[Geometric-mathematical analysis of errors during transfer to the articulator and their practical consequences. II]. With the aid of a computer faults are examined which result in the occlusal surfaces when several articular parameters are simultaneously wrongly applied in laterotrusion. The calculations which are derived from the geometrical relations of the Dentatus articulator refer to a circular field of 3 mm radius on a molar tooth. Vertical deviations, which are caused by faulty setting up of a preferred articulator size, evidently depend on the angle of the joint on the balancing side and depend on the working side on the sagittal position of the model in relation to the condylar axis and on the length of the condylar axis. In the most unfavourable case, errors of the order of 1.3 mm on the balancing side and of 0.75 mm on the working side were demonstrated. As regards horizontal deviations, all articulator sizes were shown to be independent of one another. The effects of faulty setting up on periodontal strain is presented in a practical example.


[The condymeter].

[The condymeter]. The condymeter serves for the technical measurement of condylar displacement where the usual condylar position (habitual intercuspidation) is used as a relative constant. Technical fitting and calibration of the machine with the commercially available SAM-Articulator System ensures its time-saving use in diagnosis and and therapy of diseases of the temporo-mandibular joint and the neuromuscular system.


[Physical properties of highly polishable filling materials compared to composites]. Physical characteristics of three highly polishable filling materials (Estic R Microfill, Isopast and Superlux) were compared with those of current composites (Concise, Adaptic). It was shown that the materials with improved polishing qualities exhibit lower bending strength. Critical comments of practical importance are based on special temperature measurements of the material as well as of the pulp cavity. Aspects of testing and preparing the material are mentioned. Tests were further performed on the abrasion behaviour of the dental materials mentioned and the reciprocal changeable influence of the granularity of the abrasive and the quality of the working material is debated.


[Lateral radiography of the temporomandibular joint with a new radiological device]. A lateral eccentric visualization of the temporo-mandibular joint is described using a new radiological arrangement. The exposures are made with the patient sitting and project the lateral aspect of the joint on to the picture plane, giving a symmetrical picture of the condyle, fossa mandibularis, tuberculum articulare and joint space. The exposure technique distinguishes itself by its simplicity and can be used in combination with every dental X-ray machine.


[Clinical and histological studies on the effect of a surfactant containing toothpaste on the gingiva]. A clinical double blind trial on 98 volunteers was undertaken with the aid of the SB Index to show the effects on the gum of a tooth paste containing detergents. It resulted in a significant increase of the SBI after 6 weeks of a tooth paste with a high content of surface active substances. With the light microscope changes are demonstrable in the Stratum spinosum which could be identified electron-microscopically as a strong increase of bundles of filament in the cytoplasm. The contradiction between clinical and morphological findings is explained by the assumption that the greatincrease in filament bundles leads to a lowering of the adaptability of the epithelium and consequently to increased tendency to bleed, especially in response to mechanical irritation.


[Reproducibility of plaque formation in the dento-gingival region (3)]. Plaque formation in the lower front teeth was recorded in 12 volunteers during three periods of twelve daily visits. Special interest was paid to finding which areas always contained deposits and which were always completely free of plaques. The causative aspects of regular plaque formation were demonstrated. The predominant role of surface roughness is discussed. As a clinical consequence, testing the enamel surface with the sound is suggested for the prophylaxis of gingivitis and caries.


[Cleidocranial dysplasia--histological findings in the dental cementum]. Up to now only very few reports on histological studies of teeth in cleidocranial dysplasia have been published. We are presenting a 12-year old boy with this syndrome. Seven of his teeth were studied microscopically. The most relevant results are as follows: acellular cementum in all teeth, almost complete missing of cellular cementum, normal intermediate cementum. The pathogenesis of the retarded eruption of teeth in this disorder is explained as a result of eburnation of the alveolar bone as well as fibrosis of the gingiva. The therapy of choice in order to stimulate eruption is a rigid orthodontic treatment of the impacted teeth using fixed appliances.


[Intubation anesthesia as an ambulatory treatment measure in the office of the private practitioner]. During the last 8 years we have carried out 25 intubation anaesthesias in our practice with a specialist experienced in dental anaesthetics. We have prepared a report based on the problems we encountered during this close collaboration which should be a help, warning and encouragement to colleagues interested in this special form of therapy.


Structure requirements and affinity of steroids to bind with receptor for induction of differentiation of cultured mouse myeloid leukemia cells. Glucorticoid hormones induced differentiation of mouse myeloid leukemia cells. From comparison of the structure of steroids with their ability to induce phagocytic and locomotive activities, typical characters of mature macrophages and granulocytes, the simplest steroid with inducing ability was concluded to be a steroid with the structure of progesterone and one hydroxyl group at 11beta- or 21-position. The maximum induction ability seemed to require the structure of progesterone and three hydroxyl groups (at 11beta-, 17alpha-, and 21-positions). A single, 30-min pulse treatment with glucocorticoid was sufficient to induce differentiation of leukemia cells. Glucocorticoid receptors were detected in mouse myeloid leukemia cells. The binding affinity of various steroids for the cytoplasmic receptors was closely correlated with the activities of these compounds to induce differentiation of leukemia cells, suggesting that these receptors may be involved in hormonal induction of differentiation of various cells. This suggests that the binding reaction is important for differentiation of myeloid leukemia cells.


Inpatient audit simplified.

Inpatient audit simplified. One hospital's audit program identifies the necessary procedures for selecting criteria, performing audits, and conducting follow-up and shows how they can be accomplished most effectively and efficiently. In addition, it shows that a useful emergency department/outpatient audit can be conducted using data from telephone follow-up for the patients.


Medical staff bylaws: regulations or contract? St. John's Hospital Medical Staff v. St. John Regional Medical Center is the point of departure in an exploration of the issue of whether medical staff bylaws constitute a contract or are merely important regulations. Also discussed is the practical application of bylaws in day-to-day hospital operations.


Three chromosomes' (7;9;22) rearrangement and the origin of the Philadelphia chromosome. A woman with chronic myelocytic leukemia had the Philadelphia chromosome and a complex four-break--three-chromosome rearrangement. The q32 leads to q34 portion of chromosome 9 is translocated to band q22 of chromosome 7, and at the end of this segment is attached the deleted q11 leads to qter portion of chromosome 22. A review of 12 cases of the Philadelphia chromosome originating by the rearrangement of three or more chromosomes reveals that chromosomes 9 and 22 are always involved, while the third chromosome is a different one in each case. We discuss the hypothesis that the 22q segment is always specifically attached to band 9q34 wherever this portion of 9q is transposed.


Arteriovenous perfusion with the pulmonary assist membrane oxygenator. The feasibility of employing a recently developed pulmonary assist membrane oxygenator for partial respiratory support in arteriovenous perfusion was studied using small mongrel dogs rendered hypoxic by oxygen depression. The unit is employed along with the conventional ventilation techniques (such as CPAP and PEEP) when the latter alone cannot maintain adequate gas exchange in the lung. The results indicate that poorly oxygenated arterial blood can be upgraded to life-sustaining levels when the bypass flow rate through the oxygenator is in the range of 18-33% of the total cardiac output. No cardiovascular derangements were noted at these arteriovenous flow rates for perfusion periods of four to six hours. The present results demonstrate the feasibility of providing partial support to pulmonary function using the pulmonary assist membrane oxygenator.


Nutrition--phase one of the edentulous patient. Many edentulous patients are "sick patients." Often geriatric considerations are involved, as well obesity and postmenopausal problems. These patients have deficient tissues on which to build dentures. The degenerative processes which initiate the loss of teeth continue after extraction and cause further shrinkage of supporting tissues. Repeated relining and rebuilding of dentures can be avoided and happier and healthier patients maintained by (1) saturating patients with therapeutic dosages of vitamins and minerals prior to surgical intervention, (2) using scientifically evaluated liquid and semisolid diets containing the maximum quantities of nutrients and the minimum number of calories during the preoperative and postoperative periods, and (3) thereafter maintaining the patient on a high-protein, high-vitamin, high-mineral diet.


Effects of burnishing on microleakage in an amalgam system. No significant increase or decrease in microleakage occurred as a result of single or double burnishing of amalgam. When cavity varnish use was compared with no use of cavity varnish, significantly less microleakage was noted with the nonburnished control, single-burnish, and double-burnish techniques utilizing the cavity varnish. It was concluded that burnishing is not a factor in microleakage when using Optaloy, but microleakage can be reduced by the use of a cavity liner such as Copalite.


Influence of axiopulpal line angle and proximal retention on fracture strength of amalgam restorations. Proximal retentive grooves significantly increase the strength of amalgam restorations in Class II cavities. There were no remarkable differences that could have clinical significance between sharp or rounded axiopulpal line angles and rounded or rounded and sloped pulpal walls. Chromium-cobalt dies are effective for tests of fracture strength of amalgam restorations.


Prosthetic reconstruction following resection of the hard and soft palate. The restoration of the soft palate presents a challenge completely different from that of the hard palate. The mobility of the soft palate tends to interfere with velar extensions. The reduction in size of the soft palate extension to prevent impingement upon the mobile margins of the defect will lead to insufficient oronasal separation during functional activities. The solution is to construct a specially designed prosthesis to attain the maximum utilization of the remaining structures and their motility. Although each pharyngeal extension is different in shape, they give the patient an effective functional mechanism that enhances speech and swallowing.


Pharmacotherapy of masticatory system dysfunction. The decision to use drugs systemically in the management of mandibular dysfunction must be made with consideration of the information summarized in this report. It has been shown that pharmacotherapy may be effective. It has also been demonstrated that systemic pharmacotherapy frequently produces side effects which are occasionally severe. The clinician must decide on the appropriateness of therapy with the knowledge that many other conservative measures may also have excellent success rates and that pharmacotherapy is likely to be palliative rather than curative.


Rest vertical dimension determined by electromyography with biofeedback as compared to conventional methods. This study was undertaken to determine if electromyography with biofeedback can be utilized to produce a more reliable determination of rest vertical dimension than conventional methods such as phonetics and swallowing. It was found that: 1. Electromyography with biofeedback appeared to produce a more consistently reliable determination of rest vertical dimension than conventional methods when used with edentulous subjects. 2. Determinations of rest vertical dimension by individual dentists using phonetics and swallowing had wide variations in two of the five patients in a range of up to 6 mm. An error of this magnitude could easily cause an intrusion upon the interocclusal distance and resultant failure of treatment. Since this study was limited to five patients, a more expanded study is necessary to determine the validity of electromyography vs. conventional methods for determining rest vertical dimension. Both methods have questionable aspects in relation to the time of day, patient's understanding of each technique, and past dental history. However, the most critical problem of the electromyographic technique is the feasibility of its use in a private practice in light of the excessive cost of the required equipment.


Relationship between abrasive wear and microstructure of composite resins. The in vitro abrasion resistance of seven commercial composite resin restorative materials has been measured. Analysis of the composite microstructures shows that abrasion rates are dependent upon the size, hardness, and volume fraction of particles in the material. The most abrasion-resistant composites contain a high volume fraction of large, hard particles.


Chairside modification of dentures for tissue conditioning materials. The autopolymerizing acrylic resin will act as a matrix to contain and retain the tissue conditioning material. In addition, the presence of the matrix will facilitate the reestablishment of fully extended borders when the tissue conditioner requires replacement.


Technique for cranioplasty prosthesis fabrication. A technique has been described for fabrication of a cranioplasty prosthesis. The primary advantage of this technique is the duplication of the altered working cast. The technique also permits excellent reproduction of cranial contours and a positive seat of the prosthesis on the outer table of the skull. The disadvantage is the time involved in the duplicating procedure.


Serotonin receptor binding affinities of several hallucinogenic phenylalkylamine and N,N-dimethyltryptamine analogues. Hallucinogenic phenylalkylamine and N,N-dimethyltryptamine analogues are known to affect serotonergic systems both in vivo and in vitro. Using a rat stomach fundus model, the 5-HT receptor binding affinities of several of these analogues were determined and compared. The most behaviorally potent analogues examined, DOB, DOM, and 5-methoxy-N,N-dimethyltryptamine, were found to possess rather high affirmities (pA2 = 7.35, 7.12, and 7.08, respectively) for the 5-HT receptors of the model system.


Serum prealbumin, retinol-binding protein, transferrin, and albumin levels in patients with large bowel cancer. In a study of the levels of serum prealbumin (PALB), retinol-binding protein (RBP), transferrin (TF), and albumin (ALB) in patients with large bowel cancer, critical values were established as (g/liter): PALB, 0.15; RBP, 40 X 10(-3); TF, 2.0; and ALB, 30. Values consistently below these were taken as a sign of malnutrition. The proteins in this system were interrelated and tended to show a similar pattern of change. Metastatic colon cancer caused a relatively small decline of ALB compared to the mean in tumor-free patients. PALB was the most sensitive indicator of nutrition, and its levels and rates of change had a prognostic significance. A rapid fall of PALB often occurred 2--3 months prior to the patients's death; this preterminal phase in ambulant patients was frequently heralded by a progressive rise in the level of C-reactive protein in the absence of any obvious infection.


Special features of intestinal metaplasia and its relation to early gastric carcinoma in man: observation by a method in which leucine aminopeptidase activity is used. The degree of intestinal metaplasia and its macroscopic distribution in gastric mucosa were examined by a new method in which leucine aminopeptidase (LAP) activity is used to investigate the relationship between intestinal metaplasia and gastric carcinoma. Since LAP is a specific enzyme of intestinal metaplasia, the area showing positive reaction for this enzyme corresponds strictly with the zone of intestinal metaplasia examined microscopically. Almost all metaplasia was demonstrated by this method. This method was used to examine 40 human gastric carcinomas confined to the mucosa. Gastric mucosa containing differentiated tubular-type carcinoma (60%) was associated with a high degree of metaplasia in comparison with the low degree of metaplasia in poorly differentiated carcinoma (40%). Differentiated tubular-type carcinoma was closely related to intestinal metaplasia. However, all carcinomas arising from the mucosa without intestinal metaplasia (18%) were poorly differentiated. Therefore, gastric carcinomas occurring in Japanese patients are frequently and closely related to intestinal metaplasias.


Serum protein-bound carbohydrates for following the course of disease in patients with metastatic breast carcinoma. Serum protein-bound carbohydrates, L-fucose, sialic acid, D-galactose, and D-mannose, were measured as potential biologic markers in patients with breast cancer with the use of high-resolution anion exchange separation in combination with a sensitive cerate oxidimetric fluorescence detector system. For 22 randomly selected patients with proved metastatic breast cancer, both L-fucose and sialic acid levels were above the normal range in 21 patients (95%). In contrast, D-mannose was increased in the sera of 9 patients (41%), and D-galactose in 6 (27%). By comparison, the level of carcinoembryonic antigen (CEA) was elevated in 14 patients (64%). The levels for serially determined serum protein-bound carbohydrates paralleled objective changes of response or progression in 5 patients with measurable disease. As might be expected, the degree and pattern of elevation for the individual carbohydrates varied for each patient studied. Combinations of serum protein-bound carbohydrates, particularly L-fucose and sialic acid, and, in addition, CEA, appear to have promise as potential biomarkers for following the course of the disease in patients with metastatic breast cancer.


Diet in the epidemiology of cancer of the colon and rectum. We examined the diets as reported in interviews of 256 white male patients with cancer of the colon and of 330 white male patients with cancer of the rectum. Controls were 783 patients with nonneoplastic, nondigestive system diseases distributed by age similarly to the colon cancer patients and 628 patients with nonneoplastic, nondigestive diseases distributed by age like those with cancer of the rectum. We found no increase in risk for cancer of the colon or rectum regardless of the amounts of beef or other meats ingested. However, we found an increase in risk of colon cancer with decreases in the frequency with which vegetables were eaten. A study of 214 females with cancer of the colon and 182 females with cancer of the rectum yielded similar results. The decrease in risk we found associated with frequent ingestion of vegetables, and especially cabbage, Brussels sprouts, and broccoli, is consistent with the decreased numbers of tumors observed in animals challenged with carcinogens and fed compounds found in these same vegetables.


Chemical carcinogenesis and immunity: immunologic status of rats treated with methylnitrosourea. During the treatment of outbred Sprague-Dawley rats with methylnitrosourea (MNU) or the noncarcinogenic analog diphenylnitrosamine, antibody levels to teichoic acid as well as several parameters of lymphocyte and macrophage function were assessed in animals not overtly stimulated with antigen. Treatment with MNU did not appear to alter most immunologic parameters studied. Some alterations occurred in natural antibody levels, in spleen weight, and in peripheral blood differentials of rats that had received the highest carcinogen dose (4.5 mg/kg). These alterations, however, appeared to coincide with the development of tumors and were not observed in animals with premalignant lesions. Levels of chemical carcinogens that were capable of inducing tumors did not necessarily suppress the immune response.


Nonimmune and immune surveillance. II. Effect of recipient's age, tumor immunogenicity, and neonatal thymectomy on tumor growth inhibition. After a suspension of tumor pieces was grafted into newborn and adult (CBA X C57BL/6J)F1 and BALB/c mice, the growth of Lewis lung adenocarcinoma and mammary gland adenocarcinoma was inhibited in newborn mice, whereas sarcoma of the rectum (SR-1-75) grew at the same rate in newborn and adult recipients. Neonatal thymectomy stimulated the growth of hepatoma (H-2-73) in newborns. The degree of tumor growth inhibition was age-dependent: The maximum inhibition was observed in 1- to 6-day-old recipients, but later it gradually decreased. The hepatoma (H-2-73) and ovarian carcinoma (OC-1-75) with inhibited growth in newborns and the tumor (SR-1-75) with uninhibited growth had equally low immunogenicity. The data suggested that newborns possess factors that inhibit tumor growth but these factors disappear with increased age of recipients.


Biochemical and ultrastructural studies on estrogen-induced pituitary tumors in F344 rats. The hormone function, the metabolism of nucleic acids, and the ultrastructure of estrogen-induced pituitary tumors and of normal glands were examined in male F344 rats. The tumors had a high capacity for prolactin (PRL) synthesis, and the plasma levels of PRL were elevated 65-fold to 100-fold in the tumor-bearing animals. Uridine uptake and phosphorylation to nucleotides, as well as uridine incorporation into total RNA, were similar in tumors and normal glands, whereas [3H]thymidine incorporation into DNA was double in the former group as compared to the latter. After a [3H]uridine pulse, labeled RNA turnover was different in tumors and normal glands. Electron microscopy of the tumors revealed hypertrophy, degranulation, and hyperplasia of cells producing PRL with proliferation of their ergastoplasm in whorls. Other pituitary cell types were reduced in number. It is suggested that the whorl configuration caused the high rate of protein and PRL synthesis as well as the changes in RNA metabolism displayed by the tumors.


Primary culture of rat mammary epithelial cells. I. Effect of plating density, hormones, and serum on DNA synthesis. Methods for the primary culture of rat mammary epithelial cells and the response of these cells to various hormones and culture parameters are described. In addition, the techniques for subsequent retransplantation of cultured cells into cleared fat pads of isogenic hosts as a test for normalcy of cultured tissue are detailed. A characteristic and reproducible course of the culture was followed for 2 weeks, after which rat mammary epithelial cells ceased to proliferate and were eventually overgrown by fibroblast-like cells. During the initial 2 weeks in culture, mammary cells and contaminating fibroblast-like cells were responsive to polypeptide and steroid hormones and the percentage of proliferating mammary cells could be enhanced with insulin, prolactin, estradiol, progesterone, and hydrocortisone.


Pathogenesis of rat colon carcinomas induced by N-methyl-N-nitrosourea. Colon specimens were obtained from 45 young male and female inbred CDF rats between 11 and 42 weeks after they began to receive intrarectal injections of the carcinogen N-methyl-N-nitrosourea (MNU) 43--108 mg, total dose); specimens were also obtained from 26 control rats. Well-developed tumors from rats receiving MNU generally presented grossly as polyps; histologically, as invasive adenocarcinomas that had not metastasized. These tumors resembled their human counterparts. Focal epithelial atypias were found in grossly normal mucosa both from rats that had not yet developed tumors and from tumor-bearing rats. Such foci typically consisted of low columnar cells with enlarged nuclei, increased number of mitoses, cytoplasmic basophilia, and reduced cytoplasmic mucus. Serial section studies of minute foci of atypia indicated that some of them arose from normal deep crypt cells. Transitions were frequently found between atypical foci and in situ or invasive carcinomas. Less commonly, adenomatous epithelium was identified in the early lesions or within the frank adenocarcinomas. It was concluded that most invasive carcinomas in this rat model originate in foci of epithelial atypia, which are found with increasing frequency in the flat mucosa during treatment with MNU, but that some carcinomas also arise from adenomatous epithelium.


Effect of microtubule inhibitors on malignant invasion in vitro. The malignant C3H/3T3 mouse cells MO4 invaded embryonic chick heart fragments in an organotypic coculture system on semisolid medium, which mimicked malignant invasion. In this system, at a dose of 1 microgram/ml, the microtubule inhibitors colchicine, demecolcine, vincristine sulfate, vinblastine sulfate, or methyl[5-(2-thienylcarbonyl)-1H-benzimidazol-1-yl]-carbamate (Nocodazole) totally inhibited malignant invasion. At the same dose the drugs were also mitostatic, which was apparent from C-mitoses and from the absence of postmetaphase figures. At a mitostatic dose of 10 microgram/ml, 5-fluorouracil (FUra), cytosine arabinoside, or bleomycin did not interfere with malignant invasion. Combined treatment of the cocultures with the antimetabolite FUra (10 microgram/ml) plus the microtubule inhibitor Nocodazole (1 microgram/ml) completely inhibited invasion. These cocultures also showed the effective inhibition of mitosis by FUra, because Nocodazole-induced C-mitoses were absent. The reversibility of the anti-invasive effect of 4-day treatment with Nocodazole (1 microgram/ml) was demonstrated in shaker cocultures with the use of fluid medium. Our in vitro experiments indicated that cytoplasmic microtubules were involved in malignant invasion and that cell division and invasion constituted separate characteristics of malignant cells.


Male sterility in the domestic fowl (Gallus domesticus) after 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitroso-trans-urea (NSC-95441) treatment of chicks. Of 11 male baby chicks treated twice with 3 mg of 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitroso-trans-urea (once on the day of hatching and once on the day after), 9 showed absolute lack of spermatogenesis and 2 showed severe inhibition when examined at the age of 6 months. Plasma testosterone level was not altered, however, and histologic examination revealed well-organized testicular interstitial tissue with an abundance of Leydig's cells. In the lining of the seminiferous tubules only Sertoli's cells were present. The weight of the testes from treated animals was only about 10% of control weight. These findings emphasized the possibility that the nitrosoureas cause severe damage to the reproductive system in man.


Inhibition of DNA synthesis by 1,2-dimethylhydrazine and methylazoxymethanol acetate in rabbit colon mucosa in organ culture. When maintained in organ culture, colon mucosa from male New Zealand White rabbits showed a near-normal mucosal morphology and linear incorporation of [3H]thymidine into mucosal DNA up to 36 hours of incubation. Explants from the descending colon had a higher DNA synthetic activity than did other segments of the large bowel. Inhibition of DNA synthesis in colon explants by 1,2-dimethylhydrazine (DMH) and methylazoxymethanol (MAM) acetate was dose-dependent. When DNA synthesis was determined after an 18-hour incubation, MAM acetate inhibited DNA synthesis at concentrations of 50, 100, 150, and 200 microgram/ml. With the same concentration of DMH, little or no inhibition was observed. At the concentration of 200 microgram/ml, both carcinogens significantly inhibited DNA synthesis after 3 and 6 hours of incubation. With longer incubation, the inhibitory effect of DMH appeared to be reversible, whereas DNA synthesis was continuously inhibited by MAM acetate up to 18 hours of incubation. No altered uptake of [3H]thymidine by colon explants incubated in the presence of DMH or MAM acetate for 18 hours was observed. No morphologic changes were seen in colon explants treated with 200 microgram MAM acetate/ml for 18 hours. Physostigmine sulfate had no influence on MAM acetate-induced inhibition of DNA synthesis in colon explants. These in vitro observations reflected a direct action of DMH and MAM acetate on the colon mucosa and supported the possibilility that colon epithelial cells contain enzymes capable of activating DMH and MAM acetate to their alkylating carcinogens.


Evaluation of hepatocellular neoplasms in mice. Thirty C3H-AvyfB male mice from 3 to 15 months of age were killed, and the genesis of their hepatocellular neoplasms was studied. As the mice aged, the number of tumors per liver and mean diameter of the tumors increased. Histologically, the smallest tumors were composed of solid sheets of cells and were better differentiated than the largest tumors. Small foci of hepatocytes in prominent trabeculae formation was a common characteristic of large tumors. In addition, 18 metastatic hepatocellular carcinomas observed in aged C3H-AvyfB, C3HfC, C3H, C3HfB, (C3HfB x YBR)F1, or C57BL/6 mice were also included for a morphologic study of their primary and metastatic lesions. Invariably, the pulmonary metastases were composed of a well-differentiated sheet of hepatocytes that resembled those in the solid liver tumors. The smallest hepatocellular neoplasms appear to represent early stages in the development of metastatic hepatocellular carcinoma.


Enhancement of DL-ethionine-induced liver carcinogenesis in rats fed a choline-devoid diet. The effects of a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by DL-ethionine were investigated. Groups of male outbred Sprague-Dawley rats were fed a plain CD or a plain CS diet, or the same diets containing 0.05% DL-ethionine. Hepatocellular carcinomas developed in 50% of the rats fed the CD+ethionine diet for 14 weeks and in about 80% of the rats fed the same diet for 22-30 weeks. No hepatocellular carcinomas developed in rats fed the CS+ethionine diet, the plain CD diet, or the plain CS diet up to 30 weeks. The findings suggest that a CD diet alters the response of rat liver to DL-ethionine and leads to an early and enhanced induction of hepatocellular carcinoma.


Relationship between leukemogenicity and in vivo inducibility of normal differentiation in mouse myeloid leukemia cells. Leukemogenicity was studied in sensitive mouse myeloid leukemia cells that could be induced to undergo cell differentiation in vitro into mature granulocytes and macrophages by incubation with the inducer (certain proteins or glucocorticoids) and in resistant myeloid leukemia cells that could not be induced to differentiate into mature cells. Three sensitive and five resistant clones were tested. The resistant cells were much more leukemogenic than the sensitive cells, and the survival time of syngeneic mice inoculated with the sensitive cells. In a diffusion chamber in a syngeneic, inbred SL mouse without any additional manipulation or injection of inducer, the resistant cells remained undifferentiated, but the sensitive cells were induced to differentiate into mature granulocytes and macrophages and their proliferation rate decreased. These results suggested that the greater leukemogenicity of resistant cells is associated with some defect in inducibility of cell differentiation.


In vivo enhancement of 5-fluorouracil cytotoxicity to AKR leukemia cells by thymidine in mice. The spleen colony assay was used to examine the effect of thymidine (dThd) on 5-fluorouracil (FUra) cytotoxicity in two transplantable leukemias, AKR (in AKR mice) and L1210 [in (BALB/c x DBA/2)F1 mice], in vivo. A large dose of dThd (10 mg/mouse) could not rescue these cell lines from FUra toxicity. Instead, when dThd was given within 1 hour before FUra, it enhanced FUra cytotoxicity by a factor between 100 and 1,000 in AKR leukemia. That dThd increased the cytotoxicity of FUra only by a factor of 3 in L1210 leukemia suggested a different mechanism of interaction of the two drugs in the two cell lines. Examination in hybrid mice capable of supporting the growth of both leukemias showed the enhancement to be tumor related rather than host related. We also demonstrated a dose-dependent effect of dThd injection 15 minutes before FUra in AKR leukemia. Concerning the kinetics of killing of AKR leukemia colony-forming units (LCFU) following the administration of dThd 15 minutes before FUra, LCFU survival continued to decrease for 24--36 hours following drug administration.


Selective induction of melanomas in gerbils (Meriones unguiculatus) following postnatal administration of N-ethyl-N-nitrosourea. Following a single ip injection (50 mg/kg) of N-ethyl-N-nitrosourea (ENU) on postnatal day 7 or 20, 9 of 21 gerbils (43%) developed a total of 15 cutaneous melanomas. The mean survival time of tumor-bearing animals was 909 +/- 212 (SD) days. Neoplasms were preferentially located at sites with little hair and with high content of pigment cells (feet, ears, tail, or snout). Histologic studies on newborn gerbils indicated that ENU-induced malignant transformation occurred in differentiated melanocytes rather than in precursor cells of the neural crest.


Carcinogenic activity of Symphytum officinale. The carcinogenicity of Symphytum officinale L., Russian comfrey, used as a green vegetable or tonic, was studied in inbred ACI rats. Three groups of 19--28 rats each were fed comfrey leaves for 480--600 days; four additional groups of 15--24 rats were fed comfrey roots for varying lengths of time. A control group was given a normal diet. Hepatocellular adenomas were induced in all experimental groups that received the diets containing comfrey roots and leaves. Hemangioendothelial sarcoma of the liver was infrequently induced.


Inhibition of murine osteogenic sarcomas by treatment with type I or type II interferon. Interferon was used to treat C57BL/6 female mice inoculated with a continuous line of murine osteogenic sarcoma cells. A short 7-day course of 30,000--60,000 U/day of tpe I interferon either completely inhibited or delayed the appearance of tumors in experimental animals. The therapeutic efficacy of type I interferon was compared with murine serum that contained type II interferon as well as other lymphokine activity. Tumor development was strikingly inhibited in animals treated for 7 days with serum containing only 600 U of type II interferon. Inhibition of tumor development was thus achieved with 100-fold less interferon than that required with type I preparation.


Viruslike particles in hamster embryos, fetuses, and tumors. Intracisternal viruslike particles were present in outbred Syrian golden and inbred ALB/Mey Pfd hamster blastocysts, embryos, and fetuses. Their morphology was identical to that of the "spoked" viruslike particles found in hamster tumors. They were released in great numbers in the embryonal tissues until day 9 of pregnancy and were found until day 13 in the fetal tissues. Thereafter, these viruslike particles were no longer observed in the fetus. They were never recorded in normal adult hamster tissues.


Therapy of the murine plasmacytoma MOPC 104E: role of the immune response. The murine plasmacytoma MOPC 104E was susceptible to cytotoxic therapy in female inbred BALB/c mice. Palpable subcutaneous tumors (0.6--1.2 X 10(8) cells) could be cured with a single administration of cyclophosphamide (5--250 mg/kg) or localized irradiation (800--2,400 R). Clonogenic assay showed that, following minimal curative doses of cyclophosphamide or radiation, 0.5--1.5 X 10(6) tumor cells should remain viable. Control animals succumbed to progressive, invariably lethal tumor growth after they were given sc injections of 2--3 X 10(3) tumor cells. Minimal doses of cyclophosphamide, which were curative in control tumor-bearing animals, were ineffective in treating tumor-bearing animals immunosuppressed by 450 R whole-body irradiation. Subsequent experiments measured the ability of animals cured of the murine plasmacytoma to reject secondary challenge with the same tumor. These experiments demonstrated and partially quantitated the substantial role of the immune response in effecting tumor cure following radiotherapy or chemotherapy.


Pulmonary carcinogenesis in rats given implants of cigarette smoke condensate in beeswax pellets. Lungs of inbred OM/NCR and outbred Sprague-Dawley rats were given implants, through a thoracotomy, of pellets of cigarette smoke condensate (CSC) suspended in a beeswax-tricaprylin vehicle. The pellets slowly released material into the surrounding parenchyma, which resulted in a dose-related increased incidence of lung cancer, predominantly invasive and metastasizing epidermoid carcinoma. A 42% prevalence of pulmonary carcinoma was present in the highest dosage group, which received 67 mg CSC, exposing approximately 1.65 cm2 bronchiolar epithelium. Squamous metaplasia associated with the implanted site preceded the appearance of the carcinomas and was more severe, with the larger pellets having more concentrated CSC. No difference was observed in incidence of pulmonary carcinomas with the use of CSC containing high or low concentrations of nicotine. The potential value of this bioassay system were discussed.


Correlation between the ability of tumor cells to incorporate specific fatty acids and their sensitivity to killing by a specific antibody plus guinea pig complement. Line-1 diethylnitrosamine-induced guinea pig tumor cells can be rendered sensitive to killing by rabbit anti-Forssman IgM antibody plus guinea pig complement (GPC) or antitumor antibody plus GPC following prolonged incubation (17 hr ) of the cells with one of several metabolic inhibitors. Compared to control cells, these cells have been shown to be inhibited in their ability to incorporate fatty acids into complex cellular lipids, which suggested that lipid synthesis is of fundamental importance for the ability of the tumor cells to resist humoral immune killing. In this study, drug-treated cells that were rendered sensitive to killing by anti-Forssman antibody plus GPC, but not antitumor antibody plus GPC, were inhibited in their incorporation of saturated (palmitic or stearic acid), but not an unsaturated, fatty acid (linoleic acid). These data suggested that the fatty acid composition of specific lipids may also be important for the resistance of these tumor cells to killing by antibody and complement.


A theoretical definition of minimal acceptable dialysis therapy. A theoretical definition of "minimal acceptable dialysis therapy" is that which: provides optimal sodium, potassium, hydrogen, and water homeostasis, optimal calcium and phosphorus homeostasis, and nontoxic concentration profiles for all other solutes which exhibit concentration-dependent toxicity normally controlled by renal excretion; results in maximal patient well-being and rehabilitation with minimal dialysis related morbidity; is sufficiently well-defined with respect to dose to be reliably prescribed for any individual patient which requires that, for all solutes of clinical importance, the kinetic behavior and closure of mass balance be verified for individual patients. Empirically, the minimum dose of dialysis (dialyzer urea clearance, treatment time and frequency) sufficient to result in mean predialysis BUN values of 80 mg/dl with documented dietary protein intake (DPI) greater than or equal to 1.0 g/kg/day and measured protein catabolic rate (PCR) equal to DPI has been shown to be acceptable with respect to currently achieved clinical outcome parameters.


Combined technological-clinical approach to wearable dialysis. To evaluate the constraints imposed upon the design of wearable dialysis systems, prototypes using currently available hardware were applied to three different dialysis formats: 1) the wearable artificial kidney (WAK) for hemodialysis (HD), 2) reciprocating peritoneal dialysis (RPD), and 3) alternating sorbent based dialysis/diafiltration(Ds/F) with a highflux membrane. 1) WAK dialysis has undergone extensive clinical trials with results comparable to standard HD. This system, including a self-contained power source, weighs 4.5 kg. The pulsatile blood flow can be disadvantageous, and cost of disposables is high. 2) RPD is shown to be an effective PD format, with the clearance of urea averaging 29.7 (23.9 to 41.5) ml/min in 14 patients, totalling 548 RPD dialyses. 3) After four conceptual trials, the Ds/F system was used for one treatment. Mass transfer results show removal of: urea nitrogen, 15.4 g; creatinine, 1.9 g; uric acid, 1.2 g; potassium, 89.2 mEq; and a positive bicarbonate balance of 94 mEq. The design constraints of these systems were elucidated, and prototype compact delivery systems have been constructed. It is concluded that a) non-mechanical PD wearability exists and b) true wearability of HD or Ds/F systems is not yet  technologically feasible, but constraints are less rigid for Ds/F than for HD.


Two new concepts that might lead to a wearable artificial kidney. A wearable artificial kidney involving two novel components is proposed. It consists of a turbulent flow ultrafiltering shunt, which supplies 20 liters of ultrafiltrate per day to a disposable activated charcoal cartridge (where creatinine, uric acid, and other tightly bound solutes are adsorbed) and then to an artificial loop of Henle (where the urea is concentrated into 2 liters of ultrafiltrate per day and discarded) from which 18 liters of cleansed, rewarmed ultrafiltrate containing 87% of the glucose is returned to the patient.


Roux-Y intestinal bypass for administration of sorbents in uremia. In order to minimize interaction of sorbents with food and digestive secretions, an intestinal bypass was created for sorbent administration in normal and uremic rats (N = 18) and goats (N = 5). Two separate limbs of small intestine were fashioned, one for food absorption and one for sorbent function, which joined at a Roux-Y anastomosis before the cecum. Particulate sorbent suspensions were injected into the intestine via a cutaneous stoma, and were excreted with food wastes in the feces. In animals with normal kidneys, sorbent function was calculated from changes in fecal and urinary excretion. Nitrogen clearance by the intestinal bypass was 20 to 40% of normal renal clearance in rats and goats. Potassium clearance was 40% of normal renal clearance in rats, and over 100% in goats. Sorbent treatment in anephric animals caused serum urea nitrogen concentrations to stabilize at 210 mg/dl in rats, and 110 mg/dl in goats. Serum potassium concentrations stabilized at 4.5 mEq/liter in rats, and fell to 2 mEq/liter in goats. Water balance was maintained by producing a mild osmotic diarrhea. At least three substances which accumulate in renal failure--urea, potassium, and water--were removed in therapeutically significant amounts.


Isothermic and kinetic studies of uremic metabolite adsorption with Ambersorb XE-344 resin. Ambersorb XE-344 resin is specific for certain metabolites because of unique pore size distribution and surface chemistry. Affinities are greatest for molecules which are significantly aromatic or nonpolar. For clinical purposes, however, the capacity for and kinetics of adsorbing certain aliphatic nitrogenous metabolites (e.g., guanidine) may enhance dialysis treatment as well. While significant affinity for peak 7c has been demonstrated, the resin does not adsorb the steroid, glycoprotein, or polypeptide molecules thus far tested. Despite the carbonaceous nature of Ambersorb XE-344, it does not adsorb the inorganic electrolytes.


Charcoal-induced lipid reduction in uremia. Charcoal, in divided oral doses totalling 35 g/day, was administered to six patients with renal insufficiency (creatinine clearance of 0 to 45 ml/min). Significant (P less than 0.01) reductions in serum cholesterol and triglycerides were observed in the three most hyperlipidemic patients. Maximal decreases in charcoal responders, as compared with control values, were for cholesterol (43%, 23.4% and 40.4%) and for triglycerides (76%, 60.3% and 64.3%). None of the patients showed altered concentrations of BUN, serum creatinine, uric acid, or vitamin A. Because of its safety and the profundity of its hypolipidemic action, it is suggested that charcoal may find applicability in the management of azotemic diabetic and nephrotic hyperlipidemia.


Bacterial enzymes in uremia management.

Bacterial enzymes in uremia management. The theoretical background for a conservative therapeutic treatment of uremia is described, with illustrative results from preliminary clinical trials in 10 patients and 10 normal reference subjects. The proposed treatment focuses upon the patient's gastrointestinal tract--the normal site for metabolism of both exogenous (dietary) and endogenous (recycled) protein--enabling it to behave like the rumen of the cow. The objective is to induce the uremic's organism to utilize its own "waste" substances. The patient swallows enterosoluble capsules containing specifically adapted enzymes (immobilized or free) from apathogenic soil microorganisms. These are pre-adapted to convert urea, creatinine, uric acid, guanidino derivatives, and other nonprotein nitrogen compounds (NPN). The enzymes utilize many other substances, in particular ammonia, potassium, phosphorus, and several other factors potentially dangerous for the uremic. The enzymes apparently cleave vasoconstrictatory peptides in the intestines. In the course of the therapy, renoparenchymal hypertension decreased significantly, and increased again when the regimen was interrupted. The results from the present studies are in full accord with the information published in the relevant fields. The time appears ripe for large-scale trials of the therapeutic regimen outlined, especially as many commercial microbial enzymes already have a long history of safe use in food processing.


Lecithin-cholesterol acyltransferase (LCAT) activity in chronic uremia. High plasma concentrations of triglycerides and low plasma concentrations of esterified cholesterol and lysolecithin, with an impaired rate of VLDL and LDL catabolism, have been reported in chronic uremic patients. An important contribution to these abnormalitites might be an impaired activity of the (LCAT). Serum LCAT activity and cholesteryl ester clearance were determined in 11 patients with chronic renal failure and in 10 controls. LCAT activity was determined by using the serum of each patient both as a source of enzyme and as a substrate ("intrinsic" activity) and was compared with the activity determined on a standard substrate ("extrinsic activity), so as to ascertain the presence of inhibitory factors in the patients' sera. Both activityes have been found to be significantly (P less than 0.01) lower in chronic uremic patients than in controls. The cholesteryl ester clearance apparently did not respond to the stimulatory effect of hypertriglyceridemia, as observed in other cases of dislipoproteinemias. The parallel decrease of both enzyme activities makes it unlikely that it is due to the presence of "uremic toxins" inhibiting the enzyme activity. LCAT synthesis in the liver is probably reduced in chronic uremia. These results suggest that in chronic uremia the VLDL fail to cooperate in their own catabolism.


Survival in uremia due to systemic diseases. A retrospective analysis of the mortality and morbidity experience with maintenance hemodialysis as treatment for uremia in systemic disease was conducted. Between 1962 and 1977, a total of 141 patients with chronic glomerulonephritis and 120 patients with uremia caused by renal involvement in systemic disease were treated. With the exception of two patients with bacterial endocarditis who died early in their course, survival in the diagnostic categories studied ranged from acceptable, in diabetics with 29 of 53 (54.7%) patients living, to excellent in tuberculosis where all of six patients are alive and well. Overall, it is concluded that uremia in systemic disease is responsive to maintenace hemodialysis.


Anti-A and anti-B isohemagglutinins in the course of children's leukemias. The changes in titres of isohemagglutinins anti-A and anti-B were followed up in 48 leukemic children during relapses and remissions. The levels of titres were compared 1. with levels in healthy children in the same age group, 2. in the different phases of disease in the same child. Compared with healthy children, a higher frequency of low isohemagglutinin levels was found in the phases of the exacerbation of malignancy, no changes were observed in remissions. In some children a quite evident fluctuation of isohemagglutinin titres in relation to the phases of the malignant disease was found. The depression of blood group isohemagglutinins in the phases of activation of the malignant process is considered as a partial defect in IgM, analogical to the so called normoglobulinemic defect of the antibodies.


Total osteolysis of the mandibular condyle in progressive systemic sclerosis. This report calls attention to the complete resorption of the mandibular condyle in progressive systemic sclerosis (scleroderma), a previously unreported finding. This was associated with osteolysis of the ipsilateral coronoid process, both mandibular angles, and autoamputation of the fingertips. The Panorex provides a simple, effective method for studying the mandible in systemic sclerosis. Similar mandibular osteolysis with vinyl chloride exposure is noted.


Traumatic bone cyst in the maxilla.

Traumatic bone cyst in the maxilla. This report presents an unusual asymptomatic periapical radiolucency occurring around the upper right lateral incisor. Surgical intervention revealed an empty bone cavity which was diagnosed as a traumatic bone cyst. Follow-up periapical radiographs of the patient were taken over a 6-year period until complete healing of the lesion took place.


Allergy to silver amalgams.

Allergy to silver amalgams. A case of persistent periodontitis was cured by replacement of all silver amalgam restorations. The patient had a history of developing a rash and swelling whenever she used jewelry containing silver. A patch test to silver nitrate was strongly positive.


Desmoplastic fibroma (fibromatosis) of the jawbones. Report of a case and review of the literature. Desmoplastic fibroma (fibromatosis) is rarely seen as a primary tumor of bone. Its occurrence as a central lesion in the jaws is even more uncommon. The case of a 26-year-old woman with a central desmoplastic fibroma of the body of the mandible is described. The lesion manifested as a painless swelling and radiographically appeared as a well-delineated radiolucency. On exploration, the tumor was found to have infiltrated through the lingual cortex. Microscopic examination revealed invasion of muscle. The clinicopathologic features of this case and of the twenty-five similar lesions previously described in the literature are analyzed and discussed.


Oral African histoplasmosis resembling Burkitt's lymphoma. An African type of histoplasmosis with oral involvement has been described. It differs from the histoplasmosis caused by Histoplasma capsulatum. This article discusses the clinicopathologic differences and describes a case seen in Nigeria in which the patient had an oral lesion resembling Burkitt's lymphoma but also had disseminated lesions. The treatment is discussed.


Effects of tobacco smoking and age on the keratinization of palatal mucosa: a cytologic study. Cytologic smears of palatal mucosa were evaluated for degree of keratinization in 217 healthy men ranging in age from 30 to 74 years. Two slides were prepared for each participant, and each slide was read by two different pathologists. Three separate high-power fields were examined on each slide, and the percentage of orange-brown cells, either without nuclei or with pyknotic or fragmented nuclei, was recorded. A mean value was obtained for each participant. Each participant had an extensive medical and dental work-up as part of the Veterans Administration Longitudinal Study of Oral Health in Healthy Veterans. Nonsmokers and smokers were divided into five age groups. Smokers at all ages presented a greater degree of palatal keratinization than nonsmokers, but age differences in keratinization were not statistically significant.


Management of an avulsed deciduous incisor. Report of a case. The avulsed primary tooth poses problems in its treatment and management. This case report on the long-term management of an avulsed deciduous incisor produced some diagnostic findings. Reattachment of the periodontal fibers occurred after 2 weeks of splinting. The normal eruption pattern was not delayed and secondary spacing occurred. Turner's hypoplasia occurred on the succedaneous tooth, but it was minimal. Replantation of avulsed deciduous teeth should be considered as a treatment modality under ideal conditions.


Diagnostic limitations of orthopantomography with lesions of the antrum. Two case reports are presented which illustrate the disadvantage of using curved surface tomography to investigate the maxilla. This technique should be considered as a supplement to diagnostic radiography of the maxillary sinus and not as a substitute for conventional radiography.


Effect of mAs and kVp on resolution and on image contrast. Two clinical experiments were conducted to study the effect of kVp and mAs on resolution and on image contrast percentage. The resolution was measured with a "test pattern." By using a transmission densitometer, image contrast percentage was determined by a mathematical formula. In the first part of the experiment, the density of the film was kept constant by changing the kVp and mAs. In the second part of the experiment, different mAs's were chosen, and for each mAs, several kVp's were used. Five observers read the radiographs. The first experiment showed that, when the film density is kept constant, the higher the kVp, the lower the resolution and image contrast percentage; also, the higher the mAs, the higher the resolution and image contrast percentage. The second experiment showed that when the film density is not kept constant, the correlation between kVp and resolution and between kVp and image contrast percentage was the same as in the first experiment. However, there was negligible correlation between mAs and resolution and between mAs and image contrast percentage. A high positive correlation was found between resolution and image contrast percentage, but a high negative correlation was found between resolution and film density.


Grief response of parents to neonatal death and parent participation in deciding care. We determined the grief response to neonatal death of 50 mother-father pairs by administering a questionnaire and conducting a semistructured interview during the infant postmortem review. As measured by a parent grief score, maternal grief significantly exceeded paternal grief (t = 5.89, P less than .0001). Parent grief was not significantly related to birth weight, duration of life, extent of parent-infant contact, previous perinatal loss, parent age, or distance from the hospital of birth to the regional center (Pearson product-moment correlation coefficients). However, the attitudes and behavior of family, friends, and health care personnel in the hospital of birth often adversely influenced parent grieving. Of 39 mother-father pairs whose infants required respirator support, 18 participated in a group decision with their physician to withdraw respirator support when the prospects of infant survival seemed hopeless (limited respirator care group). No significant differences in parent grief scores were found (t tests) when the limited respirator care group was compared to those parents of infants who died despite uninterrupted respirator care. Our data suggest that informed parents can participate as partners with their physician in difficult infant care decision, even when death results, and adjust to their loss with healthy grieving.


Frequency distribution of mRNA and pre-mRNA in growing and differentiated Friend cells. The frequency distribution of poly(A)+-mRNA in growing and in differentiated Friend cells has been measured by mRNA-cDNA hybridization and their differences established by heterologous hybridization of mRNA of one type and cDNA of the other. It was shown that induction of Friend cells involves an increase in abundance of a small number of mRNAs, while no specific pattern of messenger disappearance could be detected. The frequency distribution of pre-mRNA was determined by hybridizing nuclear RNA with the cDNA probes complementary to mRNA. In uninduced Friend cells, it was shown that most precursor messenger sequences are present at a single frequency of about 3 molecules per nucleus, independently of their final frequency in polysomal mRNA. In induced Friend cells, the frequency distribution of pre-mRNA is more heterogeneous and correlated to some extent with the corresponding mRNA frequency distribution.


[Recurrent collapse of one lung (author's transl)]. A case of recurrent collapse of one lung is reported. It developed within 90 minutes after reexpansion of a completely collapsed lung induced by pneumothorax. There was no demonstrable cause for the recurrence of the atelectasis. Treatment was by means of a bird respirator (ventilation via a mouthpiece) with respiratory resistance. Factors possibly responsible for the secondary atelectasis are discussed.


[Multifocal chloroma (granulocytic sarcoma), without marrow involvement. One case (author's transl)]. A case of myeloblastic tumour localised successively in the ribs, lymph nodes, pericardium and the pleura but not affecting the marrow for a period of six months is reported. Such case of chloroma without marrow invasion are rare, and this opportunity is taken to review the literature.


Acetylated histone H4 is preferentially associated with template-active chromatin. Chromatin from trout testis at an early stage of development was digested with DNase II (deoxyribonucleate 3'-oligonucleotidohydrolase; EC 3.1.4.6), and the solubilized products were fractionated into Mg2+-soluble and -insoluble components. An examination of the histones from these fractions by one- and two-dimensional polyacrylamide gels showed that the highly acetylated species of histone H4 (di-, tri-, and tetra-acetylated) were associated mainly with the Mg2+-soluble material. Digestion of this chromatin fraction with pancreatic ribonuclease converted more than half of it to an insoluble state, and the acetylated H4 remained associated with the precipitated fraction. No changes in the other histones were noted, but two other basic proteins were also found to be associated with the Mg2+-soluble fraction. Since this fraction is enriched in transcribing gene sequences, it is concluded that the histone H4 of active genes is present in a highly acetylated state.


How lac repressor recognizes lac operator. Nucleotide analogs were substituted for unmodified nucleotides at specific sites in the lac operator sequence by a combination of chemical and enzymatic procedures. The nitrocellulose filter assay was used to study the interactions of these modified operators with wild-type (SQ) and tight-binding (QX86) lac repressors. These studies implicate directly the 5 methyl of thymine and the 2 amino of guanine as important operator-repressor contact sites. Furthermore, when these findings are combined with published results from other laboratories, a model for the lac operator-lac repressor interaction can be derived. Two important postulates follow from this model. (i) The repressor interacts at specific and defined sites with the N7 of guanine, the 5 methyl of thymine, the 2 amino of guanine, and the central major groove of the operator. (ii) The repressor binds to one side of the operator.


Compact oligomers and nucleosome phasing. Micrococcal nuclease (EC 3.1.4.7) digestion of histone H1- and H5-depleted chicken erythrocyte chromatin yields, in addition to 140-base-pair (bp) core particles, a series of nucleosome oligomers containing about 260 bp (compact dimer), 380 bp (compact trimer), etc. of DNA. These are postulated to represent members of a class of oligomers in which the DNA is tightly wound on stacked protein cores. The physical properties (melting, circular dichroism) as well as DNase I (EC 3.1.4.5) digestion patterns support this view. DNase I digestion of tight oligomers in which the 5' ends of the DNA have been labeled yields results consistent with this model and inconsistent with some other possible models. Several classes of such particles are postulated to exist, differing in DNA length by 10-bp increments. This may be an explanation of the 10-bp nucleosome "phasing" that has been observed in some nuclei.


Specific phosphorylation at serine-283 of alpha tropomyosin from frog skeletal and rabbit skeletal and cardiac muscle. Tropomyosin, extracted from the leg muscle of frogs that had been injected with [32P]orthophosphate, was fractionated into two components, alpha and beta, on a CM-cellulose column. Radioactivity was associated only with the alpha component. A single phosphorylation site was located at serine-283 (pentultimate at the COOH-terminal end) of the frog alpha tropomyosin. The same phosphorylated peptide was recovered in low yields from both rabbit skeletal alpha and cardiac tropomyosin. The presence of covalently bound phosphate in alpha tropomyosin and its absence in the beta component of rabbit skeletal muscle was suggested by 31P NMR spectroscopy. The amino acid sequences around the phosphorylation sites of frog and rabbit tropomyosin are identical. Because this sequence is not similar to any other known phosphorylation site in proteins, this indicates the existence of either specific kinase or phosphatase that can distinguish between alpha and beta tropomyosins. In a model proposed for the head-to-tail overlap of alpha tropomyosin molecules, one O-phosphoserine-283 residue could form a salt linkage with lysine-6 on one side of the overlap region and another with lysine-12 on the other side. This would predict a difference in the stability of polymers of phosphorylated and nonphosphorylated alphaalpha and alphabeta dimers of tropomyosin.


Functional consequences of ligand-dependent conformational changes in trypsin-solubilized and in membrane particle constrained-acetylcholinesterase. The effect of a class of ligands on the catalytic activity of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from Torpedo californica electroplax tissue has been studied via the transient reaction of a fluorophoric acetylcholine analog, 7-(N,N-dimethyl)carbamoxy-N-methylquinolinium iodide (M7C). These "peripheral" ligands inhibit the formation of a metastable carbamyl-enzyme intermediate from M7C. They induce slow isomerization to a new conformational state that shows little or no reaction with M7C. At saturating ligand concentration, the unimolecular isomerization rate constant is 0.03 +/- 0.01 sec-1, a slow rate compared to the rate of carbamylation of the active conformation. Peripheral ligands alter the distribution between reactive and unreactive conformations, thus inducing biphasic rates and amplitudes of carbamylation. The amplitudes, but not the two specific rates, are affected by the concentration of ligand. Zn2+ and d-tubocurarine are two ligands that induce the same slow isomerization rate. On the basis of this identity of function by ligands of disparate structure, we postulate the existence of only a single active conformation and a single inactive conformation (stabilized by interaction with both ligands). In the absence of ligands, the active conformation predominates. Peripheral ligands bind specifically to the inactive conformation. Alkaline earth cations such as Ca2+ and Mg2+ interact strongly and preferentially with the active conformation and drive the conformational equilibrium toward the active state. Ligand-induced inactivation is observed both with highly purified trypsin-solubilized enzyme and with enzyme bound to unfractionated membrane fragments.


Racemization in human lens: evidence of rapid insolubilization of specific polypeptides in cataract formation. After early life, the dry weight of normal human lenses increases at a relatively constant rate with time. Transformation from soluble to insoluble material appears to occur at a comparable rate, resulting in a constant amount of soluble material. However, in cataract the insolubilization rate is accelerated. These observations are supported by determination of D-aspartic acid/L-aspartic acid ratios. The abundance of D-aspartic acid increases with aging at a constant rate in the insoluble fraction of normal lenses but does not change in the soluble fraction. However, in cataractous lenses there is a significant decrease in the ratio in the insoluble fraction. Examination of polypeptides isolated from reduced and alkylated soluble and insoluble cataractous lens proteins as well as other data suggest the following additional conclusions: (i) the 10,000-dalton polypeptide in the insoluble fraction is derived in part from degradation of an already insoluble precursor; and (ii) the lowered abundance of D-aspartic acid in the insoluble fraction of cataractous lenses is primarily due to the rapid insolubilization of the 43,000- and 20,000-dalton range components.


Influenza virus proteins: identity, synthesis, and modification analyzed by two-dimensional gel electrophoresis. A modification of the two-dimensional protein electrophoresis system of O'Farrell was used to resolve influenza A virus proteins from each other and from host proteins in infected cells. Viral protein spots corresponding to the hemagglutinin proteins, neuraminidase, nucleocapsid protein, and nonstructural protein, were identified on the two-dimensional electrophoretogram. Use of the two-dimensional separation has allowed us to identify glycoprotein heterogeneity, to demonstrate directly the synthesis of neuraminidase, to analyze some viral proteins early after infection, and to demonstrate that the influenza virus NP and NS proteins are phosphorylated in infected MDCK cells.


A rationale for stabilization of oxygen-labile enzymes: application to a clostridial hydrogenase. A general procedure for stabilization of O2-labile enzymes exploiting "salting out" of oxygen from the microenvironment in the molecular layers immediately adjacent to charged surfaces of polyionic solid adsorbents has been developed. Empirical verification of this rationale is provided. The half-life of air inactivation of the O2-labile hydrogenase (EC 1.12.7.1) from Clostridium pasteurianum is increased 20- to 25-fold simply by adsorption (noncovalent binding) in dilute Tris.HCl buffer on common anion exchange supports such as DEAE-cellulose or Dowex 1-X2. Predicted increases in degree of stabilization by using more densely charged adsorbents (such as polyethyleneimine-cellulose), as well as bulkier solvent counter-anions, are found; half-lives for air inactivation for the bound hydrogenase can be increased to 3000-fold longer than that of the free enzyme. Most of the total catalytic activity, assayed as H2 evolution from dithionite mediated by methyl viologen or ferredoxin, is retained, whereas the expected suppression of H2 uptake in the reverse reaction is observed.


Histone packing in the nucleosome core particle of chromatin. The chromatin core particle DNA conformation deduced in broad outline by Finch et al. [Finch, J. T., Lutter, L. C., Rhodes, D., Brown, R. S., Rushton, B., Levitt, M. & Klug, A. (1977) Nature 269, 29-36] can be described in detail using other available experimental results. Histone binding sites compatible with the pattern of pancreatic DNase I digestion (Simpson, R. T. & Whitlock, J. P., Jr. (1976) Cell 9, 347-353; Noll, M. (1977) J. Mol. Biol. 116, 49-71; Lutter, L. C. (1977) J. Mol. Biol. 117, 53-69] lend to core particle DNA pseudosymmetry characteristic of molecular point group D(3). DNA symmetry and pseudosymmetry, in turn, imply equivalence and quasi-equivalence properties of the histone packing arrangement that support the following deductions: (i) One and only one alpha(2)beta(2) histone tetramer, presumably (H3)(2)(H4)(2), can serve as a stable subassembly within the histone octamer. (ii) There is a unique, strand-specific way to assign DNA binding domains to the arginine-rich histones (H3 and H4). (iii) Histones H3 and H4 alone should suffice to impose a supercoiled structure on DNA, as is observed experimentally, because only the tetramer can mimic a screw dislocation and thereby complement the screw symmetry of the DNA supercoil. (iv) The two slightly lysine-rich histones H2A and H2B are probably responsible, each in a different way, for dividing the eukaryotic chromatin fiber into discrete subunits. (v) The proposed arrangement of four distinct proteins appears to be a minimum formal requirement for making nucleosomes; that is, for introducing regularly spaced supercoiled DNA folds without also allowing formation of an indefinitely long (and genetically inert) DNA superhelix.


Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor. To define the mechanism of regulation of the protein kinase that is activated in heme deficiency and that inhibits initiation of protein synthesis, we have isolated and purified the heme-reversible form of the protein kinase from rabbit reticulocytes. The inhibitory activity is found in a single band after polyacrylamide gel electrophoresis under nondenaturing conditions. It migrates as a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor becomes self-phosphorylated in the presence of ATP; the phosphorylated protein and the inhibitory activity copurify. The inhibitor produces characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2); the inhibition is reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor is no longer inhibitory after incubation with 20 micron hemin. Incubation with hemin also inhibits self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiates the inhibition of protein synthesis in the subsequent incubation, as does treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of protein synthesis in the lysate due to phosphorylation of eIF-2 appear to be related. These findings suggest that hemin acts directly on the heme-reversible inhibitor.


Modulation of the conformation of a membrane glycosyltransferase by specific lipids. Fluorescence techniques were used to study conformational changes in UDPgalactose:lipopolysaccharide alpha,3-galactosyltransferase (EC 2.4.1.44). Intramolecular energy transfer was measured from the single tryptophan residue in the peptide to a pyridoxal phosphate group linked to the same peptide via a reduced Schiff's base. Significant differences in energy transfer were seen when the enzyme was studied in aqueous solution and after entry into a phospholipid-lipopolysaccharide matrix, paralleling the restoration of its catalytic activity. Further differences were seen when the structures of the phospholipids and lipopolysaccharides were changed. Application of the Förster theory indicated that the changes in energy transfer resulted from changes in distances between the chromophores and/or changes in their relative orientations. The results suggest that entry of this membrane protein into a lipid matrix induces a change in conformation of the protein and that other alternative conformations can be induced by further changes in its lipid environment.


3-Hydroxy-3-methylglutaryl coenzyme A reductase: regulation of enzymatic activity by phosphorylation and dephosphorylation. The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) [mevalonate:NADP+ oxidoreductase (CoA-acylating); EC 1.1.1.34] was inhibited by ATP+Mg2+. Inactivation of HMG-CoA reductase by ATP+Mg2+ was dependent on time, temperature, and ATP concentration. Incubation of microsomal HMG-CoA reductase with [gamma-32P]ATP+Mg2+ was associated with a reciprocal increase in [32P]protein-bound radioactivity and a decrease in enzymatic activity. Incubation of 32P-labeled microsomal HMG-CoA reductase with a partially purified cytosolic phosphatase resulted in a time-dependent reciprocal release of [32P]protein-bound radioactivity and reactivation of enzyme activity. Phosphorylation of HMG-CoA reductase was confirmed by immunoprecipitation of partially purified [gamma-32P]-ATP+Mg2+-inactivated microsomal HMG-CoA reductase with a reductase-specific antiserum. Sodium dodecyl sulfate electrophoresis of the [gamma-32P]immunoprecipitate revealed that the 32P radioactivity was located in the electrophoretic position of HMG-CoA reductase. These results established that the reversible inactivation of HMG-CoA reductase by ATP+Mg2+ was due to covalent modification of the enzyme by a phosphorylation-dephosphorylation reaction sequence. The existence of HMG-CoA reductase in interconvertible active and inactive forms provides a mechanism for the rapid short-term regulation of the pathway for cholesterol biosynthesis.


Properties of the interaction of fluoride- and guanylyl-5'-imidodiphosphate-regulatory proteins with adenylate cyclase. The mechanism of activation of adenylate cyclase by guanylyl-5'-imidodiphosphate [Gpp(NH)p] and NaF has been investigated by studying the reconstitution of Gpp(NH)p and NaF sensitivity of an enzyme rendered insensitive to these agents by differential detergent extraction of a particulate brain enzyme. Such reconstitution can be achieved by the addition of macromolecular regulatory factors from membranes of various tissues. Trypsin digestion and thermal inactivation provide evidence for the existence of two distinct regulatory functions, one capable of restoring the Gpp(NH)p response and another the NaF response. The regulatory protein(s) seem to interact with their respective activators in an easily reversible, divalent cation-independent reaction. This appears to be followed by a high-affinity interaction between the catalytic and regulatory components of adenylate cyclase in a slow, temperature-dependent, divalent cation-dependent process tha produces the persistently activated state of the enzyme. The enzyme activation can be reversed by methods that separate catalytic from regulatory components and the resulting enzyme activity can be restimulated by the reconstitution technique.


"Tension" on heme by the proximal base and ligand reactivity: conclusions drawn from model compounds for the reaction of hemoglobin. The kinetic data on model compounds of hemoglobin indicate that in oxyderivatives ligand dissociation rates are sensitive to the "tension" exerted by the proximal base on the metal-to-ligand bond; the corresponding rates for carboxy derivatives are not sensitive to the tension. It is suggested that the metal-to-ligand bond becomes weaker with increased "pull" (or tension) on Fe from the proximal base due to the steric and/or electronic interaction between the ligand, the porphyrin ring, and the proximal base. In model compounds the linear heme Fe-to-CO bound vis-a-vis the bent heme Fe-to-O2 bond probably makes such interactions less significant in carboxy derivatives. It is proposed that the kinetic alpha,beta-chain nonequivalence in Hb4(O2)4 is due to the difference in the tension in the two chains on Fe by the proximal base. The absence of alpha,beta-chain differences large enough to show up in CO dissociation rates from Hb4(CO)4 is explained on the basis of lack of sensitivity of the Fe-CO bound to tension from the proximal base. The implications of the results for the observed cooperative effects in ligand combination (for CO) and dissociation (for O2 and NO) rates of hemoglobin have also been discussed.


Evidence for the progressive nature of neoplastic transformation in vitro. The temporal acquisition of in vitro phenotypes associated with neoplasia were examined after exposure of Syrian hamster embryo cells to a chemical carcinogen. Quantitative assays measuring morphological changes, enhanced fibrinolytic activity, and anchorage independent growth were used to detect the development of transformed cells within a population of normal hamster embryo cells. Morphological transformation and enhanced fibrinolytic activity were early changes observed after treatment with benzo[alpha]-pyrene, whereas the ability to grow in semisolid agar was delayed 32-75 population doublings after carcinogen exposure. This delay was not due to selection of a small number of cells that were present early after treatment but at a level below detection, because a large percentage of the cells isolated at early passage (10(3)-fold above the level of detection) developed the potential for anchorage independent growth at later passages. This development of the anchorage independent growth phenotype was induced by the carcinogen treatment, because spontaneous transformation was rare. These observation suggest that multiple cellular changes are required for the acquisition of the capacity for anchorage independent growth and that neoplastic transformation in vitro is a progressive process through qualitatively different stages. Thus, an analogy can be drawn to the progressive nature of in vivo carcinogenesis. These results strongly justify the study of oncogenesis in cell culture as a model for neoplastic transformation in vivo.


Retention of susceptibility to mitogens after direct dansylation of viable human lymphocytes. The covalently binding fluorescent probe 5-dimethylamino-1-naphthalenesulfonyl (dansyl) chloride was affixed directly to the plasma membrane of viable human peripheral blood lymphocytes via a solid phase transfer method utilizing Sephadex G-10 as the transfer vehicle. After dansylation, lymphocytes retain maximal short-term viability. Dansyl, as the protein conjugate or as the free acid, does not appear to penetrate the cells to any significant extent. Dansylated mixed lymphocyte cultures respond to lectin mitogen stimulation for at least 72 hr. Furthermore, differential response of dansylated lymphocytes in culture to three plant lectin mitogens provides a clue to the binding loci of concanavalin A with respect to phytohemagglutinin and pokeweed mitogen on the lymphocyte surface receptors for these lectins. The ability to sustain functionally responsive dansylated lymphocytes for several days in culture suggest that such probe-tagged cells may be useful in elucidating aspects of the plasma membrane in the regulation of cell behavior.


Estrogen induction of growth factors specific for hormone-responsive mammary, pituitary, and kidney tumor cells. The problem of estrogen-promoted tumor cell growth has been studied extensively in an attempt to establish the direct mitogenic role of these steroid hormones. We have developed cell lines from three estrogen-responsive tumors or cell populations: the H-301 kidney tumor cells established from a parent estrogen-dependent hamster kidney tumor, the GH3/C14 rat pituitary tumor cell line established as a subline of the original GH3 population, and the MTW9/PL mammary cell line developed from a parent estrogen- and prolactin-responsive MT-W9A carcinogen-induced rat tumor. With all three of these cell lines, we have encountered a paradox: although estrogens are obligatory for tumor formation in vivo, no direct mitogenic effect of estrogens can be shown in culture when assayed by an increase in cell number. We have thus considered the possibility that estrogens may induce growth factors in vivo that are then responsible for tumor formation by the three cell lines described. Experiments presented in this report show that extracts of rodent uterus, kidney, or liver contain growth activity for these three tumor cell lines, that estrogen treatment causes an increase in tissue content of these activities, and that the estrogen-induced activities are specific for the estrogen-responsive cells. These studies suggest that estrogen-responsive tumor growth in vivo includes the mechanism of estrogen leads to uterus, kidney, or liver leads to specific growth factors leads to estrogen-responsive tumor cells.


Transcription patterns of amplified Dytiscus genes coding for ribosomal RNA after injection into Xenopus oocyte nuclei. Oocytes of the frog Xenopus were injected with purified circular DNA containing amplified rRNA genes of the water beetle Dytiscus. Nuclear contents of injected oocytes were spread and examined by electron microscopy. Most of the Dytiscus DNA seen in injected nuclei contained regions indensely packed with polymerases and nascent transcripts. Apparently normal, as well as abnormal, patterns of transcription were observed. By this type of experiment, it may become possible to recognize the transcribed regions and immediate transcripts of cloned DNA molecules whose activity cannot be seen by electron microscopy of normal nuclei.


Proteins conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens C-58. Membrane-associated and periplasmic proteins of Agrobacterium tumefaciens C-58 were compared with those from avirulent (nontumrigenic) derivative strains by slab and two-dimensional gel electrophoresis. Two proteins (Per-I and Per-2), with a molecular weight of 37,500 and 37,300, respectively, were detected in the supernatant  fraction of cells of strain C-58 treated with EDTA and lysozyme in which a 117-megadalton plasmid confers virulence on the organism. The same proteins are missing in an avirulent plasmid-free derivative of C-58. When this derivative is mated with C-58, the resulting transconjugants regain the large C-58 plasmid together with the restoration of virulence and the expression of Per-1 and Per-2 proteins. When the transconjugants were cured of their plasmid, they concomitantly lost their virulence and Per-1 and Per-2. The functional roles of these proteins are unknown, but they are associated with the outer membrane and periplasmic fraction of the Agrobacterium cell. If directly involved in tumorigenesis, these proteins are not the sole determinants of tumorigenicity because they are synthesized in an avirulent derivative of C-58 that carries a deletion in the plasmid in the region conferring the tumorigenic phenotype. These results strongly suggest that the Per-1 and Per-2 proteins are plasmid-coded gene products. The possible roles of these proteins in specifying host range and host-cell attachment are also discussed.


Synthesis of globin mRNA in relation to the cell cycle during induced murine erythroleukemia differentiation. The relationship between the synthesis of globin mRNA and the phase of cell cycle was examined in synchronized murine erythroleukemia cells. Cells were synchronized with respect to the cell division cycle either by culture with 2 mM thymidine or 2 mM thymidine followed by 0.5 mM hydroxyurea, which caused cells to accumulate in late G1 or early S (referred to as G1/S boundary). Cells were induced to erythroid differentiation by culture with 280 mM dimethyl sulfoxide or 4 mM hexamethylene bisacetamide. These inducers do not alter the progression of cells from the G1/S boundary through S, G2, and M, but do cause prolongation of the subsequent G1 phase. Accumulation of newly synthesized globin mRNA is first detected when cells are in this G1 phase.


Fluorescent light-induced DNA crosslinkage and chromatid breaks in mouse cells in culture. A single 20-hr exposure of mouse cells derived from embryonic or lung tissue to cool-white fluorescent light (4.6 W/m2) causes both DNA damage and chromosome aberrations including chromatid breaks, exchanges, and minutes. In Kohn's alkaline elution technique, the DNA from exposed cells elutes more slowly than that from shielded cells. Because larger molecular weight DNA elutes slower than smaller, we interpret these results to mean that the DNA in cells exposed to light is crosslinked. The estimated frequency of crosslinks is sufficient to account for the number of chromatid breaks observed. The types of chromosome aberrations produced by light indicate that the primary lesion results in chromatid rather than chromosome breaks, and the results suggest an influence of cell density in that cells in densely populated cultures showed few or no chromatid breaks after irradiation. The present results, together with observations from the literature, suggest that the DNA crosslinkage and the chromosome aberrations produced by light may be related.


Lengthening of the G1 phase is not strictly correlated with differentiation in Friend erythroleukemia cells. Friend murine erythroleukemia cells (Friend cells) undergo erythroid differentiation in vitro with an increased probability when cells are cultured in the presence of dimethyl sulfoxide (Me2SO) or other agents. Exponentially growing Friend cells, after dilution into medium containing Me2SO, underwent a transient lengthening of the G1 phase of the cell cycle before they became committed to erythroid differentiation. For nine inducing agents, a positive correlation was found between the percentage of cells that had differentiated and synthesized heme, and the percentage of progenitor cells in which a lengthened G1 phase had previously been observed. This correlation was not found, however, with two other potent inducing agents, hypoxanthine and actinomycin D. Moreover, cells that underwent a lengthened G1 phase did not always terminally differentiate. One such example was a Me2SO-resistant, variant Friend cell line (520a) grown in the presence of Me2SO. These results imply that the prolonged G1 phase, although observed with many inducers, is not a prerequisite for erythroid differentiation with all inducers.


Changes in membrane potential of human granulocytes antecede the metabolic responses to surface stimulation. Human granulocytes (polymorphonuclear leukocytes) exposed to surface stimuli [e.g., immune complexes, concanavalin A (Con A)] generate O(2).(-), undergo a respiratory burst, and secrete lysosomal enzymes. To study the earliest reaction of ligands with surface receptors of granulocytes, purified cells were exposed to bovine serum albumin-anti-albumin complexes (Fc receptors) or Con A (glycoprotein receptors). The membrane potential (DeltaPsi) was measured by distribution of the lipophilic cation [(3)H]triphenylmethyl phosphonium ion. The Nernst equation yielded a resting DeltaPsi of -26.7 mV. Beginning within 10 sec after exposure to the antigen-antibody complex or to Con A, the cells responded with a rapid hyperpolarization --> depolarization --> slow hyperpolarization. Even when phagocytosis was inhibited by cytochalasin B, the triphasic response was obtained: evidence for surface interaction. The hyperpolarization response anteceded O(2).(-) generation (continuous recording) by at least 20-30 sec. O(2).(-) generation in response to immune complexes was stimulated by Ca(2+) whereas DeltaPsi remained unchanged; lack of Ca(2+) in the medium did not inhibit the DeltaPsi response. Dissociation of membrane hyperpolarization from subsequent metabolic responses (O(2).(-) generation) was also found in the presence of steroids (hydrocortisone, methylprednisolone), which inhibited O(2).(-) generation but did not inhibit the DeltaPsi response to antigen-antibody complex. Because O(2).(-) generation could be stimulated (Ca(2+)) or depressed (steroids) without affecting DeltaPsi, the data suggest that DeltaPsi is involved in primary triggering of phagocytic cells and that metabolic stimulation is a secondary consequence of ligand-receptor interactions.


Effect of anti-spectrin antibody and ATP on deformability of resealed erythrocyte membranes. Deformability of resealed erythrocyte membranes was measured by using an ektacytometer. Divalent anti-spectrin antibody, but not monovalent anti-spectrin Fab fragments, decreased membrane deformability. Membranes resealed with MgATP were more deformable than those without MgATP. Exogenous alkaline phosphatase, which dephosphorylates spectrin, decreased membrane deformability. These results suggest that spectrin is an essential component of the system that determines erythrocyte deformability. They are consistent with the view that the role of ATP in membrane deformability is mediated through phosphorylation of the spectrin.


Alteration of myoblast phenotype by dimethyl sulfoxide. Application of dimethyl sulfoxide to proliferating L8 myoblasts (an established cell line of rat skeletal muscle) for 72 hr completely prevented fusion and induction of creatine phosphokinase (EC 2.7.3.2) activity (an indicator of muscle differentiation). The growth pattern changed from the usual sheets of randomly oriented cells to flattened, whorled monolayers of elongated fibroblast-like cells. By electron microscopy, rough endoplasmic reticulum increased and extracellular material appeared that had the morphologic and staining characteristics of collagen. After 120 hr in dimethyl sulfoxide-containing medium, the cells secreted about 6 times more collagen than untreated controls. Dimethyl sulfoxide was ineffective when applied to L8 cells just prior to fusion, and effects of dimethyl sulfoxide were not readily reversible unless treated cells were subcultured at low density.


Membrane sialic acid on target particles modulates their phagocytosis by a trypsin-sensitive mechanism on human monocytes. Monolayers of human peripheral blood monocytes in the absence of exogenous proteins ingest a variety of natural particulate activators of the human alternative complement pathway. Sheep erythrocytes, which do not ordinarily activate the human alternative complement pathway or initiate a direct monocyte phagocytic response, can be modified to exhibit both functions by the deletion or alteration of membrane sialic acid residues. Enzymatic removal of the sialic acid residues with sialidase or their conversion to heptulosonic acid derivatives by limited oxidation with NaIO4 and reduction with BH4- have equivalent dose-response effects on the capacity of the altered sheep erythrocytes to initiate the phagocytic response by human monocytes or to activate the alternative pathway in human serum. The deposition of C3b on native sheep erythrocytes had little effect on their ingestion by human monocytes, whereas the fixation of C3b on desialated sheep erythrocytes had a synergistic effect on the percentage of monocytes ingesting such a particle. The monocyte receptor essential for ingestion of desialated sheep erythrocytes or desialated sheep erythrocytes bearing C3b was inactivated by concentrations of trypsin that also prevented the monocytes from ingesting natural activators of the human alternative complement pathway, but did not alter the receptors for C3b or the Fc portion of IgG. The capacity of the nonimmune host to respond to desialated particles by initiating the monocyte ingestive process and by activating the alternative complement pathway to provide the synergy afforded by C3b deposition on that particle represents a primitive biochemical basis for differentiation of nonself from self.


Human (HLA-A and HLA-B) and murine (H-2K and H-2D) histocompatibility antigens are cell surface receptors for Semliki Forest virus. The proteins coded for by the HLA-A and HLA-B loci in man and the H-2K and H-2D loci in mice were identified as cell surface receptors for Semliki Forest virus. This conclusion is based on the following observations: (i) Water-soluble octamers of viral coat proteins inhibit the complement-dependent cytotoxicity of antibodies directed against H-2K and H-2D antigens in mouse cells. (ii) Isolated detergent-soluble HLA-A and HLA-B antigens reconstituted in lipid vesicles inhibit the binding of viral proteins to human cells (as do the water-soluble antigens to a lesser extent). (iii) Reconstituted HLA-A and HLA-B vesicles interact in solution with Semliki Forest virus (or with vesicles containing viral spike proteins), as demonstrated by coprecipitation with antisera. (iv) Complexes between viral spoke proteins and HLA-A and HLA-B antigens or H-2K and H-2D antigens can be isolated from the cell surface by utilizing affinity chromatography or immunoprecipitation.


Inhibition of dimethyl sulfoxide-stimulated Friend cell erythrodifferentiation by hydrocortisone and other steroids. Erythrodifferentiation and hemoglobin synthesis in dimethyl sulfoxide-stimulated Friend erythroleukemia cells were inhibited by hydrocortisone (HC) and four other steroids: dexamethasone, deoxycorticosterone, corticosterone, and aldosterone. The effect was specific, because no significant cytotoxicity occurred with any of these compounds at the concentrations that were inhibitory. The mechanism of action of HC was studied in detail. In the absence of dimethyl sulfoxide, it had no effect on hemoglobin levels; but, in the presence of this inducer, the synthesis of heme and globin were each inhibited by approximately 90%. There was no alteration in the synthesis of any major protein other than globin, as determined by gel electrophoresis of cell lysates. The activities of two enzymes in the heme biosynthetic pathway, delta-aminolevulinate dehydratase and uroporphyrinogen-I synthase, were inhibited by 80% and 70%, respectively. Globin mRNA induction was reduced by approximately 90%. This demonstrated that the HC inhibition of globin synthesis occurred at a pretranslational step. The dimethyl sulfoxide-induced single-stranded breaks in DNA, which have been suggested to play a role in Friend leukemia cell differentiation, were reduced in number but not eliminated. HC reduced the dimethyl sulfoxide-stimulation of virus release into the medium by approximately 50%. HC treatment in the absence of dimethyl sulfoxide doubled the production of virus.


Uridine diphosphate N-acetylglucosamine stimulates uptake of nutrients by quiescent BALB/C3T3 cells. Preincubation of quiescent BALB/C3T3 cells with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in conditioned medium resulted in a time- and concentration-dependent stimulation of uptake of 2-deoxyglucose, uridine, and alpha-aminoisobutyric acid over that seen in conditioned medium alone. The apparent Km values for 2-deoxyglucose and uridine uptake were the same for cells incubated with or without UDP-GlcNAc, whereas the Vmax values were higher for cells pretreated with the nucleotide sugar. The stimulation of uptake was specific for UDP-GlcNAc, and the other nucleotide sugars tested were ineffective; incubation of UDP-GlcNAc-pretreated cells with N-acetylglucosaminidase abolished the stimulatory effect. In all cases, the extent of stimulation of nutrient uptake was comparable to that seen with serum-stimulated cells. Incubation of quiescent cells in situ with UDP-[3H]GlcNAc led to incorporation of radioactive N-acetylglucosamine into the acid-precipitable fraction; a large fraction of the labeled amino sugar was found on the cell surface acceptors. We interpret these data to mean that the cellular acceptors of quiescent cells are "under-glycosylated," at least in terms of N-acetylglucosamine, and that stimulation of uptake of nutrients may be a consequence of restoration of the amino sugar residues on the oligosaccharide chains of acceptors on the cell surface.


Conjugation of methotrexate to poly(L-lysine) increases drug transport and overcomes drug resistance in cultured cells. Methotrexate and [(3)H]methotrexate were conjugated through a carbodiimide-catalyzed reaction to a 70,000 molecular weight poly(L-lysine) in molar ratios of approximately 13 to 1. The cellular uptake of labeled conjugate was far in excess of the uptake of free drug in cells that were either proficient or deficient in methotrexate transport. The conjugate markedly inhibited the growth of PRO(-)3 Mtx(RII) 5-3 Chinese hamster ovary cells, which are known to be drug resistant by virtue of a deficient methotrexate transport. The cells, however, were not inhibited by the same concentrations of free poly(Lys) and free drug. The 100-fold difference in drug concentration needed to inhibit the mutant cells and their corresponding wild type was totally abolished by exposing the methotrexate-resistant cells to methotrexate-poly(Lys). That the drug is carried into the resistant cells as intact drug-poly(Lys) is evident also from the fact that the conjugate is rendered inactive by brief trypsinization in vitro. Because the conjugate fails to inhibit dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP(+) oxidoreductase; EC 1.5.1.3) in vitro, it must be concluded that the strong growth inhibitory effect of the conjugate is due to the intracellular hydrolysis of its polymeric backbone, followed by the release inside the cell of a pharmacologically active form of methotrexate. Our date show that in methotrexate-resistant cells the intracellular release of active drug after uptake of conjugate is of the same order of magnitude as the uptake of free drug by transport-proficient cells and, hence, that the drug resistance due to deficient transport can be totally overcome.