcomere. Splitting may involve breaking of the thick filaments in the H zone, elongation of these filaments, and formation of both new actin filaments and Z-disk materials, Sarcomere splitting would allow longitudinal growth of muscle cells without lengthening of sarcomeres and concomitant changes in contractile properties.


Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin. Regions of plasma membrane of dictyostelium discoideum amoebae that contain concanavalin A (Con A)-receptor complexes are more resistant to disruption by Triton X-100. This resistance makes possible the isolation of Con A-associated membrane fragments in sufficient quantity and homogeneity to permit the direct biochemical and ultrastructural study of receptor-cytoskeletal interactions across the cell membrane. After specific binding of Con A to the cell surface, a large amount of the cell's actin and myosin copurifies with the plasma membrane fragments. Myosin is more loosely bound to the isolated membranes that actin and is efficiently removed by treating membranes with ATP and low ionic strength. If cells are not lysed immediately after lectin binding, all of the Con A that is bound to the cell surface is swept into a cap in a process requiring metabolic energy. When cells are lysed at different stages of cap formation, the amount of actin and myosin that copurifies with the isolated membranes remains the same. Thick and thin filaments that are attached to the protoplasmic surface of the isolated membranes underlie lectin-receptor complexes during all stages of cap formation. Once the cap is complete, the amount of actin and myosin that tightly bound to the plasma membrane is concentrated into the cap along with the Con A-receptor complexes. These results suggest that the ATP-dependent sliding of membrane-associated actin and myosin filaments is responsible for the accumulation of Con A-receptor complexes into a cap on the cell surface.


Distribution of newly formed ribosomal proteins in HeLa cell fractions. The distribution of newly formed ribosomal proteins between cytoplasmic, nucleoplasmic, and nucleolar fractions of HeLa cells was determined. All but a few of the newly formed ribosomal proteins were concentrated 10- to 50-fold in the nucleolus and two- to fivefold in the nucleoplasm. Nevertheless, substantial amounts were found in the cytoplasm. Pretreatment of cells with actinomycin D to deplete the nucleolar pool of ribosomal precursor RNA had no effect on the concentration of newly formed ribosomal proteins in the nucleus, but did lead to an increased amount in the nucleoplasm at the expense of the nucleolus.


Visualization of pores (export sites) correlated with cellulose production in the envelope of the gram-negative bacterium Acetobacter xylinum. The Gram-negative bacterium Acetobacter xylinum assembles a cellulse ribbon composed of a number of microfibrils in the longitudinal axis of its envelope. The zone of ribbon assembly was investigated by freeze-etch electron microscopy. Freeze-etching revealed, beneath the cellulose ribbons, a linear array of pores on the lipopolysaccharide membrane. These pores have a rim diameter of 120--150 A and a central hole or deepening of approximately 35 A. The axes of pore arrays closely coincide with linear arrays of 100 A particles on the E- and P-faces of the fractured lipopolysaccharide membranes. Pores and particles in the lipopolysaccharide membrane are probably congruent. The pores are hypothesized to be the export sites (penetration sites) for cellulose.


Large species differences in the pattern of snPI RNA which can distinguish ape from human. The snPI RNA species are a recently described set of molecules whose sizes range from 5S to 10S. They can be labeled in vitro in isolated nuclei and are apparently formed by an RNA polymerase I type of activity. However, in contrast to ribosomal precursor RNA, the usual polymerase I product, they are not found in the nucleolus but rather are located in the nucleoplasm. The snPI RNAs have been found in all mammalian cell types studied. The spectrum seen in gel electrophoresis is unique to each animal species studied but is essentially the same in different cell types within a species. The differences in snPI patterns are quite large between even closely related species and are clearly distinguishable in gorilla and human cells.


Fibronectin from chicken embryo fibroblasts contains covalently bound phosphate. Fibronectin isolated from cultures of chicken embryo fibroblasts (CEF) contains phosphorus linked to serine and threonine by monoester bonds. Normal and Rous sarcoma virus (RSV)-transformed cells were incubated with [32P]orthophosphate, and fibronectin was isolated from the cell surfaces and conditioned media. 32P was stably associated with fibronectin during immunoprecipitation, SDS-polyacrylamide gel electrophoresis, phospholipid solvent extraction, and hot acid but not alkaline treatment. After a limited acid hydrolysis of fibronectin, both phosphoserine and phosphothreonine were found. The specific radioactivity of the 32P-labeled fibronectin from the conditioned medium of normal CEF was higher than that from the cultures of transformed CEF.


Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells. A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.


Biosynthesis of cytochrome P-450 on membrane-bound ribosomes and its subsequent incorporation into rough and smooth microsomes in rat hepatocytes. Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis.


Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 ganglioside. Cholera toxin internalization into Neuroblastoma GERL. Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.


Synthesis, insertion into the plasma membrane, and turnover of alpha-bungarotoxin receptors in chick sympathetic neurons. alpha-Bungarotoxin was used to identify an integral membrane protein in the plasma membrane of chick sympathetic neurons. The synthesis, insertion into the plasma membrane, and turnover of the alpha-bungarotoxin receptor were studied using isotopically labeled amino acids (2H, 13C, 15N) to directly label receptor molecules. Neurons incubated in medium containing dense amino acids continued to insert unlabeled receptors from a pool of previously synthesized molecules for 2 h. Density-labeled receptors began to appear in the plasma membrane after this 2-h period. Synthesis of receptors, but not insertion into the surface, was blocked by cycloheximide (100 microgram/ml). Neither colchicine (0.05 microgram/ml) of actinomycin D (5 microgram/ml) has any effect on alpha-bungarotoxin receptor synthesis or insertion. Autoradiographic studied revealed that receptors occur on growth cones, axons, and cell bodies of single neurons and explanted ganglia. The rate of insertion of newly synthesized receptors into the plasma membrane of axons extending from explanted sympathetic ganglia was approximately the same as that into the cell body portion of the ganglion. Cytochalasin B (2 microgram/ml) rapidly distrupted growth cones but had no effect on receptor insertion. These experiments suggested that the growth cone is not the sole or even the primary site for insertion of this membrane protein. The kinetics of turnover of the alpha-bungarotoxin receptor were a first-order exponential with t 1/2 = 11 h. Neurons that had their surface receptors labeled with 125I-alpha-bungarotoxin produced [125I]iodotyrosine. This process was inhibited by low temperature (23 degrees C) and also by a metabolic inhibitor. This is interpreted as evidence that receptors turn over by a mechanism in which they are internalized and then proteolytically degraded.


Axonal transport of actin in rabbit retinal ganglion cells. We labeled proteins in the cell bodies of rabbit retinal ganglion cells with [35S]methionine and subsequently observed the appearance of radioactive actin in tissues containing the axons and synaptic terminals of these neurons, i.e., the optic nerve (ON), optic tract (OT), lateral geniculate nucleus (LGN) and the superior colliculus (SC). The temporal sequence of appearance of labeled actin (which was identified by its specific binding to DNase I, its electrophoretic mobility, and its peptide map) in these tissues indicated that actin is an axonally transported protein with a maximum transport velocity of 3.4--4.3 mm/d. The kinetics of labeling actin were similar to the kinetics of labeling two proteins (M1 and M2) which resemble myosin; these myosin-like proteins were previously found to be included in the groups of proteins (groups III and IV) transported with the third and fourth most rapid maximum velocities. The similarity in transport between actin and myosin-like proteins supports the idea that a number of proteins in the third and fourth transport groups may be functionally related by virtue of their involvement in a force-generating mechanism and suggests the possibility that these proteins may be axonally transported as a preformed force-generating unit.


Estrogen-induced membrane alterations and growth associated with proteinase activity in endometrial cells. Endometrial cells isolated from uteri of ovariectomized rats were treated in vitro with 1 X 10(-9) M estradiol-17 beta (E2beta) to analyze early changes in membrane properties during hormone-induced growth. After 30-min exposure to E2beta at 22 degrees C, cells exhibited an enhanced capacity to bind erythrocytes (hemadsorption) in the presence of concanavalin A (Con A) to 237% of the level in paired controls. Fluorescence microscopy revealted that approximately 25% of cells exposed to E2beta, but not estradiol-17 alpha (E2alpha), showed a redistribution into polar clusters of Con A-binding sites that were dispersed in random patches at the external surfaces of control cells. These hormore-induced membrane alterations were abolished by prior treatment of cells with inhibitors of thiol proteinase activity of the cathepsin B1 (CB1) type, such as leupeptin and iodoacetate. Leupeptin at 4.5 X 10(-7) M also reduced the affinity of [3H]E2beta binding to intact cells but did not influence specific binding of the hormone to macromolecular components of cytosol. A pronounced increase in the availability of endogenous CB1, But not of alkaline phosphatase, succinate, or lactate dehydrogenase, in the extracellular media was elicited within 30 min after E2beta treatment. In cells cultured in chemically defined medium for up to 48 h, E2beta, but not E2alpha, enhanced cell proliferation and stimulated [3H]thymidine incorporation into macromolecular form. These E2beta-induced effects were abolished by prior treatment of cells with liposome-entrapped leupeptin at a final concentration of 7 X 10(-8) M. The net rate of intercellular adhesion among endometrial cells was also enhanced by E2beta. This hormonal response was diminished by prior exposure to leupeptin. Fractionation of cells by selection for adhesiveness due to E2beta exposure for 30 min yielded a subpopulation of rapidly dividing cells which surpassed their less adhesive counterparts in cathepsin secretion and in Con A-mediated hemadsorption. These results indicate that leupeptin-sensitive proteinase activity may contribute to membrane and growth modifications elicited by E2beta treatment in endometrial cells.


Interaction of microtubule proteins with phospholipid vesicles. We have examined the interaction of unilamellar dimyristoyl phosphatidylcholine liposomes with the high-speed supernate of brain homogenate and with tubulin purified through one or two cycles of microtubule assembly-disassembly. Tubulin and certian high molecular weight proteins are selectively adsorbed from these mixtures onto liposomes. The composition of adsorbed proteins is similar to that obtained during corresponding cycles of microtubule assembly, suggesting the equivalency of these processes. Adsorption induces stacking and/or fusion of liposomes into multilamellar structures indicating strong protein-lipid interaction. In addition, liposome-adsorbed tubulin forms extensive intermolecular disulfide bridges that are inert to reducing agents in the aqueous medium. The observations form a basis for further study of the distribution, function, and properties of membrane-bound tubulin.


Distribution of fluorescently labeled actin in living sea urchin eggs during early development. Rabbit skeletal muscle actin was labeled with 5-iodoacetamidofluorescein (5-IAF) and purified by gel filtration, ion-exchange chromatography, and polymerization-depolymerization. The resultant fluorescent conjugates retained full biochemical activities. The labeled actin was incorporated into unfertilized eggs of Lytechinus pictus by direct microinjection and the distribution of fluorescence was investigated after fertilization through the first division cycle. The results were interpreted by comparing the images with those of control eggs injected with fluorescein isothiocyanate (FITC)-labeled ovalbumin. After fertilization of eggs containing IAF actin, the membrane-cortical regions showed dramatic increases in fluorescence intensity which were not observed in FITC ovalbumin controls. During the first division, spindle regions of both IAF-actin-injected eggs and control eggs became distinctly fluorescent. However, no distinctly fluorescent contractile ring was detected in the cleavage furrow. After cytokinesis, the surface between blastomeres containing IAF actin exhibited an increase in fluorescence intensity. These observations have been compared with those of previous studies using different methods, and the possible implications have been discussed in relation to cellular functions.


Induction of spreading during fibroblast movement. This paper describes the phenomenon of retraction-induced spreading of embryonic chick heart fibroblasts moving in culture. Measurable criteria of cell spreading (increase in area of the spreading lamella, and total spread area of the cell) are found to change predictably with retraction of a portion of the cell margin. Ruffling activity was found to increase. The leading lamella of a spread fibroblast ordinarily advances slowly, with an average area increase of approximately 21 mu2m/min. A 10- to 30-fold increase in spreading occurs within 8 s after onset of retraction at the trailing edge and then decreases slightly so that by 1 min the increase in spreading is five to tenfold. During this period, there is a linear relationship between area increase at the leading edge and area decrease at the trailing edge. During the next 10--15 min, spreading gradually decreases to normal. Although the relationship between area spreading and area retracting of fibroblasts at different phases of movement is not significantly linear, it is highly correlated (Table II). These results suggest that the rate of fibroblast spreading may be inversely related to the degree of spreading of the cell as a whole.


Temporal organization of replication in DNA fibers of mammalian cells. The extent of coordinate control over the multiple initiation events in DNA replication has been investigated in three mammalian cell lines by DNA fiber autoradiography. Quantitative estimates have been obtained of the degree of synchrony among initiations occurring on stretches of DNA. Synchrony decreases markedly with increasing distance between initiation sites in MDBK (bovine) and L929 (mouse) cells, but only slightly in muntjac cells. Possible control mechanisms for the initiation process are discussed.


Effect of phagocytosis by human polymorphonuclear leukocytes and rabbit alveolar macrophages on 2-deoxyglucose transport. 2-Deoxyglucose transport was characterized in human polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (AM). The Km was 1 mM for human PMN and 1.6 mM for rabbit AM, and the Vmax was 0.66 x 10(-3) micromoles/45 sec/10(6) PMN and 5.09 x 10(-4) micromoles/45 sec/10(6) AM. The rate of 2-deoxyglucose transport was the same before and after phagocytosis in PMN from normal individuals and three patients with chronic granulomatous disease, as well as rabbit AM. Studies of the kinetics of 2-deoxyglucose transport and intracellular fate of 2-deoxyglucose in human PMN indicate that the nature of the membrane transport system is not altered by phagocytosis. The results support the concept that the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites.


Tunicamycin-mediated depletion of insulin receptors in 3T3-L1 adipocytes. Tunicamycin, an antibiotic that inhibits protein glycosylation, elicited a rapid depletion of insulin binding activity at the surface of 3T3-L1 adipocytes. Disappearance of insulin receptors occurred more rapidly in the presence of tunicamycin than when protein synthesis was inhibited by cycloheximide and was accompanied by a diminution in sensitivity of the adipocytes to the acute effects of insulin and anti-insulin receptor antibody on hexose uptake and metabolism.


Selective inhibition of preribosomal RNA synthesis by puromycin aminonucleoside in transformed human fibroblasts: studies of the nature of the inhibition in isolated nuclei and nucleoli. Puromycin aminonucleoside selectively inhibits the synthesis of ribosomal RNA in human lung fibroblasts transformed by the oncogenic virus SV40. The mechanism of this inhibition was studied utilizing nuclei and nucleoli isolated from cells treated for 18 hours with 100 micrograms/ml of this compound. It was established that for a limited period of time nuclei and nucleoli isolated from the fibroblasts continue synthesis of RNA of size classes seen in intact cells, and that the inhibitory effect of aminonucleoside persists after isolation of these organelles. The inhibition was shown to be directed primarily to the activity of RNA polymerase I. Studies of the mechanism of this inhibition have indicated that the decreased rate of the polymerase reaction is not due to the impairment of the template function of nucleolar chromatin, and that unbound, as well as chromatin-bound, RNA polymerase I is present in both control and treated nucleoli. Analysis of the size distribution of the products of cell-free RNA synthesis showed that aminonucleoside pretreatment results in marked reduction in the synthesis of preribosomal 45S RNA, abnormal accumulation of 32S RNA, and reduced formation of mature ribosomal RNA species in the in vitro system. The data suggest that the inhibitory effect of aminonucleoside on ribosomal synthesis is due in part to a lower rate of transcription by RNA polymerase I of preribosomal RNA, and in part to its impaired maturation.


The kinetics of chick cell population aging in vitro. We have examined the kinetics of chick cell population aging in vitro using the percentage of labeled nuclei, the number of colonies formed from a low density inoculum and the number of cells/colony to monitor culture age. The results from these studies showed a gradual age-associated decline in each of the parameters which was first detected early in the culture lifespan and well in advance of changes in total cell number at confluency. Our results also indicated that each of the above parameters, in addition to the calendar time cells had been in culture, could be used to estimate the percentage of lifespan completed by the culture. A comparison of the methods used to estimate the remaining culture lifespan indicated that the percentage of labeled nuclei was the most accurate in describing cell age.


Two distinct mechanisms for ornithine decarboxylase regulation by polyamines in rat hepatoma cells. Exogenous diamines and polyamines added to rat hepatoma (HTC) cells in culture rapidly decrease ornithine decarboxylase (ODC) activity. Previous evidence has suggested that these amines set either at the level of blocking new enzyme synthesis or by the induction of a non-competitive protein inhibitor, termed antizyme, which complexes with ODC to form an inactive complex. Wth the use of HMOA cells, a recently cloned rat hepatoma cell line that has a greatly stabilized ODC, it has been possible to demonstrate that 10(-5) M of exogenous putrescine blocks the increase in ODC activity, but unlike in the parent HTC cell line, without induction of the antizyme or formation of any inactive ODC-antizyme complex. However, complete blockade of ODC at 10(-2) M putrescine is effected by induction of antizyme and formation of the ODC-antizyme complex, as now evidenced by the isolation of the active enzyme and antizyme components after Sephadex column chromatography in the presence of 250 mM NaCl. These findings indicate clearly that two polyamine-regulatory mechanisms for ODC exist and are separable in this cell line.


Pyrimidine nucleoside, pseudouridine, and modified nucleoside excretion by growing and resting fibroblasts. We are examining the relationship of RNA metabolism and de novo pyrimidine synthesis as parameters of malignant transformation. These initial experiments on normal hamster embryo fibroblasts have shown that excreted nucleosides are markers for intracellular RNA metabolism. We employed affinity chromatography to concentrate the nucleosides in the medium and sensitive column chromatographic procedures to quantitatively measure them. The excretion of pyrimidine nucleoside from hamster embryo fibroblasts in sulture was found to be dependent on the growth state of the cells, with the greatest accumulation occurring cell quiescence. The major nucleoside excretion products, uridine and cytidine, were both normal end products of RNA metabolism and the major nucleoside excretion products from cultured cells. The modified nucleosides N-1-methylguanosine, N-2-methylguanosine, N-2-dimethylguanosine, N-4-acetylcytidine, N-1-methylinosine, pseudouridine, N-1-methyladenosine, N-3-methylcytidine, and 5-methyleycytidine were found, as were several unidentified nucleosides.


Selection and characterization of a varient of murine L5178Y lymphoma resistant to local anesthetics. A varient of murine L5178Y lymphoma resistant to procaine hydrochloride (PH) was selected by exposing the cells to gradual increments of PH in the growth medium until the cell grew exponentially in the presence of 1.5 mM PH. Using cinephotomicrography, it was observed that the majority of cells that initially succumbed to PH failed to undergo successful mitosis. With respect ot chromosomal, cell size distribution and flow microfluorometric analyses, the PH-resistant cells are very similar to a spontaneous tetraploid cell line (R1T) previously cloned. The isolated cells, designated R1/P, were also found to be cross-resistant to analogues of PH, namely, lidocaine, tetracaine and dibucaine. The naturally-occurring tetraploid cell line (R1T) was also found to be more resistant to local anesthetics, although not to the same extent as R1/P cells. Since the enzyme that hydrolyzes procaine appears to be absent in all these lymphoid cell lines, the difference in resistance does not appear to depend on differences in the ability of these cells to remove the agent. It is suggested that an alteration in the structure and/or function of the plasma membrane in R1/P cells have rendered them either less sensitive to the membrane-perturbing effects of the local anesthetics or less permeable to local anesthetics molecules. The ability of local anesthetics to affect membranes and cytoskeleton structures may play a role in the genesis and/or selection of these cell variants.


Studies on a fractionated murine fibrosarcoma: proliferative potential of the separated cells. A transplantable methylcholanthrene-induced fibrosarcoma of female BALB/c mice (the MC-2 fibrosarcoma) was dissociated by combined mechanical and enzymatic means, then fractionated by isopycnic centrifugation in linear albumin gradients. In some experiments recovered cells were both cultured in soft nutrietn agar and inoculated subcutaneously into syngeneic recipients. In these experiments a highly significant correlation was observed between subsequtnt colony number and rapid growth phase tumor size suggesting identity of clonigenic and tumorigenic cells. It was consistently found that clonigenic cells were markedly depleted from the low density extremes of the cell density distribution profiles suggesting that the low density neoplastic cells had irreversibly left the growth fraction. With increasing tumor age, sequential studies showed that both total and clonigenic cell density distribution profiles were variable, showing no obvious trend, suggesting that in the age (13-35 days) and size (2-8 g) range studied growth fraction changes had little selective effect on cells of any specific density. These results imply that a marked selective depletion of low density clonigenic cells (or selective accumulation of low density non-proliferative cells) must mainly occur during an earlier phase of tumor growth. Studies on several other murine solid tumors also showed maximal depletion of clonigenic cells from the least dense fractions, suggesting that this situation may be common.


Expression of malignancy in hybrids between normal and malignant cells. Somatic cell hybrids between either normal human fibroblasts, phenotypically normal mouse fibroblasts or mouse peritoneal macrophages and HT1080 human diploid fibrosarcoma cells were studied for their ability to form tumors in nude mice. The results of this study indicate that tumorigenic behavior is expressed as a dominant trait in both human-human and mouse-human hybrid cells.


Inosine di- and triphosphate synthesis in erythrocytes and cell extracts. The ability to synthesize inosinetriphosphate was demonstrated in blood cells as well as in a variety of tissue extracts in spite of the presence of ITP pyrophohydrolase. At the expense of having sub-optimal conditions, an assay system was selected that completely repressed the hydrolyzing enzyme, thus permitting the accumulation of ITP. In an attempt to define the biosynthetic pathway of ITP, and since guanylate kinase has been implicated in the formation of ITP, the rate of synthesis of ITP and GTP in cell extracts was compared. The comparison of the specific activities of the [14C]-labeled hypoxanthine and guanine moieties of the inosine and guanosine phosphates formed during incubation with [8-14C]-inosine and [8-14C]-guanosine respectively, revealed striking differences in the relative rates of isotope incorporation. Tentative mechanisms are proposed to explain these differences. The data obtained thus far does not discard the possibility that ITP may be formed by stepwise phosphorylation and (or) by direct pyrophosphorylation of IMP.


Production of factors required for cell attachment and spreading is a constitutive property in mouse A9 cells. It is demonstrated here that cells in a suspension culture of an established mammalian cell line release non-dialyzable factors into their growth medium. These factors are capable of promoting the adhesion and spreading of these cells on a generally non-attachable substratum and also promote spreading on an adhering substrate. Evidence is presented which demonstrates that the spreading promotion activity of the condition medium is dependent on the cell density of the culture from which it was derived. Dilution of the conditioned medium results in a proportionate dilution of the spreading promotion activity. The results clearly demonstrate that the production of this spreading promotion factor is continued even in the absence of cell to substrate attachment.


Neurotrophic effect of nerve extract on development of tetrodotoxin-sensitive spike potential in skeletal muscle cells in culture. The effect of the presence of nerve extracts on the development of tetrodotoxin (TTX)-sensitive sodium channels in cultures of dissociated embryonic chick skeletal muscle cells was examined by measuring the maximum rate of rise of TTX-sensitive spike potential. The addition of the nerve extract prepared from brain or spinal cord of chick embryos to the culture medium caused an increase in the channel density. Extracts of non-neural tissues, i.e., lung, kidney, and muscle, were ineffective. Liver extract, however, produced an effect similar to the nerve extracts. These results suggest that the TTX-sensitive sodium channels in the muscle cell membrane are regulated by a diffusible chemical substance independently of innervation, and that this substance resides in neural tissues, and perhaps also in liver.


Isolation of rat hepatoma cell variants selectively resistant to dexamethasone inhibition of plasminogen activator. Glucocorticoids induce several phenotypic changes in rat hepatoma cells in tissue culture, including the inhibition of plasminogen activator activity. Variant cell lines resistant to dexamethasone inhibition of plasminogen activator activity have been isolated using an agar-fibrin overlay technique to identify colonies with fibrinolytic (plasminogen activator) activity. The variants are resistant to concentrations of dexamethasone 1,000 times that necessary to completely inhibit plasminogen activator activity in wild-type cells. The variant phenotype has been inherited in a stable manner for more than 300 generations in continuous culture in the absence of dexamethasone. These variants are unique in that the resistance is not secondary to defective or absent glucocorticoid receptors but is due to a lesion specific for regulation of plasminogen activator. Fluctuation analyses support the hypothesis that resistance to dexamethasone arises randomly and is not induced by dexamethasone. Because HTC cells are heteroploid and karyotypically highly variable, variants are thought to arise primarily by chromosomal segregation events. These variants provide a valuable tool for studying the mechanism of hormonal regulation of plasminogen activator as well as the role of proteases in hormonal regulation of membrane functions.


Steroid hormone toxicity in human fibroblasts does not correlate with high affinity receptor content. Human diploid skin fibroblasts derived from normal individuals and those with the testicular feminization syndrome (TFM) have been shown to be killed to the same degree by dihydrotestosterone in spite of the absence of high affinity cellular androgen receptors in the TFM fibroblasts. Furthermore, several different normal fibroblast strains from various anatomical sites all showed similar amounts of androgen-induced cytotoxicity even though their respective receptor contents differed by as much as ten-fold. These results suggest that steroid-induced cytotoxicity in human fibroblasts is not correlated with receptor content, unlike murine lymphoid cells in which the receptor content has been shown to be closely related to their ability to survive hormone exposure.


Regulation of surface antigen expression in Paramecium primaurelia. II. Role of the surface antigen itself. In the wild-type strains, 156 and 168, of Paramecium primaurelia, the alleles G156 and G168 expressed at medium temperature specify two immunologically distinguishable surface antigens 156G and 168G, whose phenotypic expression shows allelic exclusion, the majority of heterozygotes being phenotypically [156G] while a small minority is phenotypically [156G-168G]. At high temperature, the antigens coded by another locus, generally the D locus, are expressed. This system, displaying both intergenic and interallelic exclusion, provides favourable material to analyze the respective roles of the genome, of the antigens expressed and of the environmental conditions, in particular temperature, on the regulation of the expression of surface antigens. This analysis was carried out by studying the variations of the expression of surface antigens as a function of temperature, culture medium and previously expressed antigens in different genetic situations (a) in homozygotes: the wild-type strains 156 and 168, and the isogenized strains "G156 isogenic 168 carrying the G156 allele in a 168 genetic background; (b) in heterozygotes of the two phenotypic classes of heterozygotes, [156G] and [156G-168G]. The results show that (1) the thermal stability of the expression of a given surface antigen and its rate of re-appearance at the cell surface depend on its own specificity; (2) in heterozygotes [156G-168G], the stability of the expression of the antigen 156G is modified and "adjusted" to that of the less stable surface antigen 168G, and (3) the surface antigen itself exerts a positive control on the maintenance of its own expression. An interpretative model of "transmembranous control" is proposed to account for the regulation of the expression of surface antigens in Paramecium.


Membrane effects of tumor promoters: stimulation of sugar uptake in mammalian cell cultures. Phorbol esters stimulate 2-deoxy-D-glucose (DG) uptake in rodent and human cell cultures. The potent tumor promoting agent, 12-0-tetradecanoyl phorbol-13 acetate (TPA), induces a 12-fold stimulation in confluent 3T3 cells and 2.5-fold stimulation in HeLa cells. When a series of macrocyclic deterpenes are assayed, their relative potencies in stimulating DG uptake in 3T3 cells correlate with other known biologic effects of these compounds. On a molar basis, TPA is a much more potent stimulator of DG transport than insulin or epidermal growth factor. In HeLa cells, the ED50 value of the TPA effect is 0.2 nM. The increase in DG uptake occurs immediately after the addition of TPA, reaches a maximum at 90 minutes, persists for at least three hours after removal of TPA from the medium, and is temperature dependent. The stimulation is not inhibited by cycloheximide or actinomycin D. As in control cells, DG uptake in TPA treated cells is inhibited by p-hydroxymercuribenzoate, phyloridzin, cytochalasin B, and dexamethasone. Although the precise mechanism is not known, evidence is presented that the TPA stimulation of DG uptake is due to enhanced transport of the sugar rather than to effects on intracellular metabolism. The enhanced transport may be secondary to a more generalized change in membrane structure.


Pollen-pistil incompatibility in Petunia hybrida: changes in the pistil following compatible and incompatible intraspecific crosses. The structural events in the stigma and transmitting tissue of Petunia hybrida pistils that accompany compatible and incompatible intraspecific pollinations have been investigated in detail, together with the changes in reserve levels that also take place at this time. Many of these phenomena may be explained in terms of 3 phases of secretion by the cells in the upper regions of the transmitting tissue. The first, independent of pollination, results in the deposition of an intercellular matrix, rich in protein and carbohydrate. The second, triggered by pollination, although independent of the compatibility of the pollen grain, involves synthesis of molecules believed to be specific to the S(incompatibility)-gene system. The third phase of secretion occurs only following a compatible mating, and involves the transfer of stylar reserves to support the growth of the pollen tubes. These observations are discussed in terms of current models of the incompatibility mechanism operating in Petunia.


Unidirectional regeneration is an intrinsic property of longitudinal microtubules in Tetrahymena--an in vivo study. The cortex of Tetrahymena contains a regular array of longitudinal microtubular bands (lms) next to basal body rows running from pole to pole. The lm exhibits a predominant unidirectionality in assembly. The direction of regeneration following breakage of the microtubules is from posterior to anterior of the cell. When the lm and the accompanying basal body row are rotated 180 degrees (inverted), so that their polarity is opposite to that of the cell, the predominant direction of regeneration exhibited by the inverted lm is from anterior to posterior. This shows that the lm has an inherent direction of regeneration independent of cellular polarity. This implies that the microtubules constituting the lm have an intrinsic property which controls the direction of assembly. This finding is in accord with the in vitro demonstration using Chlamydomonas flagellar fragments. On the basis of this finding and also the possible pattern of arrangement of the microtubules constituting the lm, it is suggested that growth of the lm involves both elongation of pre-existing microtubules constituting the lm and also laying down of new ones.


Experimentally induced differentiation of slow tonic and fast twitch muscles in the chick. The anterior latissimus dorsi (ALD) muscle of chickens is a slow tonic muscle, while the posterior latissimus dorsi (PLD) is a fast twitch muscle. These muscles on opposite sides of a 3-week-old chick were removed, minced and replaced in the site of the other muscle and left to regenerate. The regenerating muscles were examined at various periods from 4 days onwards and their contractile properties were found to resemble those typical of the muscle they replaced and not the original muscle. The regenerating muscles from 8 days onwards displayed the morphological features of the control muscles in the contralateral site. By 14 days, differentiation was almost complete and neuromuscular junctions were seen. It is suggested that the physiological and morphological characteristics of a muscle are determined by its position and possibly also by its innervation.