Double primary carcinoma of the lips.

The case of a 40-year-old man who was a moderate smoker with two squamous-cell carcinomas on the lips has been presented. The importance of follow-up in such cases has been stressed. When oral cancer develops, all mucous membrane surfaces must be considered to be highly susceptible to future malignant change. Exfoliative cytology, biopsies, and histologic studies should be employed for the early detection of precancerous conditions and, if possible, to prevent the development of subsequent oral carcinomas. It is necessary to insist on frequent and regular follow-up examinations, so that any second primary lesion may be detected and treated in its early stages. It is also necessary to initiate appropriate prophylactic measures to eliminate any possible sources of carcinogenic irritation.


Dens in dente: an unusual sequela. Abbreviated case report.

Dens in dente is the result of an invagination of the enamel organ into the developing dental papilla. The full pathologic potential of this lesion is often not fully appreciated. A case is reported in which a cyst resulting from a dens in dente obliterated the maxillary sinus and necessitated root canal therapy and apicoectomies in four adjacent teeth.


A retrospective study of the prevalence and incidence of recurrent aphthous ulcers in a professional population, 1958-1971.

A 12-year retrospective investigation of 1,788 professional school students from the University of Pennsylvania was conducted. Because of changes in profession, location, marital status, and names, only 651 proband subjected were available for the follow-up investigation. Techniques of subjective evaluation of disease status (mailed schedules describing disease and color photographs) were used in making an assessment of RAU, including both current disease and histories of prior disease. The data presented suggested a trend toward decreased RAU prevalence and severity among physicians, dentists, veterinarians, and nurses as compared with 12 years previously when they were professional school students. The prevalence rate, however, was still very high, indicating that 48.3 per cent of the men and 57.2 per cent of the women had RAU at the time. It has been suggested that the student years may represent the highest levels of RAU and that special features of the life patterns of the student favor disease expression; the data from study support this hypothesis, since reduction in severity was observed in all groups.


Differential diagnosis of some simple osseous lesions associated with sickle-cell anemia.

Sickle-cell disease is characterized by chronic hemolytic anemia, rapid destruction of defective erythrocytes, with consequent circulatory disturbances. Thromboses and infarctions with resultant osseous manifestations tend to be localized in the ends of the long bones or in the medullary area, the latter often becoming apparent in the jaws. Three case reports are presented to illustrate osseous changes observed in the jaws of these sickle-cell patients. The radiographic findings demonstrate the adaptive mechanisms of bone metabolism in response to vascular infarction, infection, or both in some sickle-cell anemia patients. A differential diagnosis is suggested for osseous lesions observed in the jaws of these patients.


Glucose intolerance associated with temporomandibular joint pain-dysfunction syndrome.

In order to eliminate any disease process, it is necessary to identify and eliminate etiologic factors associated with the disease process. Reactive hypoglycemia, a common and easily treated disorder, may be a significant factor associated with development of the TMJ syndrome. Five case histories have been presented of patients who exhibited reactive hypoglycemia associated with the TMJ syndrome and who were treated with a combination of occlusal therapy and dietotherapy. Dietotherapy may have reduced the "psychic tension" associated with the TMJ symdrome.


Nuclear characteristics of buccal mucosa cells in sickle-cell anemia.

Exfoliative cytology smears from the buccal mucosa of patients with sickle-cell anemia, selected controls, and patients with other diagnosed anemias were studied for nuclear aberrations. A filar micrometer eyepiece was used to count the number of nuclei larger than 14 microns in mean diameter in a 500 cell count on each slide. Use of the Scheffe method of paired comparisons, disclosed that the number of cells larger than 14 microns in mean diameter, was significantly smaller in the control subjects, patients with microcytic anemia, and subjects with sickle-cell anemia than in the patients with megaloblastic anemia. Serum folate-deficient subjects had significantly more nuclei, per 500 cells, larger than 14 microns than control subjects or subjects on folate therapy. An explanation for the enlarged nuclei in smears from sickle-cell anemia patients is given. The possible use of this counting procedure to evaluate tissue folate status is suggested.


Aging of tissues of the roots of nonfunctional human teeth (impacted canines).

In order to examine the aging of dentin and cementum, seventy-two impacted human canines, nonfunctional and not affected by external factors, such as attrition, abrasion, caries, etc., were surgically extracted. Ground sections were prepared and the areas of sclerotic dentin and cementum were measured with the aid of a planimeter and expressed as a percentage of the total root area. It was found that there is a significant increase in sclerotic dentin and apposition of cementum with an increase in age.


Survey of tissue diagnostic services in United States dental schools, 1953-1975.

A survey of tissue diagnostic services in United States dental schools was conducted, covering the services' activities during 1975. This survey was compared to similar surveys made in 1953, 1958, and 1969. Most significantly, many more biopsies are being processed, with a great increase in the number of malignant specimens. As could be expected, higher fees are being charged by the schools for processing specimens.


A clinical and histologic observation of enamel hypoplasia in a case of epidermolysis bullosa hereditaria.

The nature of the enamel defects in a patient with epidermolysis bullosa hereditaria dystrophica was observed by light microscopy, microradiography, and scanning electron microscopy (SEM) so that by these means more detailed information could be obtained. The random distribution of the round shape of the hypoplastic defects of enamel with hypomineralization, in which there is a continuity of the incremental lines, suggested the possibility that some ameloblasts maintained hypoactivity of their secretory function during their total life span.


Central leiomyoma of the mandible.

A case of central leiomyoma of the mandible is reported, along with ultrastructural confirmation of the diagnosis. The pertinent literature is reviewed. It is probable that this lesion arose from the smooth muscle in the walls of blood vessels. To the best of our knowledge, this is only the second report of a leiomyoma central in bone.


Rationalization of electric pulp-testing methods.

A new electric pulp-testing system, the Testark, was developed in order to assess the potential of a different way of testing pulp sensitivity, thus making this electric pulp test more dependable than the conventional electric pulp-testing method. A study was conducted to evaluate the potential of the Testark system in comparison with a commercially available pulp tester. This pilot study consisted of application of three different desensitizing agents (sodium fluoride, test product. 'A', and test product 'B') to the cervical areas of hypersensitive teeth. The results were recorded with both the Testark and  the conventional pulp tester simultaneously, and the sodium fluoride and test product 'B' proved  to yield a beneficial effect by desensitizing the teeth after a single application. The level of desensitization with these agents was higher than the level obtained with the test product 'A' paste, which was utilized daily for 14 days. The results obtained with the conventional pulp tester differed by way of interpretation from those obtained with the Testark system. The Testark system proved to be easy to use and highly accurate in the data readings because of its high resolution. The Testark systemwas found to be more dependable than the commerical pulp tester, since it reduced the subjective errors which are inevitable when a commerical pulp tester is used. The Testark system is being developed further, and more studies are being conducted at the present time, in order to assess the full potential of the instrument.


In vivo study of temporary filling materials used in endodontics in anterior teeth.

The sealing efficacy of temporary endodontic filling materials was tested in vivo. The following materials were studied: Cavit, Caviton, gutta-percha, three types of zinc phosphate cement, and zinc oxide and eugenol. All the materials were tested in the access cavity of the same anterior tooth in ten different patients for a minimum of 1 week. Seepage was determined bacteriologically by culturing a cotton pellet which was sealed into the access cavity. On the basis of the quantity of microorganisms grown anaerobically, differentiation was made between no leakage, minor leakage, and gross leakage. Findings with Cavit and Caviton are essentially the same and show no or minor leakage in the vast majority of tests. Gutta-percha showed gross leakage in six out of eight tests. Phosphate cements showed no leakage in more than two thirds of the tests.


Retrograde root filling with amalgam and Cavit.

In a 3-year review of 218 teeth with retrograde root filling with amalgam orCavit, the results obtained with the former proved significantly better than those obtained with the latter. The difference seemed to be due to a better obliteration of the canal by amalgam. The obliterating effect of amalgam probably eliminates the need for revision of incomplete othograde root filling, for example, in cases with a post in the root canal. Irrespective of type of filling materials, the results were less good in cases with marginal bone loss.


Fine structure of the Panorex image.

The plane of focus free of blur margin, as well as the rate of increase of blur margin both medial and lateral to the plane of focus, has been described in the Panorex panoramic x-ray machine by means of a continuous phantom. The average of readings made at ninety-eight separate film positions yields a focaltrough thickness of 4.3 mm. if a blur margin of 10 per cent of the phantom's film image is accepted, the percentage blur increasing directly with trough thickness. The factor enlargement of objects at the plane of focus is uniform at 1.25 and increases along with percentage blur more rapidly for objects medial to the plane of focus than for objects lateral to it.


Sclerotic mandibular bone lesion.

A case of a sclerotic mandibular bone lesion interpreted as a true osteoma has been described, along with the differential diagnosis and treatment. Because the radiographic appearance is not specific for any one lesion, microscopic examination is essential to establish the definitive diagnosis.


[Lateral periodontal cysts].

The lateral periodontal cyst is a rather infrequently reported odontogenic cyst. The cyst seems to develop in direct contact to the periodontal membrane of an erupted vital tooth. This type of cyst should not be confused with a radicular cyst in a lateral position developing as a consequence of inflammation from an infected or necrotic branch of the pulp canal. Several authors do not differentiate between the lateral periodontal cyst and the gingival cyst, but it seems justified to consider the lateral periodontal cysts as a separate entity of odontogenic cysts. In this origin of the lateral periodontal cyst is reviewed and several possibilities are discussed. It is concluded that the epithelial lining of this type of cysts most probably originates from the cell rests of Malassez. Case report of one lateral periodontal cyst is presented.


[Range of systematic local treatment with fluorides in school dental health care in 1973/74 and in public health dentistry in 1974].

In 1975 data were collected on the extent to which children and teenages elible for free dental care in the School Dental Service and the Public Dental Services particpated in programmes consisting of self- or professionally administered topical applications of fluoride solutions. Results apply to the school year 1973/74 for the School Dental Service and the calendar year 1974 for the Public Dental Services. Of the reported number of persons 6--17 years of age who were eligible for free systematic dental care under School Dental Service and Public Dental Services 70 and 67 per cent respectively, rinsed, brushed or had fluoride solutions professionally applied to their teeth. Participation was greatest among children 7--13 years of age, namely 87--89 per cent. 61 to 80 per cent of those participating in a programme brushed their teeth with fluoride solutions under supervision at school while 14--38 per cent received fluoride rinses. Professionally administered topical applications were reported for 8--12 per cent of participants in these preventive programmes. Comparisons with results of a similar survey 2 years earlier suggest increasing systematic use of topical fluoride applications in mass prophylactic programmes. Information about preventive activities is not included in the routine reports on the work carried out in the School Dental Service and the Public Dental Service. On the basis of the experience gained from this survey, it is proposed that data on preventive activities should be collected routinely as an integral part of a public dental services monitoring system.


[Recurrence with gonadic localization of acute leukemia. 113 cases].

Amongst 1500 cases of acute lymphoblastic leukaemia, affecting both sexes, the authors noted 111 testicular recurrences and 2 ovarian recurrences, i.e. an incidence of 16 percent. Clinical diagnosis is easy and testicular needle biopsy was reserved for doubtful cases only. Amongst 12 lymphographies, only one showed lumbar node involvement. Testicular involvement may exist before, after or in association with a bone marrow or a meningeal relapse. In 47 cases the first signal recurrence was a testicular tumour. The majority of these recurrences occured during the first two years of the disease, but late recurrences affecting the gonads are possible. Mean survival after an isolated testicular recurrence was 14 months, identical with meningeal recurrences. General chemotherapy and local radiotherapy were in general followed by an in situ or contralateral recurrence. Bilateral irradiation at a dose of 2400 rads is the best local treatment. However, in the light of the frequency of bone marrow recurrences a short time after, intensification of general treatment is necessary.


Magnetic properties of oxyhemoglobin.

When the magnetic susceptibility of frozen aqueous solutions of human oxyhemoglobin was measured in the range between 25 and 250 K, it showed a temperature-dependent behavior typical of a thermal equilibrium between a ground singlet state and an excited triplet state for two electrons per heme, the energy separation being [2J] = 146 cm-1. By contrast, within the same temperature range, carboxyhemoglobin was found to be diamagnetic, as already reported.


Kinetic polarization deficiency in electrolyte solutions.

The prediction and experimental confirmation of a previously unsuspected kinetic effect occurring in electrolyte solutions are presented herein. Kinetic polarization deficiency may be described as a reduction, with respect to the pure solvent, in the static permittivity of the solution; the decrement in epsilon0 is shown to be proportional to the product of the dielectric relaxation time of the solvent and the low frequency conductivity of the solution. The kinetic ion-solvent interaction affects the capacitive admittance in two closely related ways: as an ion migrates, the surrounding volume elements of the liquid tend to rotate according to the laws of hydrodynamics, and although dielectric relaxation tends to restore an equilibrium polarization appropriate to the local electric field, this adjustment is not instantaneous; rather it lags behind by the dielectric relaxation time. Conversely, the force that an external field exerts on an ion does not develop its full strength instantly because the ion is driven partly by the external field and partly by the polarization that develops in response to the applied field, the polarization field evolving with a time constant that is the relaxation time for the orientation of solvent dipoles.


Chemiluminescent reactions and electrophilic oxygen donating ability of 4a-hydroperoxyflavins: general synthetic method for the preparation of N5-alkyl-1,5-dihydroflavins.

The synthesis of N5-methyl- and N5-ethyl-1,5-dihydroflavin mononucleotides is reported. These compounds show no bioluminescence activity with bacterial luciferase. This feature is interpreted in terms of steric hindrance between the N5-alkyl group and a hydrogen bonding group at the active site of the luciferase. The chemiluminescence observed on reaction of N5-alkyl-1,5-dihydroflavins with oxygen and aldehydes has been shown to occur via formation of a mixed peroxide of flavin and aldehyde and to be associated with a primary deuterium isotope effect when [1-2H]aldehyde is substituted for aldehyde. The time course for light emission has been compared for aldehyde and ketone substrates. The suggestion is entertained that the peroxide bond of 4a-hydroperoxyflavin is sufficiently polarized to allow this species to act as the oxidant per se at the active site of mixed function oxidases. The second-order rate constants for reaction of hydroperoxides with thioxane and I- are compared. 4a-Hydroperoxy-3,5-dimethyllumiflavin is shown to convert thioxane to its sulfoxide 1.8 X 10(5) times faster than t-butyl hydroperoxide.


Enkephalin: conformational analysis by means of empirical energy calculations.

Low-energy conformations of methionine-enkephalin were generated by means of an empirical method of computation. Many compact conformations, including those containing various standard bends, were of comparable energy. However, one conformation was found to have a potential energy about 5 kcal/mol (21 X 10(3) J/mol) below that of the large group of compact conformations. In this conformation, the 3-glycyl and 4-phenylalanyl residues form a bend of type II'. The conformation is stabilized by a hydrogen bond between the OH group of the 1-tyrosine side chain and the C==O group of 3-glycine or 4-phenylalanine. The phenylalanine and methionine side chains are relatively unrestricted. The conformation is consistent with published nuclear magnetic resonance parameters--coupling constants, temperature dependence of the chemical shift, and spin-lattice relaxation times. It is likely that the molecule undergoes a conformational change when it is bound to the receptor. Leucine-enkephalin appears to have the same conformation as its methionine homolog.


Characterization and synthesis of volatile compounds from the defensive secretions of some "daddy longlets" (Arachnida: Opiliones: Leiobunum spp.).

Analyses of the chief volatile constituents of the defensive secretions of three oplionids were carried out. Leiobunum nigripalpi produces three closely related C7 compounds: E-4-methyl-4-hexen-3-one(I), 4-methylhexan-3-one(II), and 4-methylhexan-3-ol(III), along with E-4-methyl-4-hepten-3-one(IV), E,E-2,4-dimethylhexa-2,4-dienal(IX), and a minor, unidentified component. L. leiopenis secretion contains E-4-methyl-4-hepten-3-one(IV), 4-methylheptan-3-one(V), E,E-2,4-dimethylhexa-2,4-dien-1-ol(VII), and E,E-2,4-dimethylhepta-2,4-dien-1-ol(VIII). L. calcar yields chiefly E-4,6-dimethyl-6-octen-3-one(VI) and E,E-2,4-dimethylhexa-2,4-dien-1-ol(VII). Six of these compounds are new natural products. The structures of these compounds, which can be regarded either as polyketide-derived or as modified isoprenoids, raise interesting biosynthetic questions.


Functional acetylcholine receptor--electroplax membrane microsacs (vesicles): purification and characterization.

Kinetic analysis of the flux of sodium ions in a heterogeneous population of acetylcholine receptor-rich microsacs (vesicles) formed by membrane fragments of electroplax indicated that functional microsacs, which on average comprise only 15% of the preparation, can be filled with 190 mM sodium chloride while nonfunctional microsacs are filled by 190 mM cesium chloride. The functional microsacs have then been successfully separated from nonfunctional microsacs on the basis of their density differences with a continuous sucrose-190 mM cesium chloride density gradient. In the presence of acetylcholine analogs all the internal sodium ions in these microsacs rapidly exchange with external ions. The efflux of sodium ions follows a single exponential decay. The isolation of functional microsacs opens up at least two new avenues of investigation of the molecular mechanism of receptor-mediated processes. The first deals with the efficiency of the process, and the second with the characterization of membrane components important in this process. The conclusions reached so far are: (i) The efficiency of the receptor-mediated process that allows inorganic ions to equilibrate across the membranes of the microsacs can adequately account for electrophysiological results obtained with muscle and nerve cells. (ii) In the receptor-rich heterogeneous population of microsacs the concentration of receptor sites in functional and nonfunctional microsacs is about the same and is therefore not the only factor determining functionality. Significant differences between functional and nonfunctional microsacs have been found so far in the concentrations of acetylcholinesterase and Na+-K+ ATPase.


Kinetic evidence for an intermediate in the deacetylation of monoacetyl-chymotrypsin.

Mono[14C]acetyl-chymotrypsin was prepared by treating alpha-chymotrypsin with a 10-fold molar excess of p-nitrophenyl[14C]acetate at pH 5, and the acetylated enzyme was isolated free of excess reagents by gel filtration. Deacetylation at pH 6.0 was followed by observing the decrease in acid-precipitable radioactivity and provided a first-order rate constant of 0.02 +/- 0.008 min-1. Reactivation of the acetylated protein was followed by continuously monitoring the appearance of esterolytic activity towards alpha-N-acetyltyrosine ethyl ester. Reactivation at pH 6.0 occurred exponentially with a first-order rate constant of 0.2 +/- 0.015 min-1, the reactivated enzyme exhibiting an apparent catalytic contant (k' cat) of 1200 +/- 60 min-1, which decreased to a value of 945 +/- 15 min-1 by an apparent first-order process with a rate constant of 0.025 +/- 0.006 min-1. These results are interpreted in terms of a two-step deacetylation of monoacetyl-chymotrypsin involving an acetylated intermediate with esterase activity.


Charge shift electrophoresis: simple method for distinguishing between amphiphilic and hydrophilic proteins in detergent solution.

Seventeen hydrophilic proteins and five amphiphilic membrane proteins were subjected to agarose gel electrophoresis in the presence of a nonionic detergent (Triton X-100), a mixture of anonionic and an anionic detergent (Triton X-100 and sodium deoxycholate), and a mixture of a nonionic and a cationic detergent (Triton X-100 and cetyltrimethylammonium bromide). The electrophoretic mobility of the hydrophilic proteins was unaffected in the three detergent mixtures. However, the mobility of the amphiphilic proteins shifted anodally in the Triton X-100-deoxycholate system and cathodally in the Triton X-100-cetyltrimethylammonium bromide system when compared to the mobility in Triton X-100 alone. The detergent-induced shift in mobility provides a simple, rapid, and sensitive method for distinguishing between hydrophilic and amphiphilic proteins.


A thermostable sequence-specific endonuclease from Thermus aquaticus.

A sequence-specific endonuclease, Taq I, of novel specificity has been partially purified from an extreme thermophile, Thermus aquaticus. The enzyme cleaves bacteriophage lambda DNA at many (greater than 30) sites and bacteriophage psiX174 RF DNA at 10 sites. The enzyme is active at temperatures up to 70 degrees. The cleavage sites on psiX174 RF DNA have been mapped. The sequence recognized and cleaved by Taq I has been shown to be the symmetrical tetranucleotide: (formula: see text).


Identification of the iron-sulfur center in trimethylamine dehydrogenase.

Trimethylamine dehydrogenase [trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7] from a facultative methylotroph bacterium has a molecular weight of 147,000 and contains two types of prosthetic groups, one a covalently bound organic chromophore of uncertain structure and the other containing iron and labile sulfur (S*). The structure of the Fe-S* center has been investigated by reactions of the enzyme with sodium mersalyl, o-xylyl-alpha,alpha'-dithiol, and p-methoxybenzenethiol in a 4:1 vol/vol hexamethylphosphoramide/water reaction medium, which destabilizes tertiary structure. Mersalyl treatment results in reduction of visible absorbance consistent with the presence of a 4-Fe center of the ferredoxin type. Reaction with thiols effects partial bleaching of the organic chromophore, as established by separate studies of a detached chromophore peptide, and results in removal (extrusion) of the core unit of the Fe-s* center in the form of the complexes [Fe4S*4(S2-o-xylyl)2]n2n- and [Fe4S*4(SC6H4OMe)4]2-, which were identified by absorption spectra. These results, in conjunction with control extrusion reactions of oxidized ferredoxins from spinach and Clostridium pasteurianum, establish that trimethylamine dehydrogenase contains one Fe4S*4 core unit most probably present as a ferredoxin-type, cysteinate-ligated cluster [Fe4S*4(S-Cys)4].


Assembly of tobacco mosaic virus in vitro: elongation of partially reconstituted RNA.

It was reported that the rate of elongation of partially reconstituted RNA, if prepared without centrifugation, showed dependency upon the state of protein aggregation like that found when free RNA was used as the nucleic acid source [Butler, P. J. G. (1974) J. Mol. Biol. 82, 333-341]. Butler concluded from this result that disks are the kinetically favored protein source for the elongation of the tobacco mosaic virus rod. We show that partially reconstituted RNA prepared without centrifugation always contains a significant amount of free RNA and conclude that the dependency of its elongation rate upon the state of protein aggregation is due to contamination of his partially reconstituted RNA preparation by free RNA. The elongation rate of partially reconstituted RNA, which had been pelleted and contained no free RNA, was found to be about the same regardless of the state of aggregation of the added protein.


A new method for sequencing DNA.

DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.


Nerve growth factor receptors on human melanoma cells in culture.

Purified mouse nerve growth factor (NGF)radiolabeled with 125I was tested for its ability to bind to a variety of different cultured cells. NGF binds readily to human and hamster melanoma cells but does not bind to many other cell lines. The three cell lines with the highest number of NGF receptors were derived from metastatic melanomas. One of these lines, A875, was studied in detail and was shown to have approximately 7x10(5) NGF receptors per cell with an association constant of 1.0x10(9) liters/mole. The use of these cells in competition binding assays permits the detection of 0.25 ng of NGF in various biologic fluids. NGF can be shown to increase the survival, but not the division, of melanoma cells maintained in medium depleted of serum growth factors. This effect of NGF as a specific "survival factor" appears analogous to its effect on cultured sympathetic ganglion cells and on other cells derived from the neural crest.


Identification of chlorophyll b in extracts of prokaryotic algae by fluorescence spectroscopy.

Solvent extracts of three different prokaryotic algae from three species of didemnid ascidians contained pigments identified, on the basis of their fluorescence excitation (E)and fluorescence emission (F)spectral maxima (measured in nm) at 77K, as chlorophyll a (E 449, F 678) and chlorophyll b (E 478, F 658). The release of algae on cutting or freezing Diplosoma virens was accompanied by a strong unidentified acid that converted these pigments to pheophytins. This unexpected finding provided further confirmation of the identity of the chlorophylls on the basis of the fluorescence spectra at 77K of pheophytin a (E 415, F 669) and pheophytin b (E 439, F 655). There was no evidence for the presence of the fluorescent bilin pigments found in other prokaryotic blue-green algae. Chlorophyll a/b ratios ranged from 2.6 to 12.0 in algae from different ascidians. The photosynthetic membranes were not organized into appressed thylakoids or grana in the algae from any of the three species of ascidians. The relationship between these observations and those in higher eukaryotic organisms is discussed.


Chemical transformation of rat cells infected with xenotropic type-C RNA virus and its suppression by virus-specific antiserum.

Fischer rat embryo cells infected with xenotropic mouse type-C RNA viruses isolated from either NIH Swiss mice or C57L inbred mice were transformed by 4-nitroquinoline oxide or 3-methylcholanthrene under conditions in which neither treatment alone sufficed to induce transformation. Transformation was dependent on the presence of actively growing virus at the time of chemical treatment. Specific antibody to xenotropic virus neutralized the virus and inhibited malignant transformation by 3-methylcholanthrene.


Pterin-6-aldehyde, a cancer cell catabolite: identification and application in diagnosis and treatment of human cancer.

Active folic acid degradation with the formation pterin-6-aldehyde is a previously undescribed characteristic of cancer cells in tissue culture. Neither normal adult epithelial and fibroblastic cells nor human amniotic cells nor mouse embryonic fibroblasts degrade folic acid to a measurable degree. Twenty-nine patients whose diagnoses were not revealed until after the test of their first morning urine for pterin-6-aldehyde was completed were studied for the presence or absence of pterin-6-aldehyde by thin-layer chromatography. Pterin-6-aldehyde was found in the urine at about 300 nmol/ml or greater only in those 13 patients with a tissue diagnosis of cancer. When the cancer was totally resected, the pterin-6-aldehyde was no longer found in the urine postoperatively. Pterin-6-aldehyde is not found in the urine of healthy patients at this level of detection unless their diets are supplemented with folic acid.


A rapid procedure for selective enrichment of photosynthetic electron transport mutants.

Metronidazole (2-methyl-5-nitroimidazole-l-ethanol) is shown to be effective for the selective enrichment of mutants of Chlamydomonas reinhardtii that posses impaired photosynthetic electron transport. More than 99.9% of wild-type cells are killed when incubated in the presence of 6-10 mM metronidazole for 24 hr under illumination of 7500 lux. Survival of wild-type cells in darkness and of mutants that are blocked at different steps in photosynthetic electron transport is nearly 100% when incubated in the presence of the drug under identical conditions. The toxicity of metronidazole is demonstrated to depend upon its reduction by photosynthetic electron transport. Light-dependent oxygen uptake mediated by metronidazole is shown to require active photosystem l in vitro and in vivo. Ferredoxin is necessary for metronidazole reduction by thylakoid membrane fractions enriched in photosystem l activity. We propose that the toxicity of metronidazole is due to the formation of lethal derivatives of the drug or to the accumulation of hydrogen peroxide, which could occur upon autooxidation of metronidazole reduced by one electron. The results indicate that mutants of C. reinhardtii, and probably other photosynthetic organisms, with any lesion in photosynthetic electron transport from the oxidizing side of photosystem ll to ferredoxin can be isolated by metronidazole treatment of mutagenized cultures.


Secretion of alpha-ecdysone by crab Y-organs in vitro.

The cultured molting glands (Y-organs) from two species of crabs secrete a product whose behavior in several chromatographic systems and whose mass spectrum are identical with those of authentic alpha-ecdysone. It appears that alpha-ecdysone is the only molting hormone secreted by the Y-organ, and that the Y-organ is the sole source of alpha-ecdysone in these animals.


Derepression of ATP sulfurylase by the sulfate analogs molybdate and selenate in cultured tobacco cells.

Molybdate and selenate are structural analogs of sulfate that inhibit synthesis of adenosine 5'-phosphosulfate by ATP sulfurylase (sulfate adenylyltransferase, ATP:sulfate adenylyltransferase, EC 2.7.7.4) in crude extracts of tobacco XD cells. Both of these anions derepress ATP sulfurylase in cells growing on sulfate, but not in cells growing on L-cysteine. However, the two anions appear to derepress by different mechanisms. Molybdate caused derepression only at concentrations that were in excess over sulfate and were sufficient to inhibit growth and protein accumulation, indicating that the derepression resulted from sulfur starvation. Selenate caused derepression at one-tenth the concentration of sulfate, a concentration of selenate that was subtoxic, while toxic levels of selenate produced far less derepression. The susceptibility of the tobacco cells to selenate toxicity was high under conditions of sulfur nutrition that derepress ATP sulfurylase, and low under conditions that repress ATP sulfurylase, in agreement with the idea that selenate acts via a functional sulfate assimilation pathway. Since it is known that selenate is incorporated into analogs of sulfur compounds, it is proposed that the tobacco cells synthesize the seleno-analog of the end product of the sulfate pathway responsible for repression, and the seleno-analog antagonizes the normal end product in the repression mechanism, the net result being derepression of ATP sulfurylase by selenate.


Appearance of a chromatin protein during the erythroid differentiation of Friend virus-transformed cells.

Chromatin proteins from erythroleukemia cells transformed by Friend virus were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A new chromosomal protein, apparent molecular weight 25,000, was shown to appear during erythroid differentiation of the cells induced by dimethyl sulfoxide or hexamethylenebisacetamide. This protein is tightly bound to the chromatin and does not appear to be related to any of the known histones. It is also demonstrated that this protein is not induced in a dimethyl sulfoxide-resistant variant of these cells.


Lymphocyte mitogenesis induced by a mammalian liver protein that specifically binds desialylated glycoproteins.

A purified rabbit liver membrane protein that binds desialylated glycoproteins has been shown to be a mitogen for human peripheral lymphocytes. The mitogenic activity is specific for desialylated thymus-derived (T)-cells. The loss of mitogenicity upon exposure of the binding protein to neuraminidase and the inhibitory potency of asialo-orosomucoid support the conclusion that the site involved in the binding of asialoglycoproteins is also responsible for the mitogenic effect on desialylated lymphocytes. The potential relevance of this phenomenon to the etiology of hepatocellular necrosis is considered.


Suppression of production of mouse 28S ribosomal RNA in mouse-human hybrids segregating mouse chromosomes.

Mouse-human somatic cell hybrids that lose (segregate) human chromosomes produce only mouse 28S ribosomal RNA even when they retain copies of the human chromosomes that contain the genes for 28S ribosomal RNA. In contrast, mouse-human hybrid cells that segregate mouse chromosomes produce only human 28S ribosomal RNA even when they have retained copies of mouse chromosomes that contain the 28S ribosomal RNA genes.


Polymorphism and the subunit structure of enzymes: a contribution to the neutralist-selectionist controversy.

The occurrence of polymorphism in a series of 87 different loci coding for enzyme structure in human populations has been related to the size and the number of subunits in the corresponding enzymes. Polymorphic and nonpolymorphic enzymes did not differ on average in subunit size. However, multimeric enzymes showed a significantly lower incidence of polymorphism than didmonomeric enzymes. A particularly low incidence of polymorphism was noted among multimeric enzymes in which interlocus molecular hybrids occur. The findings are discussed in terms of the "neutralist" and "selectionist" hypotheses of polymorphism.


Enhanced antibody affinity in sublethally irradiated mice and bone marrow chimeras.

Sublethally irradiated mice primed with dinitrophenyl (Dnp)-keyhole limpet hemocyanin immediately after irradiation or 30 days later and subsequently boosted with a second injection of antigen displayed a secondary response to Dnp characterized by antibody affinity greater than that in unirradiated controls. Also, in radiation chimeras primed with Dnp-keyhole limpet hemocyanin 120 days after syngeneic or allogeneic bone marrow transplantation the antibodies against Dnp produced after boosting were of higher affinity than the antibodies raised in normal mice. These findings are tentatively attributed to lack of suppressor thymus-derived lymphocytes (T cells) in sublethally irradiated mice and bone marrow chimeras, in which the enhanced ability to produce antibodies of high affinity may compensate for quantitative defects of the immune system.


Inhibition of proliferative and plaque-forming cell responses by human bone-marrow-derived lymphocytes from peripheral blood by antisera to the p23, 30 antigen.

A recently described technique for the polyclonal induction of plaque-forming cells from human bone-marrow derived (B) lymphocytes of peripheral blood has been used to assess the role of a human Ia-like antigen (p23,30) in differentiation of human B cells. In particular, the effects of antisera to p23,30 on the plaque-forming cells and proliferative responses of human B cells stimulated by pokeweed mitogen or soluble products of activated human thymus-derived lymphocytes (T cells) have been examined. Antisera to p23,30 eliminated the development of plaque-forming cells induced by both T cell products and pokeweed mitogen. While these antisera also abrogated B cell proliferation induced by T cell supernatants, the proliferative response generated by pokeweed mitogen was only partially reduced. It was also determined that while the p23,30 antigen continues to be expressed on fully differentiated plaque-forming cells, antisera to this determinant exert inhibitory effects on B cell differentiation only when present during the early stages of B cell cultures. These results lend further support to the analogy between p23,30 and murine Ia antigens. Moreover, they demonstrate a major role for this antigen in the early events involved in human B cell differentiation into antibody-forming cells.


Lymphocytes alloantigens associated with X-chromosome-linked immune response genes.

A new polymorphic alloantigen system controlled by loci on the X-chromosome has been identified by using antisera from F1 hybrid mice differing in their X-chromosome. These alloantigens are associated with the X-chromosome. These alloantigens are associated with the X-linked immune response genes controlling the immune response to the so-called "thymus independent antigens" such as type III pneumococcal polysaccharide, poly(I)-poly(C), and denatured DNA. They also show association with the histocompatibility locus present on the X-chromosome. They were mainly detected on a not yet characterized thymus-derived lymphocyte subpopulation. A certain similarity with the major histocompatibility complex of the mouse supports the possibility of additional I-like regions besides the I region of the histocompatibility-2 complex.


Thymosin alpha1: isolation and sequence analysis of an immunologically active thymic polypeptide.

The amino acid sequence of a biologically active polypeptide isolated from calf thymus, termed thymosin alpha1, has been determined. Thymosin alpha1 is a heat stable, highly acidic molecule composed of 28 amino acid residues. This peptide is one of several present in thymosin fraction 5 that may participate in the regulation, differentiation and function of thymus-dependent lymphocytes (T cells). A nomenclature for the family of polypeptides present in thymosin fraction 5 is suggested.


A sex-limited serum protein of Syrian hamsters: definition of female protein and regulation by testosterone.

Normal Syrian hamster females contain a serum protein not found by simple gel diffusion assay in normal adult males. This sex-limited protein was called female protein (FP). Low levels of FP were found in sera from normal weanling male hamsters. Adult male hamsters castrated or treated with diethylstilbestrol also developed serum FP, which could be suppressed by administration of testosterone. Exogenous testosterone also suppressed serum FP in adult females, and ovarian function did not appear to be critical for maintenance of serum FP. Although FP is present in significant amounts (1-2 mg/ml) in adult females, its function is presently unknown.


Characterization of C-reactive protein and the complement subcomponent C1t as homologous proteins displaying cyclic pentameric symmetry (pentraxins).

Partial amino acid sequences of rabbit C-reactive protein, a peptide derived from human C-reactive protein by cyanogen bromide cleavage, and the C1t subcomponent of the human complement component C1 have been determined. Extensive sequence homology between these proteins establish their evolutionary relationships. In addition, examination of C-reactive proteins by negative-stain electron microscopy revealed that the protein is composed of five subunits arranged in cyclic symmetry. This structure is similar to that reported for both C1t and the amyloid P-component. The extensive structural relationship suggests similar or overlapping functions and the term pentraxin is proposed to describe these homologous proteins.


Growth of cultured cells from patients with Hodgkin's disease and transplantation into nude mice.

Long-term monolayer cell cultures have been prepared from tumor nodules in spleens removed from 28 patients with Hodgkin's disease and from 84 spleens that did not have tumors from Hodgkin's disease patients, normal adult spleens, and human fetal spleens and thymuses. After 5 to 20 serial passages in culture, cells from nine of the Hodgkin's disease monolayers underwent morphologic change in vitro with transition from a spindle and reticular pattern of replication to polygonal and round cells that propagated in mosaic arrays. Four of such Hodgkin's disease monolayer cell lines were injected subcutaneously into 43 nude, athymic mice. In 36 animals (84%), neoplasms developed at the inoculation site that werel ocally destructive, capable of pulmonary metastasis, and  eventually fatal to the recipients. Transplanted tumors were not observed in 18 athymic mice injected with cultures prepared from normal human adult spleen and fetal spleen and thymus, nor were tumors seen in 16 similar animals that received fresh, noncultured Hodgkin's disease tumor tissue. On microscopic examination, xenografts derived from Hodgkin's disease cultures were pleomorphic malignant neoplasms composed of large, undifferentiated cells, resembling reticulum cell sarcoma. These neoplasms did not involve the lymphoreticular organs of mice. Chromosome studies indicated that the transplanted neoplasms were composed of human cells with an aneuploid karyotype and that monolayer cultures prepared from the heterotransplants contained a karyotype similar to that of the cultured cells prior to passage in mice. The ability of these Hodgkin's disease cell lines to produce invasive tumors with human karyotypes in nude mice is evidence of the neoplastic nature of the monolayer cells and their relationship to the malignant cell of the human disorder.


Purification and characterization of the DNA polymerase of human breast cancer particles.

Previous studies have identified human breast tumor particles possessing many of the features characteristic of RNA tumor viruses. In addition to the expected size (600 S) and density (1.16 g/ml) these include possession of an outer membrane and an inner one surrounding a "core" containing a DNA polymerase and a large-molecular-weight (70S) RNA possessing detectable homology to the RNAs of the mouse mammary tumor virus (MMTV) and of the Mason-Pfizer monkey virus (MPMV). We report here the purification and characterization of the DNA polymerase from the human breast cancer particles. Its key properties are very similar to those ofthe RNA-dependent DNA nucleotidyltransferase (reverse transcriptase) found in MMTV and MPMV. Thus like these viral enzymes, the purified human breast cancer DNA polymerase exhibits the following three features that together distinguish the known viral reverse transcriptases from normal cellular DNA polymerases: (i) a strong preference for oligo(dT)-poly(rA) over oligo(dT)-poly(dA) as a template for the synthesis of poly(dT); (ii) the acceptance of the highly specific oligo(dG)-poly(rCm) as a template for the formation of poly(dG); (iii) the ability to use a viral RNA (AMV) as a template to fashion a faithful DNA complementary copy; and (iv) its preference for Mg++ over Mn++. In summary, the data described here on the enzyme of the human breast cancer particles add further evidence of similarities to the viral agents associated with the corresponding malignancies in the mouse and monkey models. To date, an enzyme with these properties has not been detected in normal breast tissues or in benign tumors of the breast.


Opposing effects of dopaminergic to cholinergic compounds on a cerebral dopamine-activated adenylate cyclase.

In contrast to antipsychosis drugs which inhibit the dopamine-activated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of caudate nucleus, dopaminergic drugs for treatment of Parkinson's disease stimulate this cyclase. Stimulants and inhibitors of cholinergic neurons inhibited this adenylate cyclase activity competitively and specifically. Thus, the mechanism by which dopaminergic medications ameliorate the effects of Parkinson's disease includes activation of the dopamine-sensitive adenylate cyclase. Excessive activation might be present during the psychotic episodes seen in patients with parkinsonism who are overtreated. The enzymatic effects of the drugs that affect cholinergic mechanisms seem to be generally in keeping with the pharmacological reciprocity between psychoses and extrapyramidal function, except for the anticholinergic ones which inhibited this cyclase although they can be hallucinogenic.


beta-Endorphin: behavioral and analgesic activity in cats.

beta-Endorphin has been shown to possess potent behavioral and antinociceptive activities when administered intraventricularly in cats. On a molar basis, beta-endorphin is 72-96 times more potent than morphine and its actions are blocked by the specific opiate antagonist, naloxone.


Persistence of type 5 adenovirus DNA in cells transformed by temperature-sensitive mutant, H5ts125.

The characteristic of H5ts125, a temperature-sensitive DNA-minus mutant, to transform 3 to 8 times more rat embryo cells than wild-type 5 adenovirus was correlated with the persistence of an increased proportion of the viral genome in cells independently transformed at the nonpermissive (39.5 degrees) or semipermissive (36 degrees) temperature. Reassociation kinetics of the hybridization of 32P-labeled,HindIII restriction fragments of the viral genome and excess unlabeled, transformed cell DNA was used to measure the quantity of the viral genome in transformed cells. Three of four cell lines independently transformed and maintained at 36 degrees contained all regions of the viral genome; one line transformed at 39.5 degrees contained multiple copies representing all of the viral DNA; and one line contained a large proportion of the viral genome. The cell line transformed and maintained at 32 degrees contained a quantity of viral genome consistent with that usually found in cells transformed by wild-type 5 adenovirus.


Evidence for active role of cilia in sensory transduction.

Combined high-voltage electron-microscopic and electrophysiological studies strongly suggest that cilia play an active role in sensory transduction in the grasshopper proximal femoral chordotonal organ (FCO) a ciliated mechanoreceptor. The FCO of pro- and mesothoracic legs of Melanoplus bivittatus contains a group of several hundred chorodontal sensilla arranged in a near-parallel bundle and slung between the proximal femur and the knee joint. Both flexion and extension of the tibia stimulate the FCO, which appears to measure the femoro-tibial angle. The FCO's U-shaped response curve indicates that progressive flexion or extension from the resting joint angle of 90 degrees increases the response frequency of individual receptors and recruits additional units as well. Since the FCO is a purely tonic mechanoreceptor, it is possible to fix FCOs during maximum and minimum states of stimulation and electron-microscopically observed changes in the receptor's fine structure. The most conspicuous change is the production of a pronounced bend at the base of the sensory cilia in chordotonal sensilla of maximally stimulated femoral chordotonal organs.


Human somatotropin: restoration of full biological activity by noncovalent interaction of a natural and a synthetic fragment of the hormone.

The recombinant hormone obtained by noncovalent interaction of the natural NH2-terminal fragment (consisting of 134 amino acid residues) with a synthetic COOH-terminal fragment of 52 amino acids of the reduced-carbamidomethylated human somatotropin molecule is found to exhibit full biological activity of the native hormone as evidenced by the tibia test. Radioimmunoassay data indicate that the semisynthetic recombinant hormone possesses essentially full immunoreactivity as compared to the native one. In addition, circular dichroism and fluorescence emission spectra of the semisynthetic recombinant are identical to that of the undisociated, plasmin modified, reduced-carbamidomethylated hormone.


Studies on nucleic acid reassociation kinetics: rate of hybridization of excess RNA with DNA, compared to the rate of DNA renaturation.

The rate of reaction of double-stranded replicative form (RF) [3H]DNA of bacteriophage phiX174 with excess (+)strand DNA and (+)strand RNA was measured by standard methods of hydroxyapatite chromatography. The reactions follow pseudo-first-order kinetics and the observed rate constant for the RNA-DNA reaction differs less than 25% from that of the DNA-DNA reaction. The pseudo-first-order rate constants are close to the value predicted on the basis of the second-order rate constant measured in the renaturation of the double-stranded phiX RF [3H]DNA.


Nuclear magnetic resonance studies of lecithin bimolecular leaflets with incorporated fluorescent probes.

Pulse Fourier transform proton magnetic resonance techniques have been used to determine (i) the precise location of four membrane fluorescent probe molecules in lipid bilayer model membranes above the gel-to-liquid crystal transition temperature, tm; (ii) the dynamical perturbation of the host lipid molecules induced by incorporation of the probe into the bilayer. The regions of maximal perturbation induced by these probes do not necessarily coincide with their actual location in the bilayer.


Identification of a protein related to tubulin in the postsynaptic density.

The postsynaptic density is a unique subcellular organelle associated with the synaptic complex and appears as an electron-dense area immediately subjacent to the postsynaptic plasma membrane. The postsynaptic density was isolated from the synaptosomal fraction and the protein constituents were analyzed by polyacrylamide gel electrophoresis. Polypeptides closely related to tubulin were identified as a major component of the postsynaptic density on the basis of molecular weight, subunit structure, and peptide map criteria.


Anionic glycopeptides and glycosaminoglycans synthesized by embryonic neural tube and neural crest.

Anionic glycopeptides and glycosaminoglycans synthesized by embryonic neural tube (epithelium) and neural crest (mesenchymal outgrowth) developing in vitro were examined. The profile of surface glycopeptides is relatively simple (two major ones for neural crest; four major ones for neural tube). There is one major glycopeptide found in the medium which is present only in trace amounts on the cells. Both hyaluronate and chondroitin sulfate are synthesized. Hyaluronate is predominantly cell associated; chondroitin sulfate is found predominantly in the medium. It is suggested that independent sorting of these relatively few glycopeptides can result in qualitative surface differences. Morphological state (epithelial or mesenchymal) and differentiated state may be related to these differences as well as to quantitative differences in surface glycosaminoglycans.


Changes in the composition of plasma membrane proteins during differentiation of embryonic chick erythroid cell.

Erythroid cells which are homogeneous with regard to stage of maturation are naturally available from the circulation of chick embryos at various times of development. This provides a convenient system for examining the changes in plasma membrane protein composition during red cell maturation. Plasma membranes are isolated from chick embryonic erythroid cells at various stages of maturation. Extensive characterization of the isolated membranes show that they are pure and their proteins undegraded. Analyses by sodium dodecyl sulfate/polyacrylamide gel electrophoresis show that both qualitative and quantitative changes occur in membrane protein composition during the early stage of erythroid differentiation. Specific proteins of red cell membrane such as "spectrin" and band three proteins are present in low levels in early erythroblasts but increase in their relative amounts with maturation. A steady-state membrane protein composition seems to be established by the late polychromatophilic erythroblast stage.


Establishment and characterization of a strain of human adrenal tumor cells that secrete estrogen.

We report the establishment of a new cell line, designated Fang-8, which originated from a human adrenal adenocarcinoma. It has been continuously propagated during the past 20 months. The cells exhibit an epithelioid morphology, resembling the cell structure of the zona reticularis when viewed by electron microscopy. Since passage 12, the cells showed a characteristic ring-forming property in culture. The cells produce neither testosterone, mineralocorticosteroids, nor glucocorticosteroids; instead, they possess the unique function of estrogen production. Karyotypic analysis revealed a 100% aneuploid, unstable karyotype in the hyperdiploid stem line. Identification of the Y chromosome was uncertain. In view of these characteristics, Fang-8 represents a new human cell line, unlikely to be contaminated with HeLa strains, and different from another cell line derived from human adrenal cortex andenocarcinoma, namely SW-13(ATCC CCL 105).


Changes in membrane microviscosity associated with phagocytosis: effects of colchicine.

The effects of phagocytosis on plasma membrane microviscosity were studied by fluorescence depolarization techniques. It was shown that lipophilic probes are accumulated in intracellular vesicles to a significant degree in fibroblasts and neutrophils. Microviscosity was thus determined from the behavior of probes in isolated membranes. Phagocytosis of oil emulsions or polystyrene beads by rabbit polymorphonuclear leukocytes induces a marked decrease in plasma membrane microviscosity that parallels the extent of phagocytosis. Liposomes made from extracts of membrane lipid show qualitatively the same changes, indicating that the alteration of microviscosity results at least in part from changes in lipid composition. The decrease in microviscosity is abolished when colchicine is present during phagocytosis. Addition of colchicine to membranes previously isolated from control or phagocytic cells has no effect on their microviscosity. The results suggest that phagocytosis is accompanied by a microtubule-dependent reorganization of membrane lipids.


Adhesive specificity of juvenile rat and chicken liver cells and membranes.

Liver cells, isolated from either juvenile rats or chickens by a collagenase perfusion technique, reaggregated when maintained in suspension. The cells exhibited marked adhesive specificity; when suspensions contained both cell types, the aggregates consisted primarily of either rat or chicken cells. Adhesive specificity was also observed with plasma membrane fractions isolated from rat liver homogenates, and with comparable fractions from chicken liver. These membranes stimulated aggregation of the homologous but not the heterologous cell type. Other membrane fractions had little or no effect on the aggregation of the homologous cell type. These and other properties of the liver cell and membrane preparations suggest that biochemical studies on cell-cell recognition and adhesion can most effectively be conducted with cells from juvenile and adult animals.


In vitro synthesis and processing of a putative precursor for the small subunit of ribulose-1,5-bisphosphate carboxylase of Chlamydomonas reinhardtii.

Translation of polyadenylated mRNA of Chlamydomonas reinhardtii in a cell-free wheat germ system resulted in the synthesis of numerous discrete polypeptides. Among them was a species with molecular weight 20,000 that was immunoprecipitated specifically by antibodies raised against the authentic small subunit (16,500 daltons) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase(dimerizing), EC 4.1.1.39]. Since the immunoprecipitated polypeptide has a larger molecular weight by approximately 3500 than the small subunit (S) it was identified as a putative biosynthetic precursor (pS). Post-translational conversion of pS by a specific endoprotease yielded two detectable products: one apparently identical in size to S and the other, a small peptide, presumably representing the remainder of pS. The endoprotease requires sulfhydryl groups for its activity and is present in a C. reinhardtii postribosomal supernatant as well as in a free polysome fraction. The latter could account for the observation that completion of nascent chains in free polysomes yielded S but not pS. We propose that pS is an extrachloroplastic form of S and that the small peptide portion plays a role in the transfer of S into the chloroplast.


Differential effects of chemical inducers on expression of beta globin genes in murine erythroleukemia cells.

Murine erythroleukemia cells are induced to erythrodifferentiate by polar compounds such as dimethyl sulfoxide and hexamethylene bisacetamide as well as by fatty acids such as butyric acid and propionic acid. The effect of these inducers on the expression of two beta globin genes, betamaj and betamin, during the course of differentiation of the cells has been examined. After 4 days of culture with hexamethylene bisacetamide or dimethyl sulfoxide, the betamaj-containing hemoglobin (Hbmaj) predominates. By contrast, in the presence of butyric acid or propionic acid, after 4 days of culture, relatively equal amounts of Hbmaj and Hbmin are found. When cultured with dimethyl sulfoxide or hexamethylene bisacetamide, murine erythroleukemia cells synthesize more betamaj than betamin, while about equal amounts of the two globins are synthesized in the presence of butyric acid. When poly(A)-containing RNA from the cells exposed to different inducers is translated in a wheat germ cell-free system, the ratio of betamaj to betamin synthesized reflects that in whole cells. In a strain of murine erythroleukemia cells resistant to dimethyl sulfoxide (DR-10), the preferential stimulation of betamaj synthesis by hexamethylene bisacetamide of the betamin synthesis by butyric acid is more pronounced than with the dimethyl sulfoxide-sensitive cells (DS-19). These data suggest that polar compounds and fatty acids cause different expression of the betamaj and betamin genes in murine erythroleukemia cells.


Characterization of an erythroid precursor cell of high proliferative capacity in normal human peripheral blood.

We have found that the peripheral blood of normal humans contains a significant number of committed erythroid stem cells of high proliferative capacity. These erythroid stem cells closely resemble the murine erythroid burst-forming unit (BFU-E) with respect to proliferative capacity, colony morphology, and erythropoietin requirement. BFU-E were isolated from the peripheral blood of normal individuals by Ficoll-Hypaque density gradient centrifugation and cultured in vitro using the plasma culture technique. Macroscopic erythroid colonies of between 100 and 1000 cells were observed after 10-14 days of culture in the presence of either sheep or human erythropoietin at 0.5-4 units/ml. Individual colonies contained between 3 and 20 subcolonies and reached a maximum mean size of approximately 500 cells. Colony number was linearly related to the cell input, suggesting that a single cellular entity was precursor to each colony. The frequency of cells capable of giving rise to an erythroid colony was at least 100 per ml of blood in a number of individuals tested. The ability to assay significant numbers of erythroid precursor cells of high proliferative capacity from normal peripheral blood should facilitate the study of both normal erythropoiesis and of disease states affecting erythropoiesis in which marrow samples are not available on a routine basis.


Receptor diffusion on cell surfaces modulated by locally bound concanavalin A.

In order to test the anchorage modulation hypothesis, the fluorescence photobleaching recovery method was used to measure the global inhibition of cell surface receptor mobility induced in 3T3 mouse fibroblasts by local binding of platelets labeled with concanavalin A (Con A). By measuring the diffusion of antibody-labeled cell surface receptors at various points on the cell surface, two states, immobile and mobile, were distinguished in the receptor population. Bound Con A-platelets, occupying between 4% and 30% of the cell surface, decreased the diffusion coefficient of the mobile population by a factor of 6. The magnitude of this effect was independent of distance from the sites of the bound Con A-platelets, demonstrating the propagated and nonlocal properties of the modulation effect. The immobile fraction of the population was not changed by Con A-platelet binding. Modulation of the diffusion constant of mobile receptors was partially reversed by treatment with microtubule-disrupting agents such as Colcemid and Vinca alkaloids. High doses of soluble Con A induced even higher levels of modulation than Con A-platelets, but reversal by microtubule-disrupting drugs was observed. These experiments provide additional support for the anchorage modulation hypothesis and provide a measure of the nature and degree of mobility at the molecular level. They also put important constraints on the hypothesized interactions among submembranous components (microtubules and microfilaments) of surface modulating assemblies.


An extracellular inducer of asexual plasmodium formation in Physarum polycephalum.

Asexual conversion of amoebae to plasmodia was studied in the Colonia isolate of the myxomycete, Physarum polycephalum. When a culture of Colonia amoebae is grown on a bacterial lawn, a period of amoebic growth precedes the appearance of cells committed to the plasmodial state. The onset of plasmodium production appears to be related to amoebic nutrition since cultures supplied with fewer bacteria display earlier differentiation. For a period of time after differentiation is initiated, conversion of amoebae to plasmodia is rapid and proceeds as an exponential function of time. A filter-transmissible substance, apparently released by differentiating cells, is implicated in the control of this rapid conversion.


Evolutionary consequences of parthogenesis: evidence from the Warramaba virgo complex.

Comparative quantitative analyses of variability in closely related parthenogenetic and sexually reproducing species have been lacking. This paper reports results of comparative analyses of relative variability carried out on the obligate thelytokous grasshopper Warramaba virgo (Key) and three closely related sexually reproducing species. Consistent patterns of differences in variability in 14 morphometric traits were found between clones and races of the parthenogenetic species which were absent in populations and species of the related sexual forms. When variability in the parthenogenetic and sexual species was compared, the parthenogenetic taxa were shown to be at least as variable as and often more variable than the sexual species.


Dual origin of highly social behavior among bees.

A study of behavior and structure indicates that highly eusocial behavior arose twice in the bees--i.e., in the stingless bees (Meliponinae) and in the honeybees (Apinae). Morphological features demonstrate the distinctiveness of these two groups and the relationship of the latter to bumblebees (Bombini) and orchid bees (Euglossin). The social behaviors of the stingless bees and honeybees, while more or less equally elaborate, are so different as to support their independent origins. The primitive apids, along with the related Xylocopinae (in the Anthophoridae), appear to have had the potential for parasocial, subsocial, and primitively eusocial behavior and from such forms the two highly eusocial groups arose.


Assignment of the major histocompatibility complex to a region of the short arm of human chromosome 6.

Interspecific cell hybrids containing defined parts of human chromosome 6 were used for regional mapping of gene loci previously assigned to chromosome 6: the human leukocyte antigens (HLA) region, phosphoglucomutase-3 (PGM3; alpha-D-glucose-1,6-bisphosphate:alpha-D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1) and malic enzyme-1 [malic dehydrogenase(decarboxylating) (NADP+), L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40]. Human fibroblasts containing a balanced reciprocal translocation between the short arms of chromosome 1 and chromosome 6--designated t(1;6) (p3200;p2100)--were fused with an established line of Chinese hamster cells. Hybrid clones with segregating human chromosomes were studied for the presence of the translocation chromosomes 1T and 6T and their normal homologues 1 and 6. Six clones that had retained 1T, three clones with 6T, and three clones with 6 and 6T as controls, were analyzed by a microabsorption test for expression of the allelic antigens HLA-A2 and HLA-A3, both of which were present on the human parental cells. HLA-A2 segregated with the 1T translocation chromosome and HLA-A3 with the normal chromosome 6. Hybrid clones with 6T did not possess an HLA-A gene. In contrast, human PGM3 and malic enzyme-1 expression segregated with the 6T translocation chromosome. These results indicate the location of the major histocompatibility complex in region 6p2100 leads to 6pter of the short arm of chromosome 6. The genes for PGM2 and malic enzyme-1 map in region 6p2100 leads to 6qter. The HLA:PGM3 genetic map distance is 15 centimorgans in males, as established by family studies. This allows rather precise mapping of both loci because HLA is distal and PGM3 proximal to the translocation breakpoint in band 6p2100. The findings are consistent with earlier conclusions that HLA is proximal to 6p22. Quantitative correlation between the density of HLA antigens on the hybrid cell surface and the number of copies of the respective HLA gene-bearing chromosome suggests a gene dose effect for cell surface molecules, as it exists for intracellular gene products.


Levels of delta-aminolevulinate dehydratase, uroporphyrinogen-I synthase, and protoporphyrin IX in erythrocytes from anemic mutant mice.

Levels of erythrocyte delta-aminolevulinate dehydratase [ALA-dehydratase; porphobilinogen synthase; 5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24], UROPORPHYRINOGEN-I synthase [Uro-synthase; porphobilinogen ammonia-lyase (polymerizing), EC 4.3;1.8], AND PROTOPORPHYRIN IX (Proto) were measured by sensitive semimicroassays using 2-5 mul of whole blood obtained from normal and anemic mutant mice. The levels of erythrocyte ALA-dehydratase and Uro-synthase showed marked developmental changes and ALA-dehydratase was influenced by the Lv gene. Mice with overt hemolytic diseases (ja/ja, sph/sph, nb/nb, ha/ha) had 10- to 20-fold increases in ALA-dehydratase, Uro-synthase, and Proto compared with their normal controls. Mice with an iron deficiency (mk/mk) and mice with hypoplastic anemias (W/Wv, Sl/Sld, an/an) had mild to moderate increases in these parameters. Elevated enzyme activities and Proto correlated well with the number of reticulocytes. Because all mice with anemias possessed elevated levels of ALA-dehydratase, Uro-synthase, and Proto independent of differences in their genotypes, the increase in these parameters is not likely to be the result of a specific gene defect. The increased enzyme activities and Proto concentration probably reflect increased frequency of young red cells that are still active in heme biosynthesis.


Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes.

Synthetic N-formylmethionyl peptides are chemotactic attractants for human polymorphonuclear leukocytes. The well-defined structure-activity relationship of these peptides in eliciting a chemotactic response suggests that the interaction of the peptides with a specific cellular binding site may initiate chemotaxis. By using tritiated N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe), a potent chemotactic peptide with high specific radioactivity, we have directly identified binding sites on human polymorphonuclear leukocytes. Binding of fMet-Leu-[3H]Phe to polymorphonuclear leukocytes is rapid (t1/2  less than 2 min) and reversible. The equilibrium dissociation constant (KD) for the interaction of fMet-Leu-[3H-A1Phe with the binding site is 12-14 nM at 37 degrees. The number of binding sites is approximately 2000 per cell. The specificity of the binding sites for a series of N-formylmethionyl peptides exactly reflects the specificity of the chemotactic response to the peptides in that they compete for the binding sites and initiate chemotaxis with the same order of potency (fMet-Leu-Phe greater than fMet-Met-Met greater than fMet-Phe greater than fMet-Leu greater than fMet),fPhe-Met is a competitive antagonist of the chemotactic activity of N-formylmethionyl peptides and has a calculated KD of 6x10-5 M. FPhe-Met also half-maximally inhibits binding of fMet-Leu[3H]Phe binding was the highest in polymorphonuclear leukocytes. No binding of fMet-Leu-[3H]Phe to human erythrocytes could be detected. These data indicate that fMet-Leu-[3H]Phe can be used to identify binding sites for chemotactic peptides on human polymorphonuclear leukocytes. It is likely that these binding sites initiate the specific response of motile cells to N-formylmethionyl peptides.


Multispecific lymphoid cell surface receptors.

When mice are sequentially immunized with two antigens to give an oligoclonal "double-binding" antibody response, there is a concomitant increase of "double-binding" cell surface receptors on their splenic lymphocytes. Competition studies suggest that the capacity to bind the two ligands, bovine pancreatic ribonuclease (EC 3.1.4.22) and a 2,4-dinitrophenyl (DNP) derivative, is a function of the same molecules. In ribo-nuclease-primed mice, an early response to bovine gamma globulin containing an average of 60 Dnp groups per molecule is the appearance of an increasing number of cells bearing surface receptors binding both ribonuclease and Dnp. Later, these double-binding cells are diluted by cells that bind Dnp, but not ribonuclease. The analogous phenomenon is observed when the two antigens are used in reverse order. While other reports suggest that there may be several different receptors in relatively undifferentiated cells from unimmunized mice, it seems likely that cells committed to antibody production carry a predominant multispecific cell surface immunoglobulin receptor.


Immunogenicity of liposomal model membranes in mice: dependence on phospholipid composition.

This investigation demonstrated that antigenic expression in liposomal model membranes may be markedly influenced by phospholipid composition. Incorporation of dinitrophenylaminocaproylphosphatidylethanolamine (Dnp-Cap-PE) into egg lecithin/cholesterol/dicetylphosphate bilayers did not significantly enhance the response of AKR mice to this synthetic amphipathic antigen. In contrast, the immunogenicity of Dnp-Cap-PE was increased (as measured by either plaque-forming cell frequency of hemagglutination titer) after its insertion into liposomes prepared with beef sphingomyelin instead of egg lecithin. We also show that the response to the Dnp-Cap determinant can be stimulated by the presence of lipid A in the same bilayers without altering the relative immunogenic potency of the sphingomyelin and lecithin liposomes; similarly, incorporation of this mitogen into sphingomyelin liposomes produced a greater polyclonal (nonspecific) response. The response to Dnp-Cap-PE-sensitized liposomes (with or without lipid A) prepared with a series of synthetic phosphatidylcholines (distearoyl-, dipalmitoyl-, dimyristoyl-, dilauroyl-, dioleoyl-) suggests a direct correlation between liposomal immunogenicity and transition temperature of the phospholipid.


Quantitative correlation of dopamine-dependent adenylate cyclase with responses to levodopa in various mice.

We obtained 12 groups of mice with widely different neurological responses to levodopa by selecting them from different strains and submitting some of them to pretreatments. We scored the symptoms evoked by a standardized dose of levodopa in one subgroup from each group. We tested another subgroup for activation of an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by a standardized dose of dopamine added to homogenates of the caudate nuclei of the brains of these mice. Both sets of tests were performed randomly. When the accruing two sets of data were plotted against each other there emerged a straight line which fitted the data with a coefficient of correlation of 0.97 (P less than 0.0001). The dopamine-dependent activity of the adenylate cyclase of the brain was thus shown to be a determinant of the neurological responses of intact animals to a dopaminergic drug.


Analysis of human Y-chromosome-specific reiterated DNA in chromosome variants.

A number of individuals with aberrant Y chromosomes have been tested for the presence of Y-chromosome-specific reiterated DNA. These studies locate Y-chromosome-specific reiterated sequences on the long arm of the Y chromosome. Correlation with phenotype and other known Y chromosome markers establish that the Y-chromosome-specific reiterated DNA discussed here has no evident role in male determination.


Ethylphenacemide and phenacemide: conformational similarities to diphenylhydantoin and stereochemical basis of anticonvulsant activity.

The three-dimensional molecular structures of the potent anticonvulsant agents phenacemide and ethylphenacemide have been determined by single-crystal x-ray diffraction. Although these compounds possess straight-chain acetylurea groupings, in the crystalline state both molecules adopt a pseudocyclic conformation of the acetylurea chain through amide...carbonyl oxygen intramolecular hydrogen bonding. This results in spatial positioning and relative orientation of two electron-donating functional groups in these molecules similar to those of comparable atoms in diphenylhydantoin and other heterocycle-containing anticonvulsants.


Facilitation at neuromuscular junctions: contribution to habituation and dishabituation of the Aplysia gill withdrawal reflex.

The gill withdrawal reflex of Aplysia has been used as a model for studying the neuronal mechanisms of habituation, a behavioral plasticity. We have assessed the contribution of neuromuscular facilitation, an elementary synaptic plasticity, during habituation of the reflex by recording gill muscle potentials, which we show are caused by excitatory junctional potentials. These potentials show systematic frequency-dependent changes in amplitude. The gill withdrawal evoked by central motor neuron firing during each habituation trial is determined by facilitation of the excitatory junctional potentials during the trial and the facilitated state of the initial excitatory junctional potential in a trial, determined by neuron activity prior to the trial. The neuromuscular junctions, therefore, act like a frequency-dependent amplifier of central motor activity. They are fully responsive to the dynamic changes of motor neuron firing that occurs during habituation and especially after dishabituation.


Effects of early experience upon orientation sensitivity and binocularity of neurons in visual cortex of cats.

The class of neurons within the visual cortex of normal adult cats that has the smallest receptive fields (less than or equal to 2.25 degrees2) and that responds only to low rates of stimulus motion (less than or equal to 50 degrees / sec) responds preferentially to lines oriented about either the horizontal axis (+/-22.5 degrees) or the vertical axis (+/-22.5 degrees). In animals reared without exposure to patterned visual stimulation, many of these cells display orientation preferences but are activated monocularly. In contrast, in normal animals, neurons that have larger receptive fields or that respond to higher rates of stimulus motion do not exhibit a similar bias in the distribution of their orientation preferences. Cells of this type, studied in animals reared without exposure to patterned visual stimuli, are activated binocularly but do not display orientation preferences.


Mutual entrainment of bilaterally distributed circadian pacemaker.

The interactions between the bilaterally distributed components of the circadin system that controls the locomotor activity rhythm of the cockroach Leucophaea maderae were investigated in a series of surgical lesion experiments. Complete excision of one optic lobe (either right or left) or its surgical isolation from the central nervous system had no effect on the animals' ability to free-run in constant darkness nor was there any indication, as judged by postoperative pi values of any difference between left and right lobe pacemakers. However, these surgical procedures consistently resulted in a significant increase in tau over preoperative value while optic nerve section had no effect on tau. The propostion is developed that the left and right pacemakers in the two optic lobes are mutally coupled and that the compound pacemaker's period is shorter than either of its constituent pacemakers. It was also found that the integrity of either compound eye is sufficient to assure entrainment of both left and right pacemakers.


Characterization of a delta-electroencephalogram (-sleep)-inducing peptide.

A peptide that induces slow-wave (delta) and spindles electroencephalogram enhancement after intraventricular (brain) infusion has been isolated from rabbits and given the name delta-sleep-inducing peptide (DSIP). Amino acid seqeunce: Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu. This compound, five possible metabolic products (containing residues  1--8, 2--9, 2--8, 1--4, and 5--9), two nonapeptide analogues with two amino acids exchanged, and a related tripeptide (Trp-Ser-Glu) were synthesized. All nine synthetic peptides were infused intraventricularly in rabbits under double-blind conditions. A total of 58 rabbits including controls were evaluated. The electroencephalogram leads from the neocortex and the archicortex were directly fast-Fourier transformed and analyzed by a Univac 1108 computer system. Only the delta-sleep-inducing peptide (snythetic) showed significant and specific enhancement/induction of delta and spindle electroencephalogram patterns.


Calcium modulation in brain extracellular microenvironment demonstrated with ion-selective micropipette.

Changes in extracellular Ca2+ concentration were directly measured in the rat cerebellum, using an ion-selective micropipette. Extracellular K+ was measured simultaneously with a second ion-selective micropipette. The potential reference barrels of the ion electrodes also provided fast field and slow potentials. During repetitive stimulation of the parallel fiber--Purkinje cell cerebellar circuit, extracellular Ca2+ fell to about 80% of base line concentration. During the spreading depression of Leão, extracellular Ca2+ fell to about 10% of base line; decreases of this magnitude also occurred during terminal anoxia. In all cases extracellular K+ increased substantially. These results show that extracellular Ca2+ is modulated during neuronal activity in the central nervous system and that under some conditions the Ca2+ change can be extreme. Given the well-established and antagonistic effects of reduce extracellular Ca2+ on axonal excitability and synaptic transmission, these results suggest that Ca2+ modulation in the brain cell microenvironment may be a significant parameter in the behavior of neuronal ensembles.


Heavy water as a tool for study of the forces that control length of period of the 24-hour clock of the hamster.

In alternating 12-hr periods of light and darkness, start of the dark period entrains the hamster's 24-hr clock. Blinding or constant darkness frees the clock of entrainment by allowing it to run faster or slower than 24 hr. Constant light frees the clock from entrainment and permits it to run slower than 24 hr--that is, lengthening its period. Heavy water given in drinking water linearly lengthens the period of the 24-hr clock of blinded hamsters or of hamsters kept in constant darkness in direct proportion to concentration of heavy water (1--50%). Heavy water (1-35%) has very different effects on length of the periods of the 24-hr clock when given under conditions of alternating 12-hr periods of light and darkness. Under these conditions, length of the period is controlled by three factors: (i) heavy water which slows the 24-hr clock; (ii) constant light which also slows the 24-hr clock; (iii) the counteracting effects of entrainment. It is thus possible to observe the effects of all three forces simultaneously in the same animal. The clock slowed by heavy water (1--20%) showed a strong tendency to return to a 24-hr entrainment whenever possible. On a 50% concentration of heavy water, the length of period of the clock became markedly lengthened but very constant and apparently independent of all external and internal disturbances.


Mechanism of tertiary structural change in hemoglobin.

A reaction path is presented by which the effects of oxygen binding in hemoglobin are transmitted from a heme group to the surface of its subunit. Starting from the known deoxy geometry, it is shown by calculations with empirical energy functions and comparisons with available data how the change in heme geometry on ligation introduces a perturbation that leads to the tertiary structural alterations essential for cooperatively. It is found that there is little strain on the unliganded heme; instead, the reduced oxygen affinity of hemoglobin results from the strain on the liganded subunit in a tetramer with the deoxy quarternary structure.


A nucleotide sequence from a ribonuclease III processing site in bacteriophage T7 RNA.

Transcription of that portion of the bacteriophage T7 genome encoding early functions yields RNA molecules about 7500 nucleotides long representing this entire early region. These long transcripts can be cleaved in vitro by highly purified Escherichia coli ribonuclease III (endoribonuclease III; EC 3.1.4.24), yielding five messenger RNAs identical to those produced in vivo. During this reaction, a small RNA fragment called F5 RNA is released, which is specified by the region of the T7 genome between genes 1.1 and 1.3. The following sequence of 32P-labeled F5 RNA has been determined using standard RNA sequencing techniques: pU-A-A-G-G-U-C-G-C-U-C-U-C-U-A-G-G-A-G-U-G-G-C-C-U-U-A-G-Uoh. The relative contributions of sequence and structure to ribonuclease III processing signals are considered in light of these findings.


Isolation and partial characterization of a cholesterol-requiring mutant of Chinese hamster ovary cells.

A sterol-requiring mutant has been isolated from mutagenized Chinese hamster ovary cells. This mutant grows normally only when cholesterol is present in the medium. Cell lysis occurs within 3 days in the absence of cholesterol. The frequency of reversion of this mutant to prototrophic growth is low (less than or equal to 10(-6). Whole cell pulse experiments with [14C]acetate or [3H]mevalonate indicate that the rate of synthesis of digitonin-precipitable material is greatly diminished in the mutant cells as compared to that in normal Chinese hamster ovary cells. Enzyme assays in vitro with crude cell extracts show that the biosynthetic conversion of mevalonate to squalene and the conversion of squalene to lanosterol are not impaired in the mutant cells. Gas-liquid chromatographic analyses of radioactive sterol composition after whole cell pulse experiments with [3H]squalene and with [3H]anosterol suggest that the fundamental enzymatic defect of the mutant is at the stage of lanosterol demethylation. When cells were grown in serum-free medium, lanosterol and dihydrolanosterol accumulated intracellularly in the mutant cells before cell lysis occurred; neither of these two intermediary sterols was detected in the wild-type cells grown under the same condition.


Structure of human serum lipoproteins inferred from compositional analysis.

Analysis of the correlations between size and chemical composition of lipoproteins of normolipidemic human plasma shows that the structure of all circulating lipoproteins is consistent with a spherical model of radius r in which a spherical liquid core of cholesterol esters and triglycerides of radius = r --20.2 A is surrounded by a monolayer of cholesterol and phospholipids with closely hydrophobic ends on the surface of the core. The average molecular areas at this inner surface are Spl = 68.5 A2/molecule for phospholipids and Sc= 39.1 A2/molecule for cholesterol. The proteins are closely packed with the hydrophilic head groups of phospholipids at the outer surface of the particle, with S' pl = 62.7 A2/molecule for phospholipids and Saa = 15.6 A2/amino acid for proteins. The polar head group of free cholesterol does not participate in the packing of the outer layer and thus must be masked by proteins. Free cholesterol is distributed among the circulating lipoproteins--with the exception of very high density lipoprotein and perhaps chylomicrons--according to a thermodynamic equilibrium governed by the curvature of the surface of the particle.


Embryonic chicken brain and mouse neuroblastoma cells N1E-115 and N-18 contain an inhibitor of acid deoxyribonuclease.

Activity of a trypsin-sensitive acid deoxyribonuclease (DNase) inhibitor has been detected in 13- to 21-day-old embryonic chicken brains and in clonal lines of neuroblastoma cells (adrenergic, N1E-115, and neurotransmitter-inactive, N-18) grown for 48 hr after subculture. The activities of purified porcine and bovine spleen DNases were inhibited 60--75% in the presence of the inhibitor, whereas less than 10% inhibition was observed with purified pancreatic DNase. Activities of an acidic DNase and its inhibitor reached maxima in 21-day-old embryonic chicken brain. The proteins were separated by isoelectric focusing and affinity column chromatographic techniques. The pI values of the acid DNase and its inhibitor were 5.4 and 4.2, respectively.


Chromatium hydrogenase.

Previous reports of a relatively air-stable soluble hydrogenase from the photosynthetic anaerobe, Chromatium vinosum, have been substantiated. The properties of this enzyme, as seen in highly purified samples prepared by procedures that permit improved yields, are described. A possible role for flavin mediation is noted.


Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24.

Chromosomal protein A24 has a unique structure inasmuch as it contains histone 2A and a nonhistone polypeptide the sequence of which has been partially determined. Comparative analysis of the ninhydrin-insensitive amino-terminal tryptic peptides of protein A24 and histone 2A and a quantitative analysis of their carboxyl-terminal amino acid indicated that protein A24 has two amino termini and one carboxyl terminus. The amino acid sequence analysis of tryptic peptide 17' of protein A24: (see text) showed it contains tryptic peptide 17 of histone 2A, Lys-Thr-Glu-Ser-His-His-Lys. Lysine 119, the amino terminus of this peptide, which is derived from the histone 2A portion of protein A24, is linked by an isopeptide bond to the carboxyl group of a glycine residue. Accordingly, the branched structure of protein A24 proposed is: (see text).


Molecular weight and valence of the cell-surface receptor for immunoglobulin E.

The molecular weight of the active solubilized cell-surface receptor for immunoglobulin E (IgE) was measured in nonionic detergent. The diffusion coefficient was estimated by gel filtration, the partial specific volume was estimated from the differential sedimentation in sucrose gradients prepared from H2O and D2O, and the sedimentation constant was estimated from the same centrifugation experiments. The receptor has an apparent molecular weight of 130,000. Its high partial specific volume (0.81 cm3/g) suggests that bound detergent contributes significantly to the mass. The molecular weights of the receptor-IgE complex and of unbound IgE determined similarly were 310,000 and 200,000 respectively, clearly showing that the receptor is univalent. The implications of these results for the subunit structure of the receptor, receptor-membrane integration, and a possible mechanism of receptor triggering are discussed.


Specific gonadotropin binding to Pseudomonas maltophilia.

Binding of 125I-labeled human chorionic gonadotropin to Pseudomonas maltophilia is dependent on time, temperature, and pH and the binding to this procaryotic species is hormone-specific and saturable. The equilibrium dissociation constant is 2.3 X 10(-9) M. There are no cooperative interactions between binding sites (Hill coefficient, 1.05). The number of sites is estimaated as 240 fmol/100 mug of protein. NaCl and KCl, at concentrations from 1 to 10 mM, have no effect on binding. Divalent cations (Mg2+ and Ca2+) and 1 mM EDTA inhibit hormone binding. Binding is destroyed by heat or by treatment with Pronase of alpha-chymotrypsin and is increased by phospholipase C. Binding of the labeled gonadotropin is not observed with other gram-negative organisms--e.g., Escherichia coli, Pseudomonas testosteroni, Pseudomonas aeruginosa, Enterobacter aerogenes, or Enterobacter cloacae.


Triple-helix formation on ribosome-bound nascent chains of procollagen: deuterium-hydrogen exchange studies.

Polyribosomes containing nascent [3H]proline-labeled collagen chains were isolated from chick embryo fibroblasts in culture. These nascent chains were nearly completely hydroxylated, as indicated by the presence of [3H]hydroxyproline and high hydroxyproline/proline ratios. The polyribosomes were suspended in D2O at 15 degrees and the infrared spectrum was determined using a reference cell containing collagen-depleted polyribosomes in D2O, matched to equal RNA content. The amide I and amide II bands were observed. When the polyribosomes were heated in D2O at 44 degrees in the infrared cells, the N--D amide II absorbance at 1480 cm-1 increased markedly, indicating that H leads to D exchange had occurred. Collagen-depleted polyribosomes showed no such changes in absorbance at 1450-1480 cm-1 upon heating. Polyribosomes recovered from the infrared cells after treatment at 44 degrees and cooling still contained collagen, as indicated by their [3H]hydroxyproline content. These data indicate that nascent collagen bound to the polyribosomes can assume a hydrogen-bonded structure. Taken with prior data showing that the nascent collagen was also resistant to pepsin digestion, it is suggested that the collagen examined is in triple-helix conformation. Because the nascent polyribosome-bound collagen is nearly fully hydroxylated, it must be considered that triple-helix formation can occur between nascent chains while they are attached to the endoplasmic reticulum surface and that chain association and triple-helix formation in vivo may well occur before rather than after release.


Assembly of biologically active proheads of bacteriophage lambda in vitro.

Bacteriophage lambda DNA can be packaged in vitro into preformed proheads to generate plaque-forming units. This complex set of reactions is initiated when lambda DNA is mixed with the product of the phage A gene, and proheads. Because proheads are an essential early reactant, the system has potential as an assay for the formation of biologically active proheads. When extracts of cells infected with certain lambda head mutants (for example, B--, C--, Nu3--, and E--) are used as the prohead donor, plaque-forming units are not produced. However, when extracts of E- - and Nu3- - infected cells are first reacted together the combination provides prohead-donor activity to the in vitro packaging system. In vitro assembled, biologically active proheads have the same sedimentation properties and electron micrsocopic appearance as "wild-type" proheads isolated from lambdaA-D- -infected cells. Centrifugation analysis shows that the Nu3- extract contributes gpE, the major capsid protein, to the reaction in the form of monomers or small polymers.


Tryptic peptide analysis of normal and mutant forms of hypoxanthine phosphoribosyltransferase from HeLa cells.

Hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) can be purified 5-to 10,000-fold from extracts of HeLa (human) cells by a three-step procedure consisting of high-speed centrifugation, adsorption to Sepharose-conjugated HPRT antibody, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Purified enzyme labeled in vivo with radioactive lysine, arginine, or methionine was digested with trypsin and the tryptic peptides were separated by column chromatography on Bio-Rad cation exchanger Aminex A-5. Less than 50 ng (2 pmol) of HPRT is required to produce a tryptic peptide pattern. A methionine-labeled peptide was identified as the COOH-terminus because it was not labeled with either lysine or arginine. We have compared the tryptic peptide patterns of normal HeLaHPRT and a crossreacting HPRT protein lacking enzyme activity from HeLa mutant H23 [Milman et al. (1976) Proc. Natl. Acad. Sci. USA 73, 4589--4593]. The mutant protein has a new lysine-labeled peptide, but the chromatography patterns of arginine- or methionine-labeled peptides appear identical to those of the normal protein. The appearance in the H23 mutant HPRT protein of a new tryptic peptide provides strong evidence for a mutation in the HPRT structural gene. The tryptic peptide patterns were used to determine the total number of residues of labeled amino acid in the protein, and the values are reasonably consistent with those determined by conventional amino acid analysis pf erythrocyte HPRT.


Regulation of cholesterol biosynthesis by normal and leukemic (L2C) guinea pig lymphocytes.

The cholesterol production of guinea pig leukemic (L2C) lymphocytes preceeds at greater than 30 times the rate found in normal cells. Fatty acid biosynthesis is also enhanced in L2C cells. Exposure of L2C cells to cholesterol/lecithin liposomes does not depress their sterol biosynthesis, in contrast to the behavior of normal lymphocytes [Philippot, J.R., Cooper, A.G. & Wallach, D. F. H. (1975) Biochim. Biophys. Acta 406, 161-166]. However, 25-hydroxycholesterol, an inhibitor of hydroxymethylglutaryl-CoA reductase (NADPH) [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], the rate limiting enzyme in cholesterogenesis, and 25-hydroxycholecalciferol, a biologically potent form of vitamin D3, block sterol biosynthesis of both normal and L2C lymphocytes [Philippot, j.r., cooper, A.G. & Wallach, D.F.H. (1976) Biochem. Biophys. Res. Commun. 72, 1035-1041]. Moreover, both cell types exchange cholesterol equivalently with cholesterol/lecithin liposomes. The only difference in sterol biosynthesis observed between the two cell types is in the temperature response of the enzyme. Arrhenius plots of this enzyme activity exhibit a prominent discontinuity at about 24 degrees in the case of normal cells, but none in the case of L2C. The activation energies for L2C cells and normal cells, above the normal cell transition temperature, were not significantly different. All of the data suggest that the regulatory defect in L2C lymphocytes arises from a deficiency in these cells' internal membranes.