Differential utilization of proteins in saliva from caries-active and caries-free subjects as growth substrates by plaque-forming streptococci.

Mixed or parotid saliva from caries-active individuals consistently supported better growth of Streptococcus mutans (type c) than that from caries-free individuals. Electrophoretic studies revealed that certain proteins in caries-active salivas were susceptible to microbial attack, but similar proteins in caries-free salivas were refractory.


Morphological and functional characterization of isolated human parotid acinar cells.

A method for the isolation of human parotid acinar cells using enzymatic dispersion and milk mechanical forces has been developed. The isolated acinar cells are morphologically and functionally intact, thus suitable for studies of the cellular physiology of secretion.


Recognition and treatment of patients with chronic orofacial pain.

Patients with chronic orofacial pain must be treated with methods different from those used with patients with acute pain. If different methods are not used, the characteristics of chronic pain may become firmly entrenched. Dentists should be aware of the various methods of treatment for this separate pain entity.


Clinical criteria in diagnosis of early squamous cell carcinoma of the lower lip.

Squamous cell carcinoma of the lower lip may be unsuspected and undiagnosed in its early clinical stages and may appear in occult and noncharacteristic forms. Specific risk factors and characteristics of lesions were sought after clinical and histologic data were correlated in a survey of patients with precancerous and cancerous lesions of the lower lip.


The anatomy of the maxillary molar furcal plane crown preparation.

The location and condition of the pulpal tissues under remaining dentin must be considered when a crown is used for restoration of a maxillary molar prepared to the furcations. This problem is examined by identification of basic landmarks. The study also discusses the furcal plane and the root trunk and relates the discussion to a three-part dissection of a crown preparation of a maxillary molar.


Benign cementoblastoma.

A case of benign cementoblastoma in a 20-year-old man has been reviewed. The lesion was typical and was associated with the mandibular right first molar. The patient initially reported symptoms related to that tooth. Radiographically, the lesion was 1.5 cm in diameter and had a peripheral radiolucent border. Histopathologic examination showed a well-circumscribed mass of cementum. Excision of the tumor with removal of the associated tooth resulted in normal osseous healing and no evidence of recurrence after one year.


Surgical repair of a double lip.

Double lip, an accessory fold of redundant mucous membrane inside the vermilion border, is differentiated from other chronic enlargements of the lip, and treatment for this congenital anomaly is outlined.


The interaction of erythromycin and theophylline in the asthmatic dental patient.

Possible interaction of erythromycin with theophylline is a problem in the dental treatment of asthmatic patients. This interaction can be circumvented by using other antibiotics or adjusting the dose of theophylline.


Antibody responses to influenza immunization of children with acute lymphoblastic leukemia.

Antibody responses of two doses of a bivalent influenza vaccine containing A/Victoria/75 (A/Vic/75) and A/New Jersey/76 (A/NJ/76) viral antigens were studied in 22 children receiving maintenance chemotherapy for acute lymphoblastic leukemia (ALL), 16 children no longer receiving therapy for ALL, and 50 sibling controls. Before immunization, the three groups showed no difference in titer of antibody to either antigen. After the first immunization, children off therapy showed significantly higher titers to A/NJ/76 than did either sibling controls of children receiving therapy (P less than 0.01). After the second immunization, children off therapy showed significantly higher antibody titers to both antigens than did children receiving therapy or controls (P less than 0.01 for both A/NJ/76 and A/Vic/75). Antibody titers of children receiving therapy were not significantly different from those of controls. A year later, there were no significant differences in antibody titers among the groups. Thus, children with ALL who are receiving chemotherapy respond normally to two doses of influenza vaccine, whereas children off therapy manifest abnormally high titers of antibody to both influenza virus antigens.


The intracellular enzymatic response of neutrophils and lymphocytes in patients with precancerous states and cancer of the larynx.

In patients with precancerous states and cancer of the larynx prior to and after radiotherapy exhibit the decreased activity of neutrophil beta-glucuronidase. Moreover patients treated by radiotherapy before the age of 6 to 9 years demonstrate deficiency of N-acetyl-beta-glucosaminidase in the above cells. The main finding in lymphocytes of the patients studied was in the appearance by diffusion of the above enzymes and of acid phosphatase in the cytoplasm, reflecting their release from lysosomes and immunological mobilization of these cells. The authors discuss the possible role of neutrophil enzymatic deficiency in lowering the antitumour cytotoxic effect of these cells.


Oral cancer and sex, Why do females do better?

Analysis of a group of 194 patients with squamous cell carcinoma of the oral cavity has shown that the survival figures for females is significantly better than that for males. The material has been analyzed in an attempt to explain this difference. The females in this series did not attend at an earlier stage, at a younger age or with more highly differentiated tumours. The site distribution was similar for the two sexes but the improved survival for females was found particularly with tumours of the cheek and commissure region and the lower alveolus. Various possible explanations are discussed and it is suggested that the host response of females to intra-oral cancer is different to that of males.


Incidence of lymph node metastasis in elective (prophylactic) neck dissection for oral carcinoma.

For cancers of the tongue, floor of mouth, mandibular gingiva and buccal mucosa, in which the widest diameter is greater than 2 cm, we perform neck dissection in continuity with resection of the primary growth as a part of the planned therapy,regardles of the clinical state of the cervical lymph nodes. Whenever the lesion is so situated that an in-continuity neck dissection cannot be performed (cancer of the palate) it may be wise to delay neck dissection until the lymph nodes become clinically apparent.


Complete remission of a squamous cell cancer of the mandible by chemotherapy. A case report.

This is a report of complete remission with intra-arterial Methotrexate chemotherapy in a patient with a large fungating squamous cell carcinoma of the left mandible.


Progress in the diagnosis of craniofacial injuries and tumours by computer tomography.

This paper tries to demonstrate the diagnostic possibilities  and limitations of CT in the facial skeleton. Our results stem from the examination of benign and malignant processes in 250 patients. This paper should demonstrate the different characteristics of the pictures obtained by CT and conventional polytomography. The main purpose of CT is not the diagnosis of fractures - here pluridirectional tomography is far superior - but the demonstration of space-occupying processes of benign and malignant tumours. CT cannot presently be applied to the investigation of different histopathological lesions.


Study of Biopsies from lesions of the oral mucosa by scanning electron microscopy (S.E.M.).

Surface epithelial cells from 55 biopsies including lichen planus, leukoplakia and squamous cell carcinoma were studied by scanning electron microscopy. The surface structures of the various oral lesions demonstrated morphological differences. After more information has been collected, the method of SEM may be of value for the recognition of pre- and early neoplastic lesions in the oral cavity.


Surgical access to multicystic lesions, by sagittal splitting of the lower jaw.

A case is presented of a large, multiloculated primordial cyst in the lower jaw. In treatment planning of the lesion, a hemimandibulectomy had been seriously considered, but seemed to be too agressive a therapy for a non-malignant lesion. However, the recurrence rate of multiloculated cysts made a meticulous enucleation necessary. By employing a sagittal splitting of the right half of the mandible a good surgical access was provided and the cysts could be removed very thoroughly. Recurrence was not observed during a 3 years follow up period.


Cusps or no cusps--that is not the question.

Current views of the significance of cusps to oral health range from an emphasis on meticulous recusping of the restored teeth to a recommendation of gradual prophylactic elimination of natural cusps by grinding. Cusps, their morphology and mutual arrangement are associated with masticatory function. The adaptive value of efficient mastication has diminished along with advancing cultural evolution. Industrialization marks a turning point as seen from the almost total disappearance of functional tooth wear. Restoring cusps to achieve maximum structural efficiency thus lacks a rational basis. But the occlusal morphology is also associated with the swallow reflex: retrusive occlusal interferences tend to disturb it. These interferences can be avoided by establishing the freedom in centric or by restoring the occlusion to the retruded position. The prophylactic grinding of teeth also lacks a rational basis. The consequences of artificial wear are not likely to be the same as those of functional wear because the function has changed, the oral environment is different. It seems advisable, therefore, to try to eliminate only unquestionably harmful factors, and to leave the rest of the morphology as unchanged as possible.


The effect of placement of cervical margins of class II amalgam restorations on plaque accumulation and gingival health.

The effects or restoring early proximal carious lesions were investigated in a series of thirty-six surfaces. In eighteen lesions the cervical margins of the restorations extended subgingivally, and in eighteen the margins were located supragingivally. Three months after restoration, plaque accumulation was not significantly different from pre-restoration levels. The degree of gingival inflammation showed a significant improvement after restoration where cervical margins were finished supragingivally (P less than 0.5). Where margins were finished subgingivally a highly significant deterioration in gingival health occurred (P less than 0.1).


Processing factors affecting the clarity of a rapid-curing clear acrylic resin.

The difficulty in repeatedly producing unblemished, clear acrylic resin in the dental laboratory has hindered its wider use, despite its many advantages over coloured material. Recently, rapid-cure dental acrylics have been introduced, which are available in both clear and coloured forms. This investigation examined various factors which may influence the production of unblemished, rapid-curing, clear acrylic resin. Utilizing a quantitative assessment of clarity, the most important factor influencing the clarity of the resin is shown to be the choice of separating medium. Tin-foil produces extremely high clarity, but alginate mould separator causes surface blanching. However, this surface blanching can be removed by polishing. Porosity, caused by too rapid curing, and stone model dryness are of only secondary importance. Possible water contamination of the monomer liquid due to accidental exposure only affects clarity at very high levels of contamination.


The Langerhans cell density of palatal epithelium in denture and non-denture wearers, as correlated with other parameters of the palatal mucosa.

A quantitative histological investigation was carried out on biopsy specimens taken from patients suffering from denture sore mouth. The results were compared with those obtained in investigations on denture and non-denture wearers. The sections were studied by standardized quantitative morphometric methods. After 4 years the denture bearing palatal epithelium from both groups, normal denture wearers and the patients suffering from denture sore mouth did not show changes in mean thickness of the epithelium as compared with the controls. The mitotic index in denture bearing epithelium from patients suffering from denture sore mouth was three times lower than in the epithelium of the normal denture wearers. The number of Langerhans cells correlated with the mitotic indices of the group of denture wearers and non-denture wearers. The group of denture sore mouth patients showing a low mitotic index showed a high number of Langerhans cells in their palatal epithelium. The three groups of patients investigated did not show differences in density of mast cells in the lamina propria of their palatal epithelium.


Biological testing of dental restorative materials in vitro--a review.

In vitro methods for the biological evaluation of dental restorative materials are discussed, with particular reference to the type of cells used, the physical state of the test material, the method of establishing cell-material contact, and the criteria of cytotoxicity. It is postulated that in vitro methods should provide a reliable assessment of the toxicity of dental restorative materials, but since the current in vitro methods do not mimic in vivo conditions sufficiently closely, the correlation between in vitro and in vivo results is at present poor. The development of in vitro methods more closely simulating in vivo circumstances is advocated.


Limits of perceptibility of condyle displacements on temporomandibular joint radiographs.

The present study investigated to what extent condyle positions have to be different from each other in order to be clearly detectable under clinical conditions on serial radiographs of the temporomandibular joints in identical projection. For that purpose of ten test subjects serial radiographs in optimal lateral oblique and identical projection were made with various positions of the joint. The joint was brought to these positions by displacement of the mandible to an exactly known extent with a special device. Evaluation showed that mean differences between condyle positions of 0.61 +/- 0.16 mm can be recognized clearly. It was statistically proved that this value did not depend upon the experience of an examiner in reading radiographs but upon the technical quality and the interpretability of the radiographs as well as upon the radiographic dimension of the posterior joint space.


Cervical enamel: a danger zone.

The British Standards Institutions's document (1974) recommending toothpaste specifications states that 'There is no evidence that any toothpaste ever produces a level of abrasion causing harm to dental enamel'. This paper questions the assumption that the enamel crown, especially at the cervical margin, is able to cope with modern dietary insult and a 'good' oral hygiene regimen, regardless of the enamel abrasivity of the dentifrice used. Once the enamel crown is breached the underlying dentine is sensitive and vulnerable to physical damage. In this work the potential rates of erosive and abrasive loss are related to the thickness of the protective enamel and to the relevance of fluoride treatments for adults. It is suggested that improvements in dental care and an increased tooth life expectancy now make the loss of tooth tissues at the cervical margin more important than is apparent at first sight.


The reproducibility of movement parameters of the empty open-close-clench cycle in man and their dependency on the frequency of movements.

The reproducibility of the empty open-close-clench cycle has been investigated. Four subjects were asked three times to perform thirty empty open-close-clench movements at each frequency of 1/2, 1 and 2 cycles s-1. To describe such a series of thirty movements a set of nine parameters were selected. By reference to these parameters it was found that the subjects were not always able to exactly reproduce a series of thirty movements at a repeat test. The movement-parameters strongly depended on the frequency of the movements. On increasing the frequency the durations of time in which a movement cycle was divided decreased, while the maximum jaw speeds during opening and closing and the jaw speed just before tooth contact increased. The parameter values chosen to describe the smoothness of the movements performed, indicated that the smoother movements were executed at the higher frequencies.


Hereditary angioneurotic edema: report of case.

A case report in which the management of a patient with hereditary angioneurotic edema requiring oral surgery is presented. A review of the literature with emphasis on the clinical features, etiology, diagnosis, and treatment is also presented.


Monophase extraskeletal fixation.

The monophase appliance for extraskeletal fixation of facial fractures is discussed. The technique for its implementation is presented with a discussion of its indications and considerations. Reports of three cases demonstrate clinical applications of this one-step method of extraskeletal fixation.


Loiasis: report of three cases and literature review.

Loiasis is a tropical infection caused by the microfilarial nematode Loa loa. Increased numbers of cases of loiasis have been diagnosed outside of the endemic areas in Western and Central Africa because of widespread international travel. The author presents three cases of loiasis discovered at Howard University followed by a review of the literature.


Metastases to the larynx.

Four patients with metastases to the larynx are reported. The pathways of metastatic invasion of the larynx are discussed and illustrated with whole organ laryngeal sections from these cases. Although three of the four cases were clinically silent, all could have been detected on indirect laryngoscopy. The true incidence of laryngeal metastases appears to be much higher than clinically suspected, and indirect laryngoscopy of all patients "at risk" should be considered.


Virus production and hemoglobin synthesis in variant lines of dimethyl sulfoxide-treated Friend erythroleukemia cells.

Variant Friend erythroleukemia cell clones were compared in regard to their response to dimethyl sulfoxide and in their abilities to synthesize virus and hemoglobin. Clear evidence was obtained that cellular growth is required for virus production. The effects of dimethyl sulfoxide on virus production were not observed in cell lines that were resistant to growth perturbation by the compound. Studies of cell variants that were defective in either hemoglobin or virus synthesis indicate that these activities are independently regulated.


Electron microscopic analysis of partially replicated bacteriophage T7 DNA.

Partially replicated bacteriophage T7 DNA was isolated from Escherichia coli infected with UV-irradiated T7 bacteriophage and was analyzed by electron microscopy. The analysis determined the distribution of eye forms and forks in the partially replicated molecules. Eye forms and forks in unit length molecules were aligned with respect to the left end of the T7 genome, and segments were scored for replication in each molecule. The resulting histogram showed that only the left 25 to 30% of the molecules was replicated. Several different origins of DNA replication were used to initiate replication in the UV-irradiated experiments in which 32P-labeled progeny DNA from UV-irradiated phage was annealed with ordered restriction fragments of T7 DNA (K. B. Burck and R. C. Miller, Jr., Proc. Natl. Acad. Sci. U.S.A. 75:6144--6148, 1978). Both analyses support partial-replica hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460--476, 1961; Doermann et al., J. Cell. comp. Physiol. 45[Suppl.]:51--74, 1955) as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin to a site of UV damage, and those regions of the genome which replicate most efficiently are rescued most efficiently by a coinfecting phage. In addition, photoreactivation studies support the hypothesis that thymine dimers are the major UV damage blocking cross-reactivation in the right end of the T7 genome.


Lung cancer in Louisiana: death certificate analysis.

In a search for etiologic clues, a review was made of death certificates of residents in a cluster of Louisiana parishes, mainly in the southern part of the state, where lung cancer mortality was high. A comparison of the statements on occupation for 3,327 patients with lung cancer and those of 3,327 controls (matched by sex, race, age, and parish of residence) during 1960-75 revealed an approximately twofold excess risk associated with transportation equipment manufacture, mainly shipbuilding, and the fishing industry. Smaller elevations of lung cancer risk were found among older men who had been employed in petroleum exploration and production and among male and female residents of towns where the petroleum industry was a major employer. In addition, Acadian ancestry was associated with a higher risk of lung cancer among older male and female residents.


Immune response to melanoma extracts in three melanoma-prone families.

Immune reactivity to melanoma extracts was measured by the leukocyte adherence inhibition (LAI) test in 40 members of 3 melanoma-prone families. The melanoma patients had a wide range of responsiveness to the extract, the highest responder being a 10-year survivor. As a group, family members (including spouses) without disease had significantly elevated LAI responses compared to those of unrelated controls (P less than 0.01). Within the families, members with close exposure to melanoma patients for 10 years or more had a significantly higher response to melanoma antigen than did members with 0-5 years of close exposure (P less than 0.05). Responses of spouses and members at high risk of developing melanoma (B-K mole syndrome) also correlated with length of exposure to patients, which suggests that the elevated LAI response was not genetically determined. The high frequency of positive responses to melanoma antigens in these families, particularly in spouses, suggests the presence of transmissible melanoma-associated material capable of immunizing persons in contact with melanoma patients.


Urinary excretion of estrone, estradiol, and estriol in postmenopausal women with primary breast cancer.

The daily excretion of estrone, estradiol, and estriol was determined for 22 normal women and 35 women with primary breast cancer. The excretion of the hormones (measured in microgram/24 hr) in the breast cancer group was elevated and showed a statistical significance of P less than 0.001. The same wide difference between the 2 groups was also noted when excretion was expressed in terms of the body area of the individuals and when women of similar ages were compared.


Studies on metastases. I. Role of sensitization and immunosuppression.

A normally nonmetastatic tumor metastasized to the regional axillary lymph nodes following severe immunosuppression of inbred Swiss mice. The extent of metastasis was dependent on the severity of lymphocyte depletion and the initial number of tumor cells injected. Sensitization of the host to the tumor was important in prevention of metastasis: Metastasis was induced only if the immunosuppressive treatment occurred within 3 days of tumor inoculation. Antitumor immunity was adoptively transferred by the splenocytes from animals given immunosuppressive treatment with X-rays or hydrocortisone acetate on day 14. Though spleen cells from animals given both immunosuppressants did not have this property, that no metastasis was observed even in doubly immunosuppressed mice indicated sensitization to tumor cells. Lymphocyte number was important also: Injection of splenocytes from normal to tumor-bearing animals significantly reduced the occurrence of metastases.


Role of photosensitization and oxygen in chromosome stability and "spontaneous" malignant transformation in culture.

Visible light and oxygen enhanced both chromosome instability and malignant transformation of mouse cells in culture. Nine cell lines were initiated from 8 pools of 10- to 13-day C3H embryos. Each cell line was divided into sublines, which were either maintained shielded from light or were exposed for 3 or 24 hours to fluorescent light (approximately 150 foot-candles) two or three times weekly. Cultures of the sublines were also maintained with either a gaseous phase of 0-1% oxygen or atmospheric (18%) oxygen. Each line was monitored for cytologic manifestations of malignant neoplastic transformation, and 8 lines were monitored for chromosome alterations. Seven lines were assayed for tumorigenicity by intraocular implantation into syngeneic hosts. Repeated light exposure and/or high oxygen increased the frequency of minute chromosomes, which result from chromatid breaks, and also increased the rate of shift from diploid to heteroploid state. Four cell lines showed no cytologic changes indicative of neoplastic change during the test period. Two of these were assayed in vivo and failed to grow as tumors. In the remaining 6 lines, cytologically neoplastic colonies appeared earlier or more abundantly in the light-exposed cultures and/or those gassed with high oxygen. In 3 of these lines, tumors developed only from the light-exposed cultures; in the other 2, tumor latency periods were significantly shorter in the cultures exposed to light or gassed with atmospheric oxygen.


Persistence and growth of rat liver neoplastic nodules following cessation of carcinogen exposure.

Altered (hyperplastic) foci and neoplastic (hyperplastic) nodules identified by their resistance to iron accumulation were induced in the livers of F344 rats by limited feeding of N-2-fluorenylacetamide for three, four, or five cycles of 4-week intervals separated by 1 week of a basal diet. Foci were present by the end of three feeding cycles and increased in number with further carcinogen exposure. No nodules were present at the end of three or four cycles, but they appeared at later intervals after removal of the carcinogen. Nodules were present at the end of five cycles of feeding and increased in number later. Thus nodules were found to be persistent and to have the progressive growth ability in situ that is characteristic of neoplasms.


Effects of cyanate, thiocyanate, and amygdalin on metabolite uptake in normal and neoplastic tissues of the rat.

Thiocyanate was found to resemble cyanate in its inhibitory effects on [3H]thymidine incorporation and the uptake of [32P]phosphate and [3H]amino acids in transplanted tumors of the BUF rat. The capacity to inhibit metabolite uptake in hepatomas and a colon tumor under conditions in which uptake was unchanged or increased in host liver was concluded to be a common feature of the action of cyanate and thiocyanate. Inhibition of [32P]phosphate uptake and [3H]thymidine incorporation into DNA of tumors was also observed after treatment of rats with amygdalin. With this drug, however, the action on tumors and livers of host rats was similar.


Improved remission induction rate with D-ZAPO but unimproved remission duration with addition of immunotherapy to chemotherapy in previously untreated children with ANLL.

In 163 children with acute nonlymphocytic leukemia (ANLL), a D-ZAPO induction program consisting of daunomycin, 5-azacytidine, cytosine arabinoside, prednisone, and vincristine resulted in a remission rate of 71.8%. Immunologic therapy was employed during maintenance with the aim of prolonging remission and improving survival. The administration of immunotherapy consisting of a mixture of bacillus Calmette-Guérin (BCG) and allogenic acute myelomonocytic leukemic cells injected intradermally on day 14 of each of the first three monthly cycles of 6-thioguanine for ten days, 5-azacytidine and cytosine arabinoside for four days, and vincristine for one day did not improve remission duration or survival compared to that due to chemotherapy alone. Important prognostic factors identified in this study included a remission induction rate significantly better for females than males (P = 0.04), for children between the ages of 5 and 10 years compared to those greater than this age group (P = 0.01), and a prolonged remission duration (P = 0.04), and survival (P less than 0.01) for patients with initial white blood counts of less than 20 x 10(9)/liter.


Hypothalamic-pituitary dysfunction following CNS prophylaxis in acute lymphocytic leukemia: correlation with CT scan abnormalities.

In an attempt to identify possible adverse effects of CNS prophylaxis (cranial radiation and intrathecal chemotherapy), we examined hypothalamic-pituitary function in 23 patients with acute lymphocytic leukemia (ALL). Of 18 patients who had received both cranial radiation and intrathecal chemotherapy, nine had abnormally low growth hormone responses to insulin-induced hypoglycemia (less than 7.0 ng/ml). Seven of the nine patients with abnormally low growth hormone responses also manifested ventricular dilatation on computed tomography (CT) brain scans, whereas only one of the nine patients with normal growth hormone responses demonstrated this CT scan finding (P = 0.015). The remaining patients, who had not received cranial radiation, had normal growth hormone responses and normal CT scans. There is significant correlation between ventricular dilatation on CT and abnormally low peak growth hormone responses following CNS prophylaxis in ALL.


Phosphorylase isoenzymes in normal and myophosphorylase-deficient human heart.

Phosphorylase isoenzymes were studied by acrylamide-slab electrophoresis in normal tissues and in the heart of a child with a fatal infantile form of myophosphorylase deficiency. Of the three bands present in normal human heart, two were missing in the patient's heart: the slow "muscle" isoenzyme and the intermediate band. Only the fast "cardiac" isoenzyme remained. When extracts of normal skeletal muscle and the patient's heart were mixed in appropriate conditions, the intermediate band reappeared in the electropherogram. Phosphorylase activity in extracts of the patient's heart was not inhibited by antibodies against purified enzyme from mature human muscle, whereas normal human heart phosphorylase was inhibited by approximately 50%. These results suggest that the intermediate band of human heart phosphorylase is a hybrid of skeletal and cardiac muscle isoenzymes. Retained activity of the cardiac isoenzyme may explain why patients genetically lacking skeletal muscle phosphorylase do not have clinical heart disease.


[Reduced uptake of 99mTc methylenediphosphonate by bone irradiated for neoplastic lesions].

Some cases of more or less evident reduction in 99mTc MDP take-up by bone subjected to cobalt therapy for skeletal and extraskeletal neoplastic diseases are presented. On the basis of acquired experience, stress is laid on the diagnostic and prognostic importance of correct assessment of bone scintigraphic changes after irradiation treatment. Some pathogenetic theories in this phenomenon are discussed.


Investigation of the vascular effect of newer local anesthetics and vasoconstrictors.

Changes in blood flow can be measured with the aid of infrared thermography and make possible a comparative assessment of the vascular effect of vasoconstrictors and local anesthetics. With Adrenalin, vasoconstriction sets in immediately following infiltration; with POR-8, a sufficient vasoconstriction is observed only after 10 to 15 minutes. In both products, ischemia subsides after 60 minutes. Adrenalin then reverses the reaction in reactive hyperemia after 150 minutes, while the tissue infiltrated with POR-8 returns to normal ater 120 minutes. When combining a local anesthetic with a vasoconstrictor, the intrinsic vascular effect has to be considered. We experimentally proved that Novocain (procaine), Hostacain (butanilicaine), and on a lower scale Xylocaine (lignocaine) have a vasodilator effect. Citanest (prilocaine) demonstrates no vasodilatory effect. Carbocaine (mepivacaine) produces a mild vasoconstriction.


Etiology and treatment of idiopathic trigeminal and atypical facial neuralgias.

In a series of sixteen patients with idiopathic trigeminal neuralgia and twenty-one patients with atypical facial neuralgia, it was found that the painful phenomena associated with both disorders were, in nearly all instances, closely related to the presence of maxillary or mandibular bone cavities at previous tooth extraction sites. Standard oral surgical procedures for curettage of the cavities, together with administration of antibiotics, were employed in the successful treatment of both the trigeminal and atypical facial neuralgias, with complete pain remissions for periods varying from 2 months (for most recently treated cases) up to 9 years. The observations and results of this study suggest that dental and oral disorders may play a role in the genesis of trigeminal and atypical facial neuralgias.


Clinical and histologic studies on electrogalvanically induced oral white lesions.

Among 1,128 patients with oral leukoplakia and 326 patients with oral lichen planus included in long-term follow-up studies, thirty-two patients showed lesions of the oral mucosa which could be attributed to electrogalvanism. The material consisted of twenty female and twelve male patients. The clinical diagnosis was leukoplakia in sixteen patients and oral lichen planus in the remaining sixteen patients. Histopathologic characteristics were analyzed and correlated to the clinical findings. In five cases malignant transformation developed. The term electrogalvanic white lesions is suggested as a common denominator for these mucosal changes, which seem to take the shape of lichen planus as often as that of leukoplakia.


Ultrastructural characterization of intermediate cells of mucoepidermoid carcinoma of the parotid.

Five cases of mucoepidermoid carcinoma of the parotid gland were studied ultrastructurally. In addition to epidermoid cells (tonofilament-rich) and mucous cells, three forms of intermediate cells were identified: mitochondria-rich cells, glycogen-rich cells, and ribosome-rich cells. Transitional forms of all cell types were observed. Ultrastructurally, cells rich in glycogen appeared to be the "clear cells" by light microscopy; cells rich in ribosomes appeared basophilic under light microscopy. Mitochondria-rich cells were lightly eosinophilic under light microscopy.


Focal myositis of the perioral musculature.

Focal myositis, a benign inflammatory pseudotumor, is a relatively newly defined clinicopathologic entity which arises as a rapidly enlarging swelling within an isolated skeletal muscle. It occurs most often in the lower extremity, and we are reporting what we believe to be the first cases of perioral involvement. There is no apparent age or sex preference. Focal myositis must be differentiated from neoplasm, nodular pseudosarcomatous fascilitis, proliferative myositis, myositis ossificans, polymyositis, and, in the oral region, salivary gland lesions and hypertrophic branchial myopathy. No lesions have recurred.


The effects of overfilled polyethylene tube intraosseous implants in rats.

Polyethylene tubes obturated flush at one end overfilled 2 mm. at the opposite end with unset Grossman's cement and gutta-percha were implanted in rat tibias. Microscopic evaluation of the specimens revealed that (1) the gutta-percha, Grossman's sealer, and polyethylene tubing are well tolerated by rat intraosseous tissue and (2) a canal overextended 2 mm. beyond the apex with Grossman's cement and gutta-percha produces an early mild to moderate inflammatory response which resolves by the end of 30 day's. The overfillings did not significantly compromise the healing of rat intraosseous tissue.


Determination of tooth length with a standardized paralleling technique and calibrated radiographic measuring film.

A simple radiographic measuring method is presented. Fifty teeth to be extracted were radiographed by means of a standardized paralleling technique and a preprinted intraoral radiographic film. The measuring grid was conveyed to the film as a latent pattern by a stenciling process prior to the x-ray exposure. The distance between the measuring lines was adjusted in accordance with the image magnification. The length of the teeth was read directly on the completed radiograph. The extracted teeth were measured by means of a sliding caliper. A statistical analysis of the data indicated that the present method is more accurate than other clinical methods commonly used for determination of tooth lengths.


Rubber-base impression material as a foreign body. Report of a case.

This case history describes the problems that may arise when foreign materials are injected subperiosteally during a dental procedure. The diagnosis might have been more difficult if the material had not been sufficiently radiopaque to be detected on a radiograph.


Role of mammalian RNase inhibitor in cell-free protein synthesis.

Addition of the human placental RNase inhibitor at 10 mu/ml to a mixture of wheat germ extract and translation components, prior to the addition of mRNA from dog pancreas or influenza virus-infected cells, resulted in a significant increase in the yield of proteins synthesized. Analysis of the translation products by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the inhibitor preferentially increased the yield of the larger proteins. In the presence of the inhibitor, yields of the preprocarboxypeptidases were increased 4.5-fold and yields of preamylase were increased 15-fold. Incubation of the wheat germ extract or individual translation components with dog pancreas mRNA, with or without the placental inhibitor, indicated significant RNase contamination among the fractions. Two other in vitro protein synthesis systems-the reticulocyte lysate system and the Krebs ascites system-were found to contain latent RNase activity (RNase in complex with the inhibitor) and an excess of RNase inhibitor. The addition of placental RNase inhibitor did not increase the yield in these systems, except in those cases in which the RNase contamination approached the amount of endogenous inhibitor. When used during the isolation of rat liver cell fractions, the placental inhibitor increased the yield (as measured by A(260)) of rough microsomes and detached polysomes by 24% and 4.6-fold, respectively. Analysis of translation products indicated that detached polysomes isolated in the presence of the inhibitor were intact; those isolated in the absence of inhibitor were degraded.


Theory of protein-lipid and protein-protein interactions in bilayer membranes.

A model for protein-lipid interactions in bilayer membranes where the proteins are very dilute is extended to higher protein concentration, where appreciable lipid-mediated protein-protein interactions occur. It is found that proteins may change the lipid phase transition temperature and that they weaken the phase transition. There exists a critical protein concentration above which the sharp lipid phase transition is abolished. The model also qualitatively reproduces several experimental observations on the physical behavior of bilayers formed from mixtures of cholesterol and phosphatidylcholines.


Low stereoselectivity in methylacetylene and cyclopropene reductions by nitrogenase.

The stereochemistry of reductions catalyzed by nitrogenase in 2H2O has been investigated by using allene, methylacetylene, and cyclopropene as substrates. Deuterium labeling patterns in the reduction products were determined by mass spectroscopy, infrared spectroscopy, 2H-decoupled 220-MHz 1H NMR, and 1H-decoupled 30.7-MHz 2H NMR. Reduction of allene gave pure [2,3-2H2]propene. Reduction of methyl acetylene gave a 1.8:1.0 mixture of [cis- and [trans-1,2-2H2]propene. (Similar reduction of acetylene reportedly gave virtually all [cis-1,2-2H2]ethylene.) Reduction of cyclopropene gave [cis-1,2-2H2]cyclopropane and a mixture of [2H2]propenes. The major propene 2H2 isomers formed were [trans-1,3-2H2]-propene (approximately 2), [cis-1,3-2H2]propene (approximately 1) and [2,3-2H2]propene (approximately 1). Cyclopropene appears to be unique as a nitrogenase substrate in that it simultaneously undergoes parallel reductions, one of which proceeds with high stereoselectivity while the other proceeds with low stereoselectivity. The weakly selective stereochemistry observed in these reductions is not consistent with a completely concerted dual proton-dual electron transfer mechanism. The results provide a basis to probe stereochemical effects in nitrogenase and in biomimetic model systems.


Interferon, double-stranded RNA, and RNA degradation: activation of an endonuclease by (2'-5')An.

Among the mediators of interferon action are one enzyme that is activated by double-stranded RNA to convert ATP to (2'-5')An and a second enzyme, an endonuclease, that is activated by (2'-5')An to cleave single-stranded RNA. The binding of (2'-5')An to the endonuclease (partially purified from mouse Ehrlich ascites tumor cells) is revealed by its retention on nitrocellulose filters. This can serve as the basis for an assay of the enzyme. Activation of the enzyme is reversible and is lost upon removal of (2'-5')An:gel filtration of activated endonuclease on Sephacryl S-200 results in an inactive enzyme. The enzyme can be activated again, however, by addition of (2'-5')An. The elution volume of the nonactivated endonuclease from Sephadex G-200 indicates that its molecular weight is 185,000, unusually large for a nuclease. The elution volume of the maximally activated endonuclease from Sephadex G-200 equilibrated with (2'-5')An is not detectably different from that of enzyme that had not been previously activated that was passed through Sephadex G-200 not equilibrated with (2'-5')An. This indicates that the activation does not result in a large change in the size or conformation of the enzyme.


Effect of tetanus toxin on the accumulation of the permeant lipophilic cation tetraphenylphosphonium by guinea pig brain synaptosomes.

Accumulation of the permeant lipophilic cation [(3)H]tetraphenylphosphonium (TPP(+)) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K(+) and by veratridine. A further 1:10 to 1:15 decrease in TPP(+) uptake occurs under nitrogen and in the presence of mitochondrial inhibitors such as oligomycin, whereas starvation and succinate supplementation have no effect. These data indicate that, in analogy to intact neurons, there is an electrical potential (DeltaPsi, interior negative) of -60 to -80 mV across the synaptosomal membrane that is due primarily to a K(+) diffusion gradient (K(+) (in)-->K(+) (out)). The data also indicate that mitochondria entrapped within the synaptosome but not free mitochondria make a large contribution to the TPP(+) concentration gradients observed. Conditions are defined in which tetanus toxin binds specifically and immediately to synaptosomes in media used to measure TPP(+) uptake. Under these conditions tetanus toxin induces dose-dependent changes in TPP(+) uptake that are blocked by antitoxin and not mimicked by biologically inactivated toxin preparations. The effect of tetanus toxin on TPP(+) uptake is not evident in the presence of 193 mM K(+) or veratridine but remains under conditions known to abolish the mitochondrial DeltaPsi. Moreover, tetanus toxin has no effect on TPP(+) uptake by isolated synaptosomal mitochondria. The results thus define an in vitro action of tetanus toxin on the synaptosomal membrane that can be correlated with biological potency in vivo and is consistent with the in vivo effects of tetanus toxin on neuronal transmission.


Comparison of bovine and mouse pituitary glycoprotein hormone pre-alpha subunits synthesized in vitro.

Poly(A)-containing RNA isolated from bovine and mouse pituitaries and a mouse pituitary thyrotropic tumor was translated in a wheat germ cell-free biosynthetic system. A precursor of the glycoprotein hormone alpha subunit, "pre-alpha," was immunoprecipitated from the translation mixtures with antiserum against bovine luteinizing hormone (LH; lutropin) alpha. The specificity of the immunoprecipitation was shown by competition with authentic bovine LHalpha and lack of competition with bovine thyroid-stimulating hormone (TSH; thyrotropin) beta. Bovine and mouse pre-alpha subunits migrated identically in sodium dodecyl sulfate gradient polyacrylamide slab gels with an apparent molecular weight of about 17,000. Pre-alpha comprised 2-3% and 20-30% of the total proteins translated with pituitary and pituitary tumor mRNA, respectively. Microanalysis of amino acid sequence of the pre-alpha subunits containing various radiolabeled amino acids gave the following partial sequence for mouse tumor pre-alpha: [Formula: see text] Met was also found in positions 1, 14, and 17 in mouse pituitary pre-alpha but only in residue 1 of the bovine pituitary pre-alpha subunit. Leu was found in identical positions in bovine pituitary pre-alpha, with an additional Leu in position 17. Leu in the common positions (12, 15, 19, and 22) has also been found in human choriogonadotropin pre-alpha subunit [Birken, S., Fetherston, J., Desmond, J., Canfield, R. & Boime, I. (1978) Biochem. Biophys. Res. Commun. 85, 1247-1253]. The data demonstrate that pituitary as well as placental glycoprotein hormone alpha subunits are synthesized with an amino-terminal hydrophobic extension, in accord with the "signal hypothesis" for secreted proteins. Furthermore, the positions of the hydrophobic amino acid Leu have been strictly conserved in pre-alpha subunits from various species and in two different tissues, the pituitary and placenta.


Effects of ethanol on alanine metabolism in perfused mouse liver studied by 13C NMR.

Time courses of 13C labeling from alanine and ethanol in perfused mouse livers have been followed by NMR. The enrichment at specific carbons of glucose, glutamate, glutamine, aspartate, acetate, acetoacetate, beta-hydroxybutyrate, and lactate has been measured. The specific labeling of glutamate in the presence of labeled alanine and labeled or unlabeled ethanol shows that, under these conditions, alanine enters the tricarboxylic acid cycle almost exclusively through pyruvate carboxylation, whereas ethanol is the exclusive source of acetyl-CoA. In the absence of ethanol, the alanine label flows through both paths. By comparing the scrambling of 13C between C3 and C2 of glutamate it is possible to estimate the mitochondrial fumarase activity; the C6-to-C5 ratios in glucose give the additional scrambling by cytosolic fumarase activity. In addition, the C6-to-C1 and C5-to-C2 ratios in glucose show that there is about 15% flux through the pentose cycle. Finally, the C4-to-C2 ratios in glutamine and glutamate are unequal at any time (the glutamine labels reflect the label distribution in glutamate measured 1 hr earlier), providing a method for studying flow through glutamine synthetase in situ.


Interferon action: induction of specific proteins in mouse and human cells by homologous interferons.

Treatment of mouse (Ehrlich ascites tumor and L929) and human (FS4, GM258, etc.) cells with homologous interferons results in the induction of several proteins. Extracts obtained from cells labeled with [35S]methionine in the absence or presence of interferon were fractionated on poly(I) . poly(C)-agarose columns. The proteins retained on the columns revealed, upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, three protein bands in mouse cells (Mr 120,000; 80,000; and 67,000) and two in human cells (Mr 120,000 and 80,000) which were detected in the extracts of interferon-treated but not of untreated cells. These proteins were retained on double-stranded RNA [poly(I) . poly(C)-agarose] columns but very poorly, if at all, on single-stranded RNA [poly(I)- or poly(C)-agarose] columns, suggesting that they have an affinity for double-stranded RNA. In addition, interferon treatment of human fibroblasts greatly increased the labeling of three other protein bands (Mr 88,000; 67,000; and 56,000) which were detected in whole extracts but were not appreciably retained on poly(I) . poly(C)-agarose columns. The appearance of the induced proteins was blocked by actinomycin D if added together with interferon, indicating that transcription of certain genetic information is required. The possible correlation between the induced proteins described here and the elevated levels of certain enzymes in interferon-treated cells (a protein kinase and 2'-5'-oligoadenylate synthetase) is at present unclear.


Structure of the human G gamma-A gamma-delta-beta-globin gene locus.

We have constructed a physical map of the human G gamma-, A gamma-, delta-, and beta-globin genes. The previously described maps of the fetal and adult beta-like globin genes have been linked to one another by identification of a DNA fragment, generated by BamHI, that contains part of each of the A gamma- and delta-globin genes. The map obtained, which spans more than 40 kilobases, shows the following intergene distances: between G gamma and A gamma, 3500 base pairs; between A gamma and delta, 13,500 base pairs; and between delta and beta, 5500 base pairs. All genes are transcribed from the same DNA strand.


Translation and characterization of messenger RNAs in differentiating chicken cartilage.

Total RNA, prepared from chicken limb bud cultures undergoing differentiation to cartilage, has been translated in a wheat germ cell-free protein-synthesizing system. Antibodies against chondroitin sulfate proteoglycan core protein immunoprecipitate a single component which migrates as a protein of 340,000 daltons in sodium dodecyl sulfate/polyacrylamide gels. The messenger RNA for this protein sediments at approximately 27 S in 70% formamide or aqueous sucrose gradients. The 340,000-dalton protein is present in cell-free translation products directed by RNA prepared from limb bud cultures and sternae and is absent in cell-free translation directed by RNA prepared from embryonic calvaria or liver. The level of synthesis of this protein is greatly reduced when RNA prepared from limb bud cultures inhibited from differentiation by BrdUrd is used. (Pre)pro alpha 1(I), -alpha 2(I), and -alpha 1(II) collagen bands have been identified on gels by electrophoresis of collagenase-digested or immunoprecipitated cell-free translation products directed by RNA from differentiating limb bud cultures, embryonic sternae, and embryonic calvaria.


Micronuclei of Tetrahymena contain two types of histone H3.

Evidence is presented that micronuclei of Tetrahymena thermophila contain significant amounts of two types of histone H3. One is indistinguishable from that found in macronuclei and the other is unique to micronuclei. The micronucleus-specific H3 has a slightly faster mobility than the common H3 in three different gel systems (both of these species were artifactually lost during procedures for histone preparation in previous studies). Both micronuclear H3s appear to contain a single cysteine residue and are present in sucrose gradient-purified nucleosomes. Acid extracts from micronuclei also contain three prominent high molecular weight proteins that also were lost during previous procedures. These proteins are present in extracts from oligomers but are not observed in extracts from mononucleosomes, suggesting that they may be associated with linker regions between nucleosomes.


Studies of binding of prekallikrein and Factor XI to high molecular weight kininogen and its light chain.

Prekallikrein and Factor XI have been reported to circulate as complexes with the coagulation cofactor high molecular weight (HMW)-kininogen. In this study we have shown that native HMW-kininogen possesses a strong binding site for prekallikrein and Factor XI with association constants of 3.4 x 10(7) M-1 and 4.2 x 10(8) M-1, respectively. The diminished binding of prekallikrein relative to Factor XI may, in part, account for the ability of kallikrein to leave the surface and interact with other molecules of Hageman factor and HMW-kininogen. Prekallikrein and Factor XI appear to compete for binding to HMW-kininogen, suggesting a single (or closely overlapping) binding site(s). The purified light chain derived from kinin-free HMW-kininogen is shown to compete with native MHW-kininogen for binding to Hageman factor substrates and direct binding of the isolated light chain to prekalikrein and Factor XI is demonstrated. This binding of the light chain to prekallikrein and Factor XI appears to be essential to the function of HMW-kininogen as a coagulation cofactor and further digestion of the light chain with excess kallikrein destroys its coagulant activity.


Peptide maps comparing subunits of maize chloroplast and type II nuclear DNA-dependent RNA polymerases.

Both one- and two-dimensional peptide mapping techniques have been used to compare the 180-kilodalton subunits from maize chloroplast and type II nuclear DNA-dependent RNA polymerases (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). Despite their similar molecular mass, these 180-kilodalton subunits are not the same. Also, two-dimensional tryptic maps of the 160-, 43-, and 28-kilodalton polypeptides from maize type II nuclear RNA polymerase and of the 140-, 42-, and 27-kilodalton polypeptides from maize chloroplast RNA polymerase show that each of these six polypeptides is unique.


Segment 8 of the influenza virus genome is unique in coding for two polypeptides.

In previous studies we showed that a ninth polypeptide with a molecular weight of approximately 11,000 (NS2) found in influenza virus-infected cells was unique, that it could be synthesized in vitro, and that its expression in vivo required early protein synthesis. On the basis of these results we suggested that one of the eight genome RNA segments of influenza virus codes for two polypeptides [Lamb, R.A., Etkind, P.R. & Choppin, P.W. (1978) Virology 91, 60-78]. We describe here differences in the electrophoretic mobility of the NS2 polypeptides of different strains of influenza A virus. These results provided further evidence that NS2 is virus coded and also made possible genetic studies using recombinants between two virus strains (HK and PR8) whose NS2 polypeptides differ. These studies showed that the gene for NS2 reassorts with that of the nonstructural polypeptide NS1, which is coded by genome segment 8. A mRNA for NS2 has been separated from that of NS1 and the other viral polypeptides by centrifugation and has been translated in vitro. Hybridization of genome segment 8 to the total mRNAs from infected cells specifically prevented the synthesis of NS2 and NS1. These results indicate that influenza virus genome segment 8 is transcribed into two separate mRNAs that code for two polypeptides, NS1 and NS2. Possible mechanisms for the transcription of the two mRNAs from either contiguous or overlapping genes are discussed.


Insulin receptor: covalent labeling and identification of subunits.

Two methods were used to label insulin receptors covalently with 125I. In the first, an aryl azide derivative of insulin, 125I-labeled 4-azido-2-nitrophenyl-insulin, was synthesized and used to photolabel the binding region of the insulin receptor in rat liver membranes and human placenta membranes. In the second, insulin receptors were purified from rat liver membranes and labeled with 125I by use of chloramine-T; this method presumably has no specificity for the binding region of the receptor. The proteins labeled by both methods were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis after or without reduction by dithiothreitol. The photoaffinity label specifically labeled a single band in both liver and placenta that had an apparent molecular weight of 135,000 after reduction. A band with similar mobility was present in the chloramine-T-labeled preparation, which also contained a second major band with an apparent molecular weight of 45,000. Without reduction, both methods resulted in a single labeled band with an apparent molecular weight of about 310,000. These results indicate that the insulin receptor of both liver and placenta has a subunit of molecular weight 135,000 that binds insulin and that the receptor may be composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds.


Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation.

We describe a simple procedure for isolating specific mRNAs and for mapping them to the regions of the DNA from which they originate. The method involves hybridization of total cytoplasmic RNA to restriction fragments of DNA that have been fractionated in agarose gels and immobilized on nitrocellulose filters. The hybridization-selected RNAs are eluted and translated in a cell-free system in order to identify their encoded polypeptides. Optimal hybridization and filter washing conditions are given for selection of mRNAs from adenovirus 2-infected cells and transformed cells.


Inhibition of amino acid transport into lymphoid cells by the glutamine analog L-2-amino-4-oxo-5-chloropentanoate.

Transport of L-glutamine and of the chloroketone glutamine analog L-2-amino-4-oxo-5-chloropentanoate into lymphoid cells is mediated by the same system. Arginine and a number of other amino acids (e.g., glutamate, aspartate, and lysine) are transported to a much lesser extent by this system. However, after uptake of the chloroketone into the cells, the transport of glutamine, arginine, and other amino acids is markedly inhibited, due evidently to reaction of the chloroketone with intracellular components that are involved in amino acid transport. The chloroketone acts more effectively on growing than on resting cells. Treatment of lymphoid cells with the chloroketone or with L-chloro-2,4-dinitrobenzene leads to rapid and complete depletion of intracellular glutathione without affecting cell viability. These reagents appear to be useful experimental tools for studies of glutathione function and metabolism.


13C NMR quantitation of polymer in deoxyhemoglobin S gels.

13C/1H magnetic double-resonance spectroscopy has been used to quantitate the amount of polymerized hemoglobin S in deoxygenated gels at 30 degrees C, for samples whose hemoglobin concentration range from 21 to 32 g/dl. Scalar- and dipolar-decoupled spectra and a 13C proton-enhanced dipolar-decoupled spectrum were recorded for each sample as was a scalar-decoupled spectrum for a matching oxyhemoglobin S control. The difference between the oxyhemoglobin S and deoxyhemoglobin S scalar-decoupled spectra was used to determine the polymer fraction, and this value was compared with the polymer fraction determined by using ultracentrifugation sedimentation on the same sample (assuming a two-phase model). The polymer fraction value determined by uncorrected sedimentation averaged 0.15 more than the value obtained from NMR. The discrepancy between the two techniques was largely removed when the analysis of the sedimentation data included a correction for depletion of hemoglobin in the supernatant or sol phase due to sedimentation of free molecules. The best fit to both the sedimentation and NMR data was obtained by using a solubility of deoxyhemoglobin S at 30 degrees C of 17.3 +/- 1 g/dl. These results indicate that the NMR techniques, which do not require separation of the sample into a sol phase and a pellet phase, provide quantitative information about the deoxyhemoglobin S polymer and will be useful for studies of sickle erythrocytes.


Highly active template-specific RNA-dependent RNA polymerase from barley leaves infected with brome mosaic virus.

The extraction of a template-dependent and template-specific RNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from a eukaryotic source is described. The enzyme, extracted from barley leaves infected with brome mosaic virus (BMV), is capable of incorporating high levels of radioactivity into trichloroacetic acid-insoluble products. The purification procedure included solubilization with nonionic detergent and precipitation with polyethylene glycol. The enzyme was more than 50 times more active than was a comparable preparation from mock-inoculated leaves and was stimulated more than 15-fold by the addition of BMV RNA to the reaction. Other viral RNA templates were less than 25% as efficient as was BMV RNA in stimulating UMP incorporation; poly(A), tRNA, and mRNA gave little stimulation and rRNA was inactive. Autoradiographic analysis after electrophoretic separation of the radioactive products from reaction mixtures containing BMV RNA template revealed prominent bands that coelectrophoresed with replicative forms of BMV RNAs. When BMV RNA template was enriched in RNA3 or RNA4, larger proportions of the products were replicative forms of RNA3 or RNA4, respectively.


Transmethylation and transguanylylation in 5'-RNA capping system isolated from rat liver nuclei.

Rat liver nuclei were isolated and sonicated for extraction in order to study the capping of RNA. The guanosine 7-methyltransferase was purified from the extract by hydroxylapatite column chromatography with stepwise addition of phosphate buffer. It was assayed by using as methyl acceptor synthetic G(5')ppp(5')G and S-adenosylmethionine as donor. The enzyme appeared in a sharp peak at 160 mM. The same peak fraction was subsequently found to contain the enzyme that guanylylates short synthetic polynucleotides and low molecular weight yeast RNA as acceptors. The two enzymatic activities were separated on Sephadex G-150 chromatography, yielding guanylyltransferase and guanosine 7-methyltransferase with molecular weights of approximately 65,000 and 130,000 respectively. Guanylyltransferase was further purified by CM-Sephadex chromatography, whereby G-7-methyltransferase was completely removed. Dithiothreitol was essential for guanylylation, and 2 mM Mn2+ (optimum) was twice as active as 8 mM Mg2+ (optimum). The alpha-32P of [32P]GTP, but not its beta- or gamma-32P was incorporated into the cap structure. By using unlabeled GTP with [beta-32P]ppGpCpC-poly(A2,U2,G) as acceptor, [beta'-32P]-GpppG... was formed. Our purified transguanylylation enzyme was found to catalyze a [32P]pyrophosphate exchange with GTP, which may be useful as a rapid assay for transguanylylation.


Tryptic peptide analysis and NH2-terminal amino acid sequences of polyhedrins of two baculoviruses from Orgyia pseudotsugata.

Comparative analysis of the tryptic peptides and terminal amino acid sequence was made on polyhedrins from two genetically different baculoviruses that are naturally pathogenic for the same insect host. Comparison of the tryptic peptides of the nucleopolyhedrosis bundle virus and nucleopolyhedrosis single-rod virus of Orgyia pseudotsugata by means of cation-exchange resins indicated that the proteins have a closely related amino acid sequence. The NH(2)-terminal amino acid sequence of polyhedrins from the two viruses differed in only 4 out of 34 amino acids. The nucleopolyhedrosis bundle virus and the nucleopolyhedrosis single-rod virus also differed in 4 and 5 out of 34 terminal amino acids, respectively, from the sequence reported for polyhedrin of a baculovirus of Bombyx mori [Serebryani, S. B., Levitina, T. L., Kautsman, M. L., Radavski, Y. L., Gusak, N. M., Ovander, M. N., Sucharenko, N. V. & Kozlov, E. A. (1977) J. Invertebr. Pathol. 30, 442-443]. In addition, the nucleopolyhedrosis single-rod virus had two amino acids (Met-Tyr) on the NH(2) terminus that were not present on the terminus of nucleopolyhedrosis bundle virus or B. mori baculovirus polyhedrin. Approximately half (six) of the total tyrosine residues are clustered in the terminal 20 amino acids of the polyhedrins. Secondary structures predicted from the primary sequence suggest that the tyrosines are clustered in two areas. This nonrandom distribution and the pK(a) of about 10 for tyrosine may be related to the alkali solubility of the polyhedrin.


Identification of iron-sulfur centers in the iron-molybdenum proteins of nitrogenase.

The core extrusion method has been applied to the determination of the type ([2Fe-2S], [4Fe-4S]) and number of iron-sulfur centers in the FeMo proteins of the nitrogenases from Clostridium pasteurianum and Azotobacter vinelandii. The method involves extrusion with o-xylyl-alpha, alpha'-dithiol, ligand exchange of the extrusion products with p-CF3C6H4SH (RFSH), and identification and quantitation of the resultant [FenSn(SRF)4]2- complexes (n = 2,4) by 19F NMR spectroscopy. In hexamethylphosphoramide/water, 4:1 (vol/vol), 49-56% of the Fe content was extruded as [Fe4S4(SRF)4]2-, corresponding to 3,4-4.0 Fe4S4 cores per alpha 2 beta 2 subunit complex. The extruded iron does not arise from the FeMo cofactor, separate examination of which detected no extrusion products, and corresponds to 90-103% of noncofactor iron. No significant quantity of Fe2S2 cores was extruded. These results indicate the presence of four [4Fe-4S] centers per alpha 2 beta 2 subunit complex in preparations undepleted in iron. There are two main structural populations of iron atoms in these proteins, those in the cubane-type Fe4S4 cores and those in the FeMo cofactor.


Comparison of amino acid sequence of bovine coagulation Factor IX (Christmas Factor) with that of other vitamin K-dependent plasma proteins.

The amino acid sequence of bovine blood coagulation Factor IX (Christmas Factor) is presented and compared with the sequences of other vitamin K-dependent plasma proteins and pancreatic trypsinogen. The 416-residue sequence of Factor IX was determined largely by automated Edman degradation of two large segments, containing 181 and 235 residues, isolated after activating Factor IX with a protease from Russell's viper venom. Subfragments of the two segments were produced by enzymatic digestion and by chemical cleavage of methionyl, tryptophyl, and asparaginyl-glycyl bonds. Comparison of the amino acid sequences of Factor IX, Factor X, and Protein C demonstrates that they are homologous throughout. Their homology with prothrombin, however, is restricted to the amino-terminal region, which is rich in gamma-carboxyglutamic acid, and the carboxyl-terminal region, which represents the catalytic domain of these proteins and corresponds to that of pancreatic serine proteases.


Reassessment of histone gene expression during cell cycle in human cells by using homologous H4 histone cDNA.

The representation of H4 histone mRNA sequences in RNAs isolated from G1 and S phase HeLa cells was assessed by use of a homologous H4 histone cDNA. S phase cells were obtained by double thymidine block, and G1 cells were obtained by double thymidine block or mitotic selective detachment. Nuclear and cytoplasmic RNAs from S phase cells hybridized with H4 histone cDNA as did nuclear and cytoplasmic RNAs from G1 cells synchronized by double thymidine block. In contrast, significant levels of hybridization were not observed between H4 histone cDNA and nuclear, polysomal, or postpolysomal cytoplasmic RNAs of G1 cells synchronized by mitotic selective detachment. Double thymidine block yields a G1 cell population containing 20-25% S phase cells whereas the G1 population obtained by mitotic detachment contains less than 0.1% S phase cells. The ability of H4 histone cDNA to hybridize with the RNAs from G1 cells obtained after release from double thymidine block can therefore be explained by the presence of S phase cells in such a G1 population--an artifact of the synchronization procedure. We interpret these results to be consistent with the presence of H4 histone mRNA sequences during the S but not G1 phase of the cell cycle in continuously dividing HeLa S3 cells.


Acidic polypeptides can assemble both histones and chromatin in vitro at physiological ionic strength.

We provide evidence that nucleosomes can assemble in vitro at physiological ionic strength (0.1-0.2 M NaCl/10 mM Tris . HCl, pH 8.0) in the absence of "assembly factors" and that poly(glutamic acid) greatly facilitates chromatin assembly under these conditions. We also show that in the presence of either poly(glutamic acid) or poly(aspartic acid), core histones assemble into octamers at physiological ionic strength. We suggest that it is a property of histones to assemble into octamers upon their interaction with macromolecules containing regions of high negative charge density, and we discuss several implications of this property.


In vitro production of 1,25-dihydroxyvitamin D3 by rat placental tissue.

Weanling female rats were fed a vitamin D-deficient diet for 4 months until they reached maturity. They were mated with normal, vitamin D-replete male rats and, at 20 days of pregnancy, the female rats were killed and their placentae were removed, homogenzied, and incubated with 25-hydroxyvitamin D3. The incubation mixtures were extracted and the extracts were subjected to Sephadex LH-20 column chromatography followed by high-pressure liquid chromatography. The 1,25-dihydroxyvitamin D3 region of the high-pressure liquid chromatogram was recycled to purity and the structure of the product was identified as 1,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry and by mass spectrometry. Thus it is now evident that placenta, in addition to renal tissue, is capable of converting 25-hydroxyvitamin D3 to the hormonal form of vitamin D, 1,25-dihydroxyvitamin D3.


Amino acid sequence of bacteriorhodopsin.

The complete primary structure of the purple membrane protein bacteriorhodopsin, which contains 248 amino acid residues, has been determined. Methods used for separation of the hydrophobic fragments included gel permeation and reverse-phase high-pressure liquid chromatography in organic solvents. The amino acid sequence was determined by a combination of automatic Edman degradation and mass spectrometric methods. The total sequence was derived by ordering of the CNBr fragments on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry and by analysis of N-bromosuccinimide fragments containing overlaps between CNBr fragments. The present sequence differs from that recently reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and several amino acid assignments.


Localization of transcribed regions on extrachromosomal ribosomal RNA genes of Tetrahymena thermophila by R-loop mapping.

R-loop hybridization and electron microscopy were used to map the RNA transcription products of the extrachromosomal rRNA genes of Tetrahymena thermophila. The mature 17S and 26S rRNAs and the nuclear 35S pre-rRNA and pre-26S rRNA were located with a precision of approximately 100 base pairs. A 370-base pair intervening sequence was found in the 26S coding region. It has the same size and relative location as that found in Tetrahymena pigmentosa [Wild, M.A. & Gall, J.G. (1979) Cell 16, 565-573]. One class of R-loop structures formed by nuclear pre-rRNA provided preliminary evidence for a primary transcript that contains the intervening sequence. The results suggested a processing scheme in which splicing of the intervening sequences is followed by a series of strand cleavages to give the mature 17S and 26S rRNAs. Analysis of the data also showed that RNA . DNA and DNA . DNA duplexes, when mounted for electron microscopy by the R-loop procedure, have the same length per base pair within 4%.


Essential role for mevalonate synthesis in DNA replication.

The relationship between 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity [mevalonate:NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34] and DNA synthesis was studied in synchronized cultures of BHK-21 cells. During a 24-hr period of cell replication, two phases of accelerated thymidine incorporation into DNA corresponding to two S phases of the cell cycle occurred. A marked increase in activity of HMG CoA reductase was consistently observed at or just prior to each of these peaks of DNA synthesis. Moreover, when HMG CoA reductase activity was suppressed by the competitive inhibitor compactin, the normal S-phase burst of DNA synthesis was specifically and totally prevented. Finally, the compactin-induced inhibition of DNA synthesis could be completely reversed within minutes by the addition of mevalonate, the product of the HMG CoA reductase reaction. By contrast, addition of cholesterol-rich lipoproteins had no effect upon DNA synthesis in compactin-treated cells. These data demonstrate that HMG CoA reductase activity, and therefore the production of mevalonate, plays an essential role in the synthesis of DNA specifically during the S phase of the cell cycle. Moreover, the results indicate that this function of mevalonate in regulating DNA replication is independent of its conversion to cholesterol.


On the origin of the contractile force in skeletal muscle.

Analysis of the early tension responses after abrupt step changes in the length of isometrically contracting skeletal muscle shows that the magnitude of the recovery tension (T2) and the time dependence of this process at various step displacements give a good correlation with the behavior expected for a helix-coil transition in the subfragment-2 (S-2) region of myosin. The "instantaneous" tension response (to T1) after the step change in length appears to have its origin in compliance within the coil region of S-2, which is formed through helix melting at the moment of force generation.


Protein synthesis in rabbit reticulocytes: mechanism of protein synthesis inhibition by heme-regulated inhibitor.

Partially purified Met-tRNAf binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography. Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNAf and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg2+ and dissociated by Co-eIF-2B at 5 mM Mg2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-e-IF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNAf binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNAf . 40S . AUG complex to the 60S ribosomal subunit to form Met-tRNAf-80S . AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.


Lectin-like activity of components extracted from human glomerular basement membrane.

Different fractions of human glomerular basement membranes have been isolated by enzymatic and chemical methods. These fractions were analyzed for their chemical structure and biological activity. The hypothesis that human glomerular basement membrane glycopeptide fractions related to collagenous sequences could act as a lectin-like substance was explored; in fact, extracellular glycoprotein factors play a role in cell-cell interactions and cell adhesion. These collagenous glycopeptides agglutinate human transformed or embryonic cells within 2 hr. The cell agglutination is inhibited by the following sugars: N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. In addition, the rapid cytoagglutination is followed by a cell spreading effect after a further 20-hr incubation. These data led to the postulate that the maintenance of tissue differentiation is governed by the interaction of cells with peculiar sites of the basement membrane consisting of a proteolysis-resistant association between collagen and matrix glycoprotein.


A study of the adipose conversion of suspended 3T3 cells by using glycerophosphate dehydrogenase as differentiation marker.

The adipose conversion of 3T3 cells has been examined in stabilized suspension cultures. In 3T3-F442A cells, glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: NAD(+) 2-oxidoreductase, EC 1.1.1.8), a key enzyme in triglyceride synthesis, increases in specific activity by more than 5000-fold and can be used as a sensitive and precise measure of the conversion. The conversion depends on an adipogenic factor present in the serum, and this factor can be assayed by the cellular enzyme response. If the cells are growing at the time they receive the adipogenic factor, the enzyme response does not become detectable until after 3 days, during which the cells first enter a resting state. If the cells are resting at the time they receive the adipogenic factor, the enzyme activity begins to increase in 24 hr or less. Only resting cells seem susceptible to the reprogramming of their differentiated state necessary for the adipose conversion. Once the conversion begins, the increase in enzyme activity is exponential over at least 2 orders of magnitude. When cells in a resting state begin the adipose conversion, their biosynthetic processes are accelerated: the rate of protein synthesis increases, they accumulate cell protein, and they may replicate their DNA and divide. The cell multiplication is not essential for adipose conversion but is a form of clonal selection that increases the proportion of adipose cells relative to nonadipose cells.


Synthesis and processing of human chorionic gonadotropin subunits in cultured choriocarcinoma cells.

Pulse and pulse-chase experiments have identified the presence of partially glycosylated precursors of the alpha and beta subunits of human chorionic gonadotropin (hCG) in cultured JAR choriocarcinoma cells. The alpha subunit precursor has an apparent molecular weight (by sodium dodecyl sulfate/polyacrylamide gel electrophoresis) of 18,000 (compared to 22,000 for fully processed alpha subunit); the beta subunit precursor has an apparent molecular weight of 24,000 (fully processed, 34,000). Both of these precursors appear to have an intracellular half-life of at least 1 hr and to contain the mannose core but not the terminal carbohydrate sequences. Fully processed alpha and beta subunits do not accumulate intracellularly, indicating that further processing of the precursors is followed by rapid secretion.


Rapid changes in the synthesis of specific ovarian granulosa cell proteins induced by human choriogonadotropin.

Total ovarian granulosa cell proteins were labeled in vitro with [35S]methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis. Of the 100-150 distinct proteins resolved by this method, 1-2% demonstrated an observable change in their synthesis during the early phases of luteinization after hormone administration. Two specific proteins first appeared as early as 30 min after treatment with human choriogonadotropin, while four additional new proteins became apparent at 3 hr. These changes in the synthesis of specific proteins occurred far in advance of the morphological changes associated with luteinization, which occur 24-72 hr later, and thus may play an important role in this process of hormonally induced cell differentiation in the rat ovary.


Regulation of normal differentiation in mouse and human myeloid leukemic cells by phorbol esters and the mechanism of tumor promotion.

The control of cell multiplication and differentiation by tumor-promoting phorbol esters including 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied with different clones of mouse myeloid leukemic cells, a line of human myeloid leukemic cells, and normal mouse bone marrow myeloblasts. TPA induced normal cell differentiation in one of the mouse leukemic clones and this was mediated by induction of the protein inducer of differentiation to macrophages or granulocytes (MGI) in the cells that then differentiated. Other mouse clones were not induced to differentiate by TPA. In one of these clones, TPA induced cell susceptibility to externally added MGI. This effect was not due to a general induction of susceptibility to all compounds because TPA did not induce susceptibility to lypopolysaccharide or dexamethasone in this clone. In the human leukemic cell line, TPA also induced differentiation with the induction of MGI activity and enhanced susceptibility to added MGI. It is suggested that the clonal differences in induction of MGI activity and increased susceptibility to MGI may be associated with differences in receptors for TPA and the ability of TPA to modify receptors for MGI. Studies with normal bone marrow cells have indicated that TPA stimulated MGI activity and also increased susceptibility of normal myeloblasts to induction of multiplication by MGI. The ability of different phorbol esters to produce these effects on normal myeloblasts and myeloid leukemic cells paralleled their ability to act as tumor promoters. The results indicate that a tumor promoter such as TPA can induce the production of and increase cell susceptibility to a normal regulator of cell multiplication and differentiation. TPA has pleiotropic effects. It is suggested that, by these mechanisms, TPA may thus act as a tumor promoter by increasing cell multiplication in initiated cells, induce differentiation in some cells, or inhibit differentiation in other cells, depending on which molecules are being regulated in the TPA-treated cells.


Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes.

Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3'-dioctadecylindocarbocyanine) had a mean (+/-SD) diffusion coefficient of (1.7 +/- 0.3) x 10(-8) cm(2)/sec, with essentially all of the probe mobile in the membrane. We detected little or no effect on the diffusion of this probe due to the presence of microvilli. Its diffusion was slightly restricted in capped regions. No differences in lipid probe mobility were detected between T and B cells. Fifty to 90% of the detectable sIg and Thy-1 antigen was free to move in the plane of the membrane with diffusion coefficients of approximately 3 x 10(-10) cm(2)/sec; the remainder was immobile. Crosslinking of sIg with anti-Ig antibodies (in the presence of azide to inhibit capping) completely immobilized sIg at high concentrations but failed to do so at low concentrations. Thy-1 antigen could not be immobilized with an IgG rabbit anti-mouse brain reagent without an additional layer of crosslinking antibody. In parallel labelings (in the absence of azide), capping of sIg and Thy-1 antigen was observed only under crosslinking conditions sufficient to immobilize the membrane antigen. Sodium azide, colchicine, and cytochalasin B had no measurable effect on lipid probe, sIg, or Thy-1 diffusion.


Synthesis and turnover of globin mRNA in murine erythroleukemia cells induced with hemin.

When murine erythroleukemia (MEL) cells are induced with hemin, they carry out several early functions of the erythroid program. However, they do not become committed to terminal differentiation nor do they become benzidine positive. This is in contrast to MEL cells induced with dimethyl sulfoxide (Me(2)SO) which undergo a more complete program of erythroid differentiation. In order to determine the relationship between commitment and various events in the erythroid program, we compared the induction of MEL cells with hemin and with Me(2)SO. The amount of globin mRNA accumulated in inducing MEL cells and the rate of its synthesis and turnover were quantitated. Although MEL cells induced with hemin accumulated significantly less globin mRNA than did cells induced with Me(2)SO, the rate of synthesis of globin mRNA was the same in fully induced cells, irrespective of inducer. Therefore, there is no evidence that induction with hemin produces an early program that is different or altered from that which is part of Me(2)SO induction. MEL cells induced with Me(2)SO specifically destabilize their globin mRNA after 4 days of induction. This raises the question of whether this destabilization of globin mRNA is an independently programmed late event, as suggested by the time of its occurrence, or, alternatively, whether it might be the inevitable consequence of an early event(s). For instance, destabilization might be linked to the synthesis or translation of globin mRNA. Because MEL cells induced with hemin do not destabilize their globin mRNA, we have concluded that this turnover of globin mRNA is a late event, occurring only in a committed cell population.


Nucleosome core histone complex isolated gently and rapidly in 2 M NaCl is octameric.

A relatively stable specific complex of the chromatin core histones H2A, H2B, H3, and H4 has been obtained in 2 M NaCl/25 mM sodium phosphate buffer, pH 7.0. The histone core complex has an apparent specific volume of 0.73 ml/g. Its sedimentation coefficient was dependent on rotor speed (angular velocity, omega) and attained different stable values at low and high rotor speeds. The drop in sedimentation coefficient occurred sharply between omega 2 values of about 9 x 10(6) and 1.1 x 10(7) (radians/sec)2. The s020,w corresponding to zero angular velocity (1 atmosphere pressure) was 6.6 S +/- (SEM) 0.1 S. At high rotor speeds the value decreased to 3.8 S +/- 0.1 S. The core complex has a diffusion coefficient, D20,w, of 5.4 x 10(-7) cm2/sec and a molecular weight of 108,000 +/- (SD) 2500.


Biogenesis of intestinal plasma membrane: posttranslational route and cleavage of sucrase-isomaltase.

The biosynthesis in vivo of rat intestinal sucrase-isomaltase [a complex of sucrose alpha-glucohydrolase, EC 3.2.1.48, and oligo-1,6-glucosidase (dextrin 6-alpha-D-glucanohydrolase), EC 3.2.1.10] has been studied by following the incorporation of L-[6-(3)H]fucose into the enzyme with time. Immunoprecipitation of sucrase-isomaltase from Triton-X-100-solubilized Golgi or basolateral membranes and subsequent polyacrylamide gel electrophoresis revealed the presence of an immunoreactive glycoprotein with an apparent molecular weight approximately twice that of the separated sucrase-isomaltase subunits, but no active subunits were found in these membranes. This glycoprotein was also found in the microvillus membrane in addition to the subunits of sucrase-isomaltase. Kinetic studies showed a maximal labeling of this glycoprotein in Golgi membranes at 15 min, in basolateral membranes at 30 min, and in microvillus membranes at 45 min and a half-life of less than 30 min in each membrane. However, the radioactivity of the sucrase-isomaltase subunits in the microvillus membrane reached a plateau after 60 min. These data suggest that sucrase-isomaltase is synthesized as a one-chain polypeptide precursor that is split into the subunits after its transfer to the microvillus membrane. Elastase (EC 3.4.21.11), but not trypsin (EC 3.4.21.4) or alpha-chymotrypsin (EC 3.4.21.1), split the putative precursor into two polypeptides that had electrophoretic behaviors similar to those of the active enzyme subunits. These studies suggest that pancreatic proteases may play an important role in the late posttranslational processing of sucrase-isomaltase in vivo.


Purification of two spectrin-binding proteins: biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bands 2.1 and 4.1.

Two peripheral proteins of the human erythrocyte membrane that are capable of forming a stable complex with spectrin have been purified. The proteins, band 2.1 (Mr 210,000) and band 4.1 (Mr 82,000), are water soluble and exist as monomers in solution. Both exhibit strong, specific binding to purified spectrin molecules as determined by cosedimentation in sucrose gradients and both enhance binding to spectrin-depleted, inside-out vesicles that have been stripped of bands 2.1 and 4.1. Rotary replicas of bound material reveal site-specific associations among native, but not heat-denatured, molecules.


Contractile protein isozymes in muscle development: identification of an embryonic form of myosin heavy chain.

The nature of the myosin heavy chain in embryonic muscle tissue, cultured muscle cells, and several adult muscles was investigated. After denaturation with sodium dodecyl sulfate, purified rat myosins were subjected to partial proteolytic cleavage or immunological analysis using microcomplement fixation. Three types of myosin heavy chains could be demonstrated by both approaches. Whereas adult muscles contain fast- or slow-type myosin heavy chains, embryonic tissue and cultured muscle cells harbor a distinct embryonic form. The existence of this distinct form further characterizes the isozymic transitions of contractile proteins during muscle development.


Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2-acetylaminofluorene.

A knowledge of the biological characteristics of carcinogen-induced hyperplastic nodules of rat liver may be important in the understanding of cancer development. Although its biological role remains to be elucidated, the level of microsomal epoxide hydrase (epoxide hydrolase, EC 3.3.2.3) is 5- to 7-fold greater in hyperplastic nodules nodules induced by feeding the hepatocarcinogen 2-acetylaminofluorene than in liver of control rats. After removal of the carcinogen from the diet, the high level of the enzyme is maintained in those nodules that persist and in the hepatocellular carcinomas that subsequently develop. The availability of antibody to the epoxide hydrase made it possible to use electron microscopic immunocytochemistry to localize this enzyme in the cells of hyperplastic nodules. The immunocytochemical procedure provides direct visual evidence for the presence of this enzyme in smooth endoplasmic reticulum and also in rough endoplasmic reticulum (including the nuclear envelope) of the nodule's parenchymal cells.


Long-term culture of capillary endothelial cells.

Capillary endothelial cells from rats, calves, and humans, have been carried in long-term culture. Bovine capillary endothelial cells have been cloned and maintained by serial passage for longer than 8 months. This prolonged culture was accomplished by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching cells in primary culture. Cultured bovine capillary endothelial cells produce Factor VIII antigen and angiotensin-converting enzyme, but do not have Weibel-Palade bodies. Human cells do contain Weibel-Palade bodies. Capillary endothelial cells are distinguished from aortic endothelial cells by their requirement for conditioned medium. Bovine capillary endothelial cells in regular medium grow slowly with a mean doubling time of 67 hr and eventually die. In tumor-conditioned medium, these cells grow rapidly with a doubling time of 28 hr and continue to proliferate for as long as the tumor-conditioned medium is present. In contrast, bovine aortic endothelial cells grow as rapidly in regular medium as in tumor-conditioned medium. This method allows the production of pure capillary endothelial cells that may prove useful for studies of tumor angiogenesis, metastatic mechanisms, and the role of capillary endothelium in other pathologic states.


Gene mutations (de novo) found in electrophoretic studies of blood protein of infants with anomalous development.

Twelve proteins of enzymic and nonenzymic nature in blood samples of infants that deviate from the average population in physical development (50 premature and 177 full-term infants with rough and multiple developmental defects) were studied by electrophoresis in polyacrylamide and starch gels. The control group consisted of 500 normal newborns. In infants with developmental disorders, the frequency of rare electrophoretic protein variants was found to be about one order of magnitude higher than in the control. It has been shown for at least five cases that such variants are de novo mutations. According to these data the mutation rate is approximately 2 x 10(-3) per locus per generation for the group selected and approximately 6 x 10(-5) for the total population. Despite the fact that further specification of the estimations found is required, we consider the results obtained as evidence in favor of the efficiency of the earlier substantiated monitoring model of gene mutations in the human population [Dubinin, N.P. & Altukhov, Yu. P. (1977) in Genetic Consequences of Environmental Pollution, ed. Dubinin, N.P. (Mysl, Moscow), pp. 14-45]. This approach, which infers electrophoretic screening of blood proteins in a specially selected group of newborns, makes it possible to reduce the size of samples needed for statistically reliable estimations of the alteration of mutation rate.


Assignment of the gene for cytoplasmic superoxide dismutase (Sod-1) to a region of chromosome 16 and of Hprt to a region of the X chromosome in the mouse.

In the search for homologous chromosome regions in man and mouse, the locus for cytoplasmic superoxide dismutase (SOD-1; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is of particular interest. In man, the SOD-1 gene occupies the same subregion of chromosome 21 that causes Down syndrome when present in triplicate. Although not obviously implicated in the pathogenesis, SOD-1 is considered to be a biochemical marker for this aneuploid condition. Using a set of 29 mouse-Chinese hamster somatic cell hybrids, we assign Sod-1 to mouse chromosome 16. Isoelectric focusing permits distinction between mouse and Chinese hamster isozymes, and trypsin/Giemsa banding distinguishes mouse from Chinese hamster chromosomes. The mouse fibroblasts used were derived from a male mouse carrying Searle's T(X;16)16H reciprocal translocation in which chromosomes X and 16 have exchanged parts. Analysis of informative hybrids leads to regional assignment of Sod-1 to the distal half of mouse chromosome 16 (16B4 --> ter). Because the Chinese hamster cell line (380) used for cell hybridization is deficient in hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), that part of the mouse X chromosome carrying the complementing Hprt gene can be identified by selection in hypoxanthine/aminopterin/thymidine medium and counterselection in 8-azaguanine. Mouse Hprt is on the X(T) translocation product containing the proximal region X cen --> XD.


Late replication in an X-autosome translocation in the mouse: correlation with genetic inactivation and evidence for selective effects during embryogenesis.

A technique involving 5-bromodeoxyuridine, 33258 Hoechst, and fluorescence microscopy has been used to analyze replication kinetics in cells from embryonic and adult mice bearing the Cattanach [T(X;7)ICt] translocation in a balanced or an unbalanced form. In balanced 9- and 13-day female embryos, the translocated X was late replicating in 28 and 22% of the cells, respectively, whereas it was late replicating in only 13% of adult cells. In contrast, in unbalanced females, the translocated X was late replicating in 62 and 70% of 9- and 13-day embryos and in 70% of adult cells. Such divergent late replication frequencies suggest the operation, during development, of selection against cells with extreme genetic imbalance. Within a late-replicating translocated X chromosome, the autosomal segment itself replicated late approximately half of the time, regardless of karyotypic balance. The late replication data are consistent with the measurements of levels of mitochondrial malic enzyme (MOD-2, whose locus is on the autosomal segment) activity in these mice [Eicher E. & Coleman, D. (1977) Genetics 85, 647-658]. The present study also shows a dissociation between the replication timing in X chromatin distal and proximal to the autosomal segment, supporting the hypothesis of at least two inactivation centers in the X chromosome.


Precise localization of human beta-globin gene complex on chromosome 11.

Cloned DNA probes were used in combination with a panel of five hybrid cell clones containing a series of different terminal deletions in human chromosome 11 to map precisely the human hemoglobin beta and delta chain structural genes contained on this chromosome. The region of deletion in each clone of the panel has been defined by biochemical, immunologic, and cytogenetic markers. DNA from clones containing successively larger terminal deletions was tested with appropriate DNA probes to determine the point on the chromosome at which DNA for these two closely linked hemoglobin genes is deleted. These genes, and by inference the closely linked G gamma and A gamma globin genes as well, have been assigned to the intraband region 11p1205 leads to 11p1208 on the short arm of chromosome 11, an interval containing approximately 4500 kilobases of DNA. The approach appears to have potential for even greater resolution and reasonably wide applicability for gene mapping.