Characteristics of Streptococcus milleri and Streptococcus mitior from infected dental root canals. Morphological and physiological characteristics of 103 streptococcal isolates obtained from positive routine cultures of material from dental root canals at filling, were studied. The majority of the isolates were those which at a primary identification not fullfilled the criteria of enterococci, Strep. sanguis, Strep. mutans or Strep. salivarius. Only a few representative strains of the latter types were included in the study as a control of the method. The resemblance of the 103 isolates to 38 reference strains were assessed with numerical methods. The isolates formed 9 clusters with reference strains occurring in all but one. Of the isolates which it was intended to identify and characterize with numerical taxonomic analysis, all but one joined one of 3 clusters and were identified as Strep. milleri or Strep. mitior (mitis). The most useful cultural characteristics to dfferentiate the two species were growth on the sulphonamide containing MC-agar and 7.5% bile blood agar by Strep. milleri and production of hydrogen peroxide by Strep. mitior. The characteristics of the strains in the nine 80-phenons are given in Table 2. Other characteristics that appeared to be of interest for differentiating Strep. milleri from Strep. mitior and these two species from other viridans streptococci are underlined in Table 2. Strep. milleri seems not earlier to have been reported to occur in infected root canals. The occurrence of Strep. milleri is in agreement with the data given on serological groups found among streptococcal isolates from routine root canal cultures. Little is known about the occurrence and distribution of Strep. milleri in the human oral cavity, which therefore warrant further investigations.


Cytogenetic and histochemical analysis of experimental oral carcinogenesis in the rat. The present investigation has shown that in chemical carcinogenesis progression of malignant epithelial changes in associated with an increased number of cells with chromosomal deviations. Each out of two oncogenic agents with different chemical characteristics had its own non-random pattern. There was usually a close relationship between such chromosomal changes and the simultaneous presence of morphological atypia (dysplasia) and metabolic atypia (aberrant G-6-P.DH activity) from moderate dysplasia and onwards during carcinogenesis. Changes in X-chromosomes, which i.a. in human code the synthesis of G-6-P.DH molecules, could perhaps explain and be reflected in deviating metabolic reactions in premalignant cells.


Metastatic melanoma of the maxilla presenting as a gingival swelling. Malignant melanoma metastatic to the gingiva has been reported only once. We present a case in which the occurrence of melanoma in the gingiva followed extraction of a periapically "abscessed" tooth. Since the initial periapical mass may well have been a metastatic tumor, particularly in a patient undergoing therapy for disseminated malignant disease, the need for biopsy of such lesions is emphasized.


The effects of thermal and electrical injury on pulpal histamine levels. The present investigation was undertaken to determine possible quantitative changes in porcine pulpal histamine levels following thermal and electrical insult. Quantitative spectrophotofluorometric assays revealed a fourfold increase in pulpal histamine levels within 30 minutes of thermal injury. These findings suggest that histamine may play a role in the initial stages of pulpal inflammation. Electrical insult was found to have no significant effect on pulpal histamine contents 30 minutes after insult. The possible implications of these findings are discussed.


Change in working length following instrumentation of molar canals. A study was undertaken to determine the reduction in working length following instrumentation of molar canals. A sample of forty maxillary and forty mandibular extracted teeth was used. The canals were measured before and after instrumentation. The greatest changes were observed in the mesiobuccal canal of the maxillary molars, which showed a mean reduction of 0.35 mm. with a range of 0.8 mm. The smallest changes were observed in the palatal canal of the maxillary molars and the distal canal of the mandibular molars. These canals showed a mean change of less than 0.1 mm. None of the changes were statistically significant.


Radiographic interpretation of so-called developmental defect of mandible. Ten cases of so-called development defect were detected on panoramic radiograms. One of them was bilocular, two were round, three were ovoid, and four were half-ovoid. Eight were in men and two were in women. These cystlike lesions are sometimes misdiagnosed as epithelium-lined cysts or benign tumors, such as ameloblastoma. For the detection of this kind of defect, panoramic radiography is available, and the lesion is differentiated from many cysts and tumors in the jaws by posteroanterior projection and occlusal films. From the published literature, some tendencies of age distribution and sex incidence were reviewed.


Warthin's tumor. Report of a case and survey of 205 salivary neoplasms. A case of Warthin's tumor with an unusual sialographic appearance is reported. The extensive pooling of the contrast media within the tumor was probably the result of communication of the cystic spaces of the tumor with the ductal system of the parotid gland. This case stimulated a re-examination of the incidence of this tumor, with a study of 205 major salivary gland tumors from pathology reports in the files of Hartford Hospital which is affiliated with the University of Connecticut Health Center in Farmington, Connecticut.


The retrognathic mandible--surgical correction. The preceding case reports demonstrate the excellent results available to the patient with a retrognathic mandible. Etiology, adverse effects, and the modalities used in evaluation of the underdeveloped mandible have been discussed. The age at which surgical intervention is undertaken needs additional input by clinicians. Several surgical procedures have been and are employed to advance the mandible. The C osteotomy is recommended since the final result has been excellent and consistent. The surgeon has constant surgical control and vision. The advanced portion of the mandible can be firmly held by intraosseous wires. There is minimak disturbance of muscle position and the inferior alveolar nerve. We have not encountered unusual loss of blood, immediate postoperative problems with the airway, infection, or unfavorable relapse. A postoperative regimen that has been found rewarding is discussed. We have come to the conclusion that most patients can tolerate lengthy surgical procedures with few adverse side effects. Our colleagues in anesthesia and nursing provide superb operative and postoperative care. When the preoperative work-up indicates that more than one procedure is indicated, we recommend that the total surgical treatment plan be carried through at one operation, although occasionally this approach may be contraindicated. We should strive for perfection and can fall short of this goal when a patient will not return for additional surgical procedures which could have been accomplished during the first operation. Although good results have been experienced with both the C osteotomy and the sagittal split osteotomy, we believe that the C osteotomy is a superior procedure. However, this does not imply that the sagittal split osteotomy should not be a part of the armamentarium of the oral surgeon. In conclusion, the C osteotomy, the sagittal split osteotomy, genioplasty, and additional procedures--such as a mandibular alveolar osteotomy to intrude supraerupted teeth--when indicated, can provide very favorable results in cases of retrognathia or micrognathia of the mandible.


Potential of elastin and collagen as initiators of in vivo calcification. The osseous repair response of the guinea pig to purified bovine elastin from ligamentum nuchae and decalcified rat femur collagen was studied by implantation of these materials into an extraction socket. A nylon mesh tube was used to carry the respective implant materials to place, and in one group of animals only the nylon tube was implanted, thereby serving as a control for the study. Neither the collagen or elastin matrix appeared to elicit an immune rejection response from the host animal. Histologic and quantitative results indicated that collagen implants accelerated the osseous repair of the extraction socket. Elastin implants, which characteristically resulted in ossicle formation, did not appear to accelerate healing, but the results were quantitatively similar to those in the experimental control animals.


Nomalike lesion in a patient with chronic lymphocytic leukemia. Review of the literature and report of a case. A case in which a nomalike lesion occurred in a patient being treated for chronic lymphocytic leukemia has been reported. Differential diagnosis of noma from other oral gangrenous processes has been described. The pathophysiology of systemic disease and blood dyscrasias contributing to the development of the lesion has been discussed with reference to management and treatment. A review of the literature is included.


A clinical study of cysts arising from mucosa of the maxillary sinus. Panorex radiographs taken during the past 5 years at the University of Kentucky College of Dentistry were reviewed for evidence of mucosal cysts of the maxillary sinus. Thirteen patients with this lesion were recalled for re-evaluation of their status. Ten of the patients had symptoms that could be related to the involved sinus. These included stuffiness, fullness, postnasal drip, gushing of yellow fluid from the nose, and headache. Radiographic examination revealed that three of the cysts had increased is size, three had decreased in size, three had not changed in size, and two had disappeared; no evaluation could be made on two. On the basis of radiographic, transillumination, history, and clinical findings, the nine cysts that could be evaluated were diagnosed as being of the nonsecretory type of mucosal cyst. We recommended periodic radiographic examination for this type of lesion. Surgical intervention is necessary only if destruction of surrounding bone has occurred or recurrence of disturbing symptoms is reported.


Florid osseous dysplasia. A clinical-pathologic study of thirty-four cases. Thirty-four patients with florid osseous dysplasia were studied. The majority were asymptomatic Negro women: Seventeen biopsy-proven simple bone cysts were found in affected quadrants of fourteen patients. Radiographs displayed a spectrum of sclerotic and ground-glass opacities limited to alveolar processes but not to root apices. Biopsy material was studied in all cases, and biochemical analyses of serum and cyst fluid were performed on some patients. Test results and skeletal radiographs indicate that the disease is limited to the jaws. Patients have remained asymptomatic with little alteration of radiographic patterns. Three cysts failed to heal following treatment, others filled with radiographically abnormal tissue. Chronic osteomyelitis may infrequently complicate the disease. These cases appear to represent the most exuberant manifestation of this reactive fibro-osseous jaw disease.


The effect of radiation-induced xerostomia on saliva and serum lysozyme and immunoglobulin levels. Saliva and serum lysozyme, immunoglobulin, albumin, and total protein levels were monitored in thirty patients with cancer of the head or neck before, during, and after radiotherapy and compared with those of a group of non-irradiated noncancer control subjects. The mean volume-based saliva lysozyme and total protein concentrations were significantly higher in the cancer patients before radiotherapy than in the control group. During radiotherapy, the mean volume-based concentrations of all protein components assayed increased as the saliva flow rate decreased. Protein-based ratios of saliva albumin, IgG, and lysozyme and the ratio of IgG/IgA increased as the xerostomia intensified. Ratios of saliva total protein and IgA to flow rate paralleled the flow rate decrease. Such increased concentrations in saliva immunoproteins were offset, however, by a greater than 93 per cent reduction in total saliva output. This reduced saliva output, therefore, resulted in an immunoprotein deficit. There were no significant differences between the mean serum lysozyme levels of the cancer and control groups at any point of comparison. The mean serum immunoglobulin concentrations in the cancer patients before radiotherapy were significantly higher than those in the control group. During radiotherapy, there was a decrease in the mean serum total protein, albumin, and immunoglobulin levels which reverted toward the pretreatment values during the postirradiation period.


Bilateral eosinophilic granuloma of the mandible. A case of extensive bilateral eosinophilic granuloma of the mandible has been reported. The pertinent clinical features have been emphasized and difficulties in diagnosis have been discussed. The case highlights the problem that faces the surgeon and pathologist when there is a superimposed chronic osteomyelitis. If the lesion had been limited to the left side of the mandible and if the possibility of eosinophilic granuloma had not been raised on histologic grounds at the time of original exploration, then a correct diagnosis might not have been reached.


Estimates of stratospheric pollution by an analytic model. With suitable choices of the height profile of eddy diffusion coefficient, the vertical flow of an inert tracer is given by an analytic solution. Odd nitrogen, or NOX, from aircraft exhausts can be regarded as such a tracer, and the amount in the stratosphere resulting from a source of a given strength can be immediately calculated. The resulting destruction of ozone is then estimated with the help of a formula obtained from earlier work.


Inhibition of citrate synthase by oleoyl-CoA: a regulatory phenomenon. Fatty acyl-CoAs are good detergents (dritical micelle concentrations = 3-4 muM) and can inhibit a number of enzymes, including some involved in fatty acid biosynthesis. The regulatory significance of fatty acyl-CoAs as negative effectors has been questioned largely because of the difficulties in distinguishing possible nonspecific detergent effects from more specific regulatory interactions with these enzymes. A new analogue of oleoyl-CoA, oleoyl-(1, N6-etheno)-CoA, is a better detergent (critical micelle concentration = 3.2 muM) than oleoyl-CoA (critical micelle concentration = 4.7 muM). This new analogue is not as good (by an order of magnitude) an inhibitor of citrate synthase [citrate oxaloacetatelyase (pro-3S-CH2-COO-vectoracetyl-CoA); EC 4.1.3.7] nor is it bound as well oleoyl-CoA. Since the only difference between these two compounds is substitution of 1,N6-ethenoadenine for the adenine of CoA, the difference in inhibition and binding implies a specific interaction between the adenine moiety of oleoyl-CoA and citrate synthase. Moreover, since oleoyl-(1,N6-etheno)CoA is a better detergent than oleoyl-CoA, the detergency of oleoyl-CoA is not the sole cause of the fatty acyl-CoA inhibition of citrate synthase. These results support a physiological role for oleoyl-CoA as a negative effector for citrate synthase. An analogous physiological role for fatty acyl-CoA as negative effectors for other enzymes seems reasonable.


Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes. The visible fluorescence of terbium(III) when bound to a calcium binding site of thermolysin is greatly enhanced with an excitation maximum at 280 nm but substitution of cobalt(II) for zinc at the active site decreases the intensity by 89.5%. Treatment with N-bromosuccinimide quenches enzyme tryptophan and Tb(III) fluorescence to a similar extent and suggests the operation of tryptophan vector Tb(III) vector Co(II) energy relay system in the enzyme. Dipoledipole radiationless energy transfer between the Tb(III) donor and the Co(II) acceptor can account for this quenching. The inherent characteristics of the metal pair limits the value of the orientation factor, K2, of the Förster equation, thereby reducing uncertainties in distance measurements by energy transfer compared with other systems. A quantum yield of 0.51 yields a value of R0, the distance for 50% energy transfer, of 19.6 A, and a distance, R, between Tb(III) and Co(II) of 13.7 A, a value identical to that measured for the distance between the active site zinc atom and calcium atom number 1 by x-ray analysis in native thermolysin crystals. The limits of confidence of this measurement are discussed. Energy transfer between two different metal atom sites of a protein provides a new class of probes to measure intramolecular distances of biological macromolecules in solution.


Isolation and characterization of two acidic proteins of 60s ribosomes from Artemia salina cysts. 60S ribosomes from encysted gastrulae of the brine shrimp Artemia salina contain two acidic proteins, which are homologous to the Escherichia coli proteins L7 and L12. The proteins were purified and characterized with respect to molecular weight, amino-acid composition, peptide maps, and their functional requirement in the elongation factor dependent binding of aminoacyl transfer RNA to the ribosome.


Lambda repressor turns off transcription of its own gene. We report transcription in vitro of the lambda repressor gene, cI, using specific restriction endonuclease fragments as templates. This transcription is repressed by lambda repressor. Moreover, we report the sequence change caused by a cI promoter mutation. This change is located between two repressor binding sites in the rightward operator (OR). Transcription studies using mutant templates indicate that repressor bound to two sites in OR regulates transcription of gene tof, and repressor bound to the remaining site(s) controls transcription of cI.


Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template. The protein products of T4 bacteriophage genes 41, 43, 45, 44, and 62 have been purified to near homogeneity using an assay which measures their stimulation of DNA synthesis in a crude lysate of Escherichia coli cells in fected by an appropriate mutant phage. When all of these proteins and T4 gene 32 protein are incubated in the presence of deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single and double-stranded DNA templates. Analysis of this in vitro system reveals most of the features attributed to in vivo DNA replication: (1) De novo DNA chain initiation is found on a single-stranded DNA template only if ribonucleoside triphosphates are present (as expected for RNA priming of Okazaki pieces on the "lagging" strand of a replication fork). (2) With single-stranded circular DNA as template, synthesis continues for many doublings. The products after extensive synthesis resemble a rolling circle as visualized in the electron microscope, with discontinuous "lagging" strand synthesis generating a long, unbranched double-stranded tail. The fact that all six mutationally identified T4 replication gene products are required for these syntheses suggests the existence of a large multienzyme complex, constituting the T4 replication apparatus.


X-ray fiber diffraction evidence for neighbor exclusion binding of a platinum metallointercalation reagent to DNA. Good quality x-ray diffraction patterns have been obtained of polycrystalline fibers containing 2-hydroxyethanethiolato(2,2'2"-terpyridine)platinum(II) bound to calf thymus DNA by intercalation. The photographs strongly support the neighbor exclusion binding model in which electron-dense platinum atoms are regularly distributed at 10.2 A intervals, every other interbase pair site.


Statistical theory of chromatography: new outlooks for affinity chromatography. We have developed further the statistical approach to chromatography initiated by Giddings and Eyring, and applied it to affinity chromatography. By means of a convenient expression of moments the convergence towards the Laplace-Gauss distribution has been established. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. A simple procedure is deduced for expressing the fundamental constants of the model in terms of purely experimental quantities. Thus, affinity chromatography can be used to determine rate constants of association and dissociation in a range considered as the domain of the stopped-flow methods.


NH---S hydrogen bonds in Peptococcus aerogenes ferredoxin, Clostridium pasteurianum rubredoxin, and Chromatium high potential iron protein. Results from refinement of the crystal structures of P. aerogenes ferredoxin and C. pasteurianum rubredoxin determined by x-ray diffraction show that there are 15-18 NH---S bonds in the former and six in the latter with lengths in the range 3.1-3.9 A. Earlier tritium exchange experiments are consistent with the presence of these hydrogen bonds in the ferredoxin structure and show that more peptide hydrogen atoms are available for exchange in apoferredoxin than in intact ferredoxin. Four types of NH---S bonds are observed and two of these are geometrically similar to the two types of 3(10) NH---O bonds. The existence of more NH---S bonds in ferredoxin than in high potential iron protein suggests why the -2 form of the Fe4S4 cluster is preferred in ferredoxin over the -1 form found in high potential iron protein. From comparison of Cys-X-Y-Cys sequences in rubredoxin, ferredoxin, and high potential iron protein we suggest that two Cys-X-Y-Cys-Z sequences, where Z may have conformation angles similar to glycine, are required to make a one-iron cluster, no more than one Cys-X-Y-Cys-Z-Gly sequence is required to form a Fe2S2 ferredoxin, and a Cys-X-Y-Cys-Gly sequence where Y has a conformation such that the cysteines bond to different iron atoms is necessary to form the tetrameric cluster.


Spike-forming model of the neural membrane: computer simulation of some perfused axon experiments. This paper is an attempt to account for the action potential spikes observed when the normal axoplasm of a squid giant neuron is replaced by a postassium-free, sodium-rich fluid, and the axon is immersed in a potassiumand sodium-free, calcium-rich bath. For this purpose a recently described model of the neural membrane [Wooldridge, D. E. (1975) Proc. Nat. Acad. Sci. USA 72, 3468-3471] is extended. Allowances are made for the flow of Ca++, Cl-, and Na+ ions through some of the membrane pore configurations, as well as for some electron conductance, all of amounts too small to be significant in the normal neuron. It is also postulated that the nature of the perfusing fluid affects the rates of some of the reactions that change the convertible gates from one configuration to another, as well as the ionic permeabilities of the resulting configurations. The result of the modifications is a single membrane model that accounts for the 3-sec, 15 muA/cm2 action potential spikes of the perfused axon as well as for the 0.5-msec, 1 mA/cm2 spikes of the normal neuron.


Structural requirements for time-dependent inhibition of prostaglandin biosynthesis by anti-inflammatory drugs. Several anti-inflammatory drugs have been examined for their ability to cause a time-dependent destruction of the fatty acid oxygenase that produces prostaglandins. All of the time-dependent inhibitors contained carboxylic acid moieties, and in addition all but one of the drugs contained a halogen atom. Structural analogs (of the time-dependent inhibitors) lacking halogen atoms were unable to cause a time-dependent destruction of the enzyme. The time-dependent property of an inhibitor was totally eliminated after methylation of the carboxylate group. Methylation did not, however, alter the ability of the inhibitors to competitively inhibit the oxygenase. Thus, the reversible binding of the agents at the active site was not appreciably dependent upon the free carboxyl group, whereas the subsequent irreversible process was.


Cytoplasmic and nuclear binding components for 1alpha25-dihydroxyvitamin D3 in chick parathyroid glands. Specific binding of 1 alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] to macromolecular components in the cytoplasm and nucleus is demonstrated in parathyroid glands of vitamin-D-deficient chicks. The interaction of 1alpha,25-(OH)2D3 with the cytoplasmic binding component is of high affinity (Kd = 3.2 X 10(-10) M) and high specificity [1alpha,25-(OH)2D3 greater than 25-hydroxyvitamin D3 greater than 1alpha-hydroxyvitamin D3 greater than vitamin D3 in competing with radioactive 1alpha,25-(OH)2D3]. Both cytoplasmic and nuclear hormone-macromolecular complexes sediment at 3.1 S in 0.3 M KC1-sucrose gradients, and agarose gel filtration of the components indicates an apparent molecular weight of 58,000. The 3.1S binding molecules are not observed in adrenal gland, testes, liver, or kidney, but similar receptors for 1alpha,25-(OH)2D3 have been found previously in intestine. Macromolecular species with a high affinity and preference for 25-hydroxyvitamin D3 [25-(OH)D3] are also identified in parathyroid cytosol and differ from the parathyroid 1alpha,25-(OH)2D3-binding component in that: (1) they sediment at 6 S in 0.3 M KC1-sucrose gradients, (2) they are observed in all tissues examined, (3) they have a higher affinity for 25-(OH)D3 than 1alpha,25-(OH)2d3, and (4) they are not found in the nucleus of the parathyroid glands, in vitro. The discovery of unique 1alpha,25-(OH)2D3-binding components in the parathyroid glands is consistent with the sterol hormone's action at this endocrin site and possible involvement in the regulation of parathyroid hormone synthesis and secretion.


Interferon activity produced by translation of human interferon messenger RNA in cell-free ribosomal systems and in Xenopus oöcytes. Translation of messenger RNA isolated from poly(rI)-poly(rC)-induced human fibroblasts in cell-free ribosomal systems and in Xenopus oocytes resulted in the production of biologically active proteins that had the properties of human fibroblast interferon. The translation in the oocytes was much more efficient, giving approximately 500 times higher titers of interferon activity than the cell-free systems. A control messenger RNA isolated from noninduced human fibroblasts, did not code for interferon synthesis in these systems. Both messenger RNA preparations stimulated [3H]amino-acid incorporation into trichloroacetic acid-insoluble material. The radioactive products and their immunoprecipitates were electrophoresed on polyacrylamide gels under denaturing conditions. The products resulting from the translation of the control (uninduced) messenger RNA in oocytes contained a major protein of approximately 45,000 molecular weight. The messenger RNA isolated from poly(rI)-poly(rC)-induced cells stimulated the synthesis of an additional 25,000 molecular weight protein that electrophoresed in the same position as human fibroblast interferon. These results suggest that human fibroblast interferon was synthesized by the translation of its messenger RNA in Xenopus oocytes and in cell-free ribosomal systems.


Search for nucleic acid sequences complementary to a murine oncornaviral genome in poly(A)-rich RNA of human leukemic cells. The presence of viral-like sequences in the RNA of various types of leukemic cells was investigated by hybridizing cellular poly(A)-containing RNA with cDNA synthesized in an endogenous system of purified Moloney murine sarcoma virus [M-MSV-(MLV)]. Poly(A)-RNA-cDNA hybrids were detected by assaying their resistance to S1 nuclease. Hybrids were found in 22 out of the 46 leukemias that were tested. None of the controls, including material obtained from buffy coats, bone marrow cells, and a continuous human cell line, was positive. Positive cases were found in all the different categories of leukemias with the exception of chronic myelogenous leukemias. There was no definite corelation between the category of leukemia and positivity. A few cases contained a very high proportion of poly(A)-RNA-cDNA hybrid.


Procollagen: biological scission of amino and carboxyl extension peptides. Procollagen, the triple-stranded precursor of chick embryo skull bone collagen, contains two pro alpha1 and one pro alpha2 chains. We find that each of these is a collagen chain with both an NH2-terminal and a COOH-terminal extension peptide. The NH2-peptide of pro alpha1 contains cysteine and differs from the NH2-peptide of pro alpha2. The three NH2-peptides are cut off, giving a disulfide-linked intermediate, named altered procollagen; then the disulfide-linked COOH-peptides, which contain cysteine and tryptophan, are cut off, leaving collagen. Procollagen, altered procollagen, and COOH-peptide were isolated. Collagenase digestion of procollagen gave both NH2- and COOH-peptides, while altered procollagen gave only COOH-peptides. The following results of sequential, in vitro labeling at 37 degrees and of specific cleavage of procollagen proved the structure: [(NH2-peptide)-collagen-(COOH-peptide)]3 with interstrand S-S links between only the COOH-peptides. (i) The COOH-peptides of pro alpha chains were labeled with [3H]proline before the remainders of the chains; (ii) [35S]cysteine appeared in the COOH-peptides of completed covalent molecules 5 min earlier than in the NH2-peptides; (iii) tadpole tail collagenase, which cuts native collagen into triple-stranded 3/4 pieces containing the NH2 termini and 1/4 pieces containing the COOH ends, cuts procollagen into 3/4 pieces with NH2-peptides attached and 1/4 pieces attached to the disulfide-linked COOH-peptides. The COOH-peptides of pro alpha 1 and pro alpha2 were labeled in a 2:1 ratio at 4 min, indicating simultaneous translation of pro alpha1 and pro alpha2.


Infectious viral DNA of murine leukemia virus. A fraction of the unintegrated viral DNA that appears early after infection of mouse cells by Moloney leukemia virus is infectious. The infectivity could be deomonstrated by an XC plaque assay of the cells exposed to DNA co-precipitated with calcium phosphate. The number of plaques deriving from closed-circular, supercoiled DNA was proportional to the concentration of added DNA, indicating that a single DNA molecule of about 5.5 X 10(6) daltons carries all the viral information. Nonsupercoiled viral DNA is also infectious; these molecules appear to be largely double-stranded and 5 to 6 X 10(6) dalthons in mass.


Effect of rotation on the diffusion-controlled rate of ligand-protein association. The rate of binding a fairly large ligand molecule to a protein is reduced below the usual diffusion-controlled rate by the requirement of a certain rotational orientation. A simple, approximate treatment of this effect is given for special cases of spherical and ellipsoidal ligands. As the center of an ellipsoidal ligand approaches a protein surface, there is an effective repulsive potential between ligand and surface owning to restricted rotation of the ligand. The frequency factor kT/h of the Eyring rate theory is replaced in these reactions involving diffusion in solution by D/Rlambda, where D = diffusion coefficient of ligand, lambda = thermal deBroglie wavelength of ligand, and R = "capture" distance around the binding site on the protein.


Analogue solution for electrical capacity of membrane covered square cylinders in square array at high concentration. Analytical solutions of Laplace equations have given the electrical characteristics of membranes and interiors of spherical, ellipsoidal, and cylindrical cells in suspensions and tissues from impedance measurements, but the underlying assumptions may be invalid above 50% volume concentrations. However, resistance measurements on several nonconducting, close-packing forms in two and three dimensions closely predicted volume concentrations up to 100% by equations derived from Maxwell and Rayleigh. Calculations of membrane capacities of cells in suspensions and tissues from extensions of theory, as developed by Fricke and by Cole, have been useful but of unknown validity at high concentrations. A resistor analogue has been used to solve the finite difference approximation to the Laplace equation for the resistance and capacity of a square array of square cylindrical cells with surface capacity. An 11 x 11 array of resistors, simulating a quarter of the unit structure, was separated into intra- and extra-cellular regions by rows of capacitors corresponding to surface membrane areas from 3 x 3 to 11 x 11 or 7.5% to 100%. The extended Rayleigh equation predicted the cell concentrations and membrane capacities to within a few percent from boundary resistance and capacity measurements at low frequencies. This single example suggests that analytical solutions for other, similar two- and three-dimensional problems may be approximated up to near 100% concentrations and that there may be analytical justifications for such analogue solutions of Laplace equations.


Three-dimensional structure of herring sperm protamine Y-I with the aid of dark field electron microscopy. High resolution electron micrographs of herring sperm protamine (clupeine) Y-I provide sufficient detail to constrain the folding of the known amino-acid sequence of the protein into a unique three-dimensional configuration. The structure consists of a loose helix of turns of various sizes held together at one edge, spread apart along the other. This form appears to represent the shape of several fish protamines.


Membrane-microtubule interactions: concanavalin A capping induced redistribution of cytoplasmic microtubules and colchicine binding proteins. The relationship between microtubules and concanavalin A surface receptors during concanavalin A capping in primary cultures of rabbit ovarian granulosa cells was examined by electron microscopic and fluorescence labeling techniques. Cells treated with concanavalin A and hemocyanin at 4 degree or 37 degree and then incubated at 37 degree for 1 hr formed large juxtanuclear caps that were observed with shadow cast replicas of the cell surface. Thin section analysis of capped cells revealed an abundance of microtubules immediately beneath the cap which were arranged approximately perpendicular to the plane of the membrane. The capping process was unaffected by the antimicrotubule agents colchicine or vinblastine. Further, vinblastine treatment of capped calls resulted in the formation of numerous paracrystals that were confined to the cytoplasm underlying the capped region of the membrane; uncapped cells displayed paracrystals that were randomly dispersed in the cytoplasm. Exposure of fixed cells to fluorescein thiocarbamyl colchicine, which localizes colchicine binding proteins, revealed an intensely fluorescent region that corresponded to the cap; this staining pattern was absent in uncapped cells. These findings indicate that concanavalin A mediated capping modifies the cytoplasmic disposition of microtubules and colchicine binding proteins. Further, it is suggested that the capped region of the plasma membrane is a preferred site of microtubule polymerization.


Differences among myosins synthesized in non-myogenic cells, presumptive myoblasts, and myoblasts. Myosins synthesized in non-myogenic cells and replicating presumptive myoblasts differ from those synthesized in postmitotic mononucleated myoblasts and myotubes. Myoblasts and myotubes synthesize the definitive light chains, MLC1 and MLC2. These light chains display different molecular weights in sodium dodecyl sulfate-polyacrylamide gels from the fibroblast light chains FLC1 and FLC2 synthesized in non-myogenic cells and presumptive myoblasts. There are immunological differences between the myosin heavy chains synthesized in myoblasts and myotubes and those synthesized in non-myogenic cells and presumptive myoblasts. Fluorescein-labeled antibodies against skeletal light meromyosin are bound only along the lateral edges of emerging and definitive A-bands. This antibody to light meromyosin is not bound to the outside of, or the microfilaments subtending, the plasma membrane in non-myogenic cells or in myoblasts or in myotubes. These findings suggest that: (1) non-myogenic cells and replicating presumptive myoblasts synthesize similar myosin heavy and light chains; (2) replicating presumptive myoblasts synthesize a different set of myosins from those synthesized by their postmitotic daughters, the myoblasts; (3) the myosins associated with the plasma membranes of non-myogenic and myogenic cells are products of structural genes distinct from those coding for the myosins for skeletal myofibrils.


Localization of cytoplasmic-membrane-associated DNA in human chromosomes. In situ hybridization was used to localize the sites of the repeated sequences of cytoplasmic-membrane-associated DNA (cmDNA) in human chromosomes. cmDNA was mainly present in the heterochromatic regions of chromosomes 9. Although cmDNA has buoyant densities in neutral and alkaline CsCl gradients similar to those of human satellite C DNA, which also is localized in the heterochromatic region of chromosomes 9, cmDNA was not homologous to satellite C DNA as determined by DNA-complementary RNA hybridization. These results are in agreement with our previous studies, which indicated that cmDNA is a unique species of human DNA.


Snythesis and differentiation of plasma proteins in cultured embryonic chicken liver cells: a system for study of regulation of protein synthesis. A new system is described for studying the control of protein synthesis. In a monolayer culture of chick embryo liver cells, plasma proteins are synthesized for three days at in vivo rates. The plasma proteins are secreted into the culture medium and without concentration are detected there simply and sensitively by a modified Laurell electronimmunoassay. Secretion of the newly synthesized plasma proteins occurs within 30 min of their synthesis. Thus, rates of synthesis of the plasma proteins can be followed readily from rates of their accumulation in the culture medium. This system has the following advantages for the study of protein synthesis: cells do not have to be disrupted for the assay; the cell population can be followed over several days; it is not necessary to label the proteins radioactively; and turnover of plasma proteins is negligible and need not be taken into account. The usefulness of the system is illustrated by a number of findings. The spectrum of plasma proteins synthesized in culture changed qualitatively and quantitatively. Albumin synthesis steadily decreased with culture time and stopped at the third day, whereas the synthesis of some new plasma proteins ("adult") was induced. These qualitative changes suggest differential gene expression in culture and a special control of albumin synthesis in vivo, different from the synthesis of the other plasma proteins. Quantitative changes in the rates of synthesis of specific plasma proteins suggest a competition among their messenger RNAs for components of the translational machinery. Insulin has a differential effect on the synthesis of specific plasma proteins at concentrations within the physiological range of the hormone.


Homology in amino-terminal sequence of precursors to pancreatic secretory proteins. Sequence determination of up to 24 amino-terminal residues of several putative precursors for dog pancreas secretory proteins, synthesized in vitro by translation of their mRNA's in the presence of radioactively labeled amino acids, revealed extensive sequence homology in the 16 amino-terminal residues. It is suggested that this common sequence constitutes a metabolically short-lived peptide extension which precedes the amino-terminal sequences of all pancreatic secretory proteins and that it functions in the transfer of these proteins across the microsomal membrane. This sequence was found to contain an unusually large percentage of hydrophobic residues.


Plasmid interactions in Staphylococcus aureus: nonadditivity of compatible plasmid DNA pools. Six different Staphylococcus aureus plasmids have been examined for compatibility relationships and their intracellular DNA pools have been measured singly and in various combinations. All six were mutually compatible, but contrary to expectation, their intracellular DNA pools were not additive; instead, there appeared to be a maximum level of extrachromosomal DNA that could be supported by the cell, and the plasmids studied approached this level individually as well as in varous combinations. One exception was encountered: a plasmid encoding kanamycin/neomycin resistance was present in a small but constant number of copies regardless of the presence of other plasmids.


Acquisition of a determinant for chloramphenicol resistance by coliphage lambda. A determinanat for chloramphenicol resistance, cam, initially detected on a resistance transfer factor (RTF) and since transferred to phage P1, may be acquired from P1 by coliphage lambda. Lambdapcam are obtained when a lambda prophage is induced in bacteria which also harbor P1 cam prophage. Lambdacam formation is not dependent upon host Rec or lambda Red recombination functions. Electron microscopic heteroduplex analysis shows that the cam locus in two lambdapcams is a 5% addition of DNA in the b2 region of lambda, not contiguous with att. The extent and nucloetide sequence of the DNA insertion in the two independent lambdapcam isolates appear to be the same though they are located at different sites within the b2 region. We conclude that the determinant for chloramphenicol resistance is contained on a unique piece of DNA which facilitates its insertion into a number of unrelated genomes.


Social structuring of mammalian populations and rate of chromosomal evolution. To test the hypothesis that the evolution of organisms is dependent to a large degree on gene rearrangement, we devised a way of estimating rates of evolutionary change in karyotype. This non-biochemical method is based on consideration of chromosomal variability within taxonomic groups having a fossil record. The results show that chromosomal evolution has been faster in placental mammals than in other vertebrates or molluscs. This finding is consistent with published evidence that placentals have also been evolving unusually fast in anatomy and way of life. However, the structural genes of placentals seem not to have experienced accelerated evolution. Possibly, therefore, anatomical evolution may be facilitated by gene rearrangement. To explain how placentals achieved this rate of chromosomal evolution, we consider the process by which a new gene arrangement becomes fixed and spreads. The structure and dynamics of placental populations may be especially favorable for this process. The key factor involved seems to be the type of social behavior which produces small effective population sizes and inbreeding. As Bush points out elsewhere, such social structuring of populations may promote rapid fixation of gene rearrangements and rapid speciation.


On the mechanism of cytolysis by complement: evidence on insertion of C5b and C7 subunits of the C5b,6,7 complex into phospholipid bilayers of erythrocyte membranes. The doughnut hypothesis of cytolysis by complement [Mayer, M. M. (1972) Proc. Nat. Acad. Sci. USA 69, 2954-2958] describes an annular structure made up of C5b-9 (complement factors C5b, C6, C7, C8, and C9) which becomes inserted in the lipid bilayer of the cell membrane, thus creating a hole. We now present initial explorations of this hypothesis. EAC1-6 and EAC1-7 (sheep erythrocytes carrying rabbit antibody and complement factors C1 through C6 or C1 through C7, respectively), prepared with either 125I-C3 or 125I-C5 were incubated with trypsin and the release of bound 125I was measured. In the case of 125I-C3, all of the radioactivity was released by trypsin from both intermediates. With 125I-C5, trypsin released all of the 125I from EAC1-6, but only 40-55% from EAC1-7. Possible reasons for resistance of the C5b subunit in EAC1-7 to tryptic digestion are discussed; in terms of the doughnut hypothesis it would be due to shielding by lipid molecules as a consequence of insertion into the lipid bilayer. In accord with this interpretation we have also found that C5b in EAC1-7, but not in EAC1-6, resists elution by 0.3 M NaC1. Similarly, we have found that 125I-C7 in EAC1-7 resists stripping by trypsin. Hence, we now propose the hypothesis that hydrophobic polypeptide chains from the C5b and the C7 subunits of C5b,6,7 complex become inserted in the phospholipid bilayer and that subsequent reactions with C8 and C9 open a channel across the membrane.


Evidence for tryosine peptide homologies in the HLA antigens system. The tetrameric HLA antigens are composed of two heavier chains which carry the alloantigenic determinants and two lighter chains identified as beta2-microglobulin. Although at least 40 different antisera are required to define the varying HLA specificities, it appears that these antigens may be closely related to each other and to the immunoglobulins. Through the use of a new electrophoretic technique, which is able to compare simultaneously the tyrosine peptides produced from radioiodinated cell surface proteins, this report gives evidence that HLA antigens of the three chromosomal loci may have similar amino-acid sequences. Since the retention of homologous tyrosine residues and a tendency for sequence preservation surrounding these residues are features of immunoglobulin structure, this may indicate that similarly conservative evolutionary mechanisms have been operative in the HLA allelelic proteins or that immunoglobulins and HLA antigens may indeed have a common evolutionary origin.


Activation of antibody Fc function by antigen-induced conformational changes. IgM antibody directed against the pheny-beta-lactoside hapten was examined for its capacity to fix complement in the presence of the hapten, monohapten-substituted antigen, and multihapten-substituted antigen. Hapten was found to have no effect; monovalent antigen induced an excellent response which could be inhibited by hapten; and multivalent antigen also induced an excellent response which was related to the number of determinants added and not to the formation of antigen-antibody aggregates. The difference between the activities of hapten and monovalent antigen was reflected in their affinities for the IgM antibody. The monovalent antigen had a lower Ka, indicating that energy from binding was used to activate the Fc complement binding sites. These data show that the expression of IgM Fc function depends on a change in Fc conformation produced by the binding of antigen at the distant Fab combining sites.


Mutation and childhood cancer: a probabilistic model for the incidence of retinoblastoma. The incidences of some childhood cancers have been shown to fit a two-mutation hypothesis for cancer initiation. According to this hypothesis, the first mutation can be either germinal or somatic while the second is always somatic. A probabilistic model involving the mean number of tumors per genetically susceptible individual is developed as a function of age and is compared with age incidence data for retinoblastoma. The change in the mean number of tumors with time is interpreted in terms of the growth of retinal cells. In patients who are not genetically susceptible, the times of occurrence of the first and second somatic mutations can be inferred from a comparison of familial and non-familial unilateral case incidences. The total incidences of hereditary and nonhereditary forms of retinoblastoma are related to germinal and somatic mutation rates. The even distribution of certain childhood cancers throughout the world suggests that their incidences are determined by spontaneous mutation rates rather than by local environmental mutagenic carcinogens.


Porphyria variegata and porphyria cutanea tarda in siblings: chemical and genetic aspects. A woman aged 54 was studied because of a severe acute porphyric (neurologic) relapse with clinical and chemical findings characteristic of porphyria variegata. During a family survey, her brother, aged 59, was found to have chemical abnormalities typical of porphyria cutanea tarda, without suggestion of neurologic manifestations. He had mild skin changes compatible with either of these forms of porphyria. The sister exhibited the protocoproporphyria of porphyria variegata, together with a large amount of fecal "x" porphyrin fraction, without demonstrable isocoproporphyrins. The brother had a uro-isocopro-type of porphyria in accord with the diagnosis of porphyria cutanea tarda, and quite at variance with the sister's findings. This occurrence of porphyria variegata and porphyria cutanea tarda in siblings is thus far unique. Certain hypotheses are considered in respect to genetic aspects of the differing prophyrias in this sibling pair.


Increased concentrations of nonesterified arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2alpha in epidermis of psoriasis. Lesional epidermis of psoriasis has a probable reduction in the cyclic AMP/cyclic GMP ratio. This altered ratio may in part be responsible for the characteristic glycogen storage, rapid cell proliferation, and reduced differentiation in lesional epidermis. The concentrations of prostaglandins E2 and F2alpha, free arachidonic acid, and 12L-hydroxy-5,8,10,14-eicosatetrawnoic acid in specimens of uninvolved and involved epidermis of psoriasis were measured with deuterium-labeled carriers and multiple ion analysis. Snap frozen specimens contained: 1.4 +/- 0.4 mug/g (wet weight) of arachidonic acid in uninvolved in contrast to 36.3 +/- 16.7 mug/g in involved epidermis (P = 0.015); less than 0.05 +/- 0.01 mug/g of hydroxyeicosatetraenoic acid in uninvolved in contrast to 4.1 +/- 1.9 mug/g in involved epidermis (P = 0.015); 23.6 +/- 5.0 ng/g of prostaglandin E2 in uninvolved in contrast to 33.1 +/- 5.7 ng/g in involved epidermis (P less than 0.01); and 21.0 +/- 4.4 ng/g of prostaglandin F2alpha in uninvolved in contrast to 39.0 +/- 5.9 ng/g in involved epidermis (P less than 0.01). The arachidonic acid and hydroxyeicosatetraenoic acid levels in involved epidermis were strongly correlated (r = 0.97). The increased levels of arachidonic acid and 12L-hydroxy-5,8,10,14-eicosatetraenoic acid in involved epidermis may have diagnostic and pathophysiological importance.


Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. About 300 carcinogens and non-carcinogens of a wide variety of chemical types have been tested for mutagenicity in the simple Salmonella/microsome test. The test uses bacteria as sensitive indicators for DNA damage, and mammalian liver extracts for metabolic conversion of carcinogens to their active mutagenic forms. Quantitative mutagenicity data from linear dose-response curves are presented: potency varies over a 10(6)-fold range. There is a high correlation between carcinogenicity and mutagenicity: 90% (156/174) of carcinogens are mutagenic in the test and despite the severe limitations inherent in defining non-carcinogenicity, few "non-carcinogens" show any degree of mutagenicity. The results also demonstrate the great utility, and define the limitations, of the test in detecting environmental carcinogens.


Demonstration of non-functional beta-globin mRNA in homozygous beta (0) thalassemia. In two Chinese patients with homozygous beta(0)-thalassemia, messenger RNAs from peripheral blood reticulocytes and the bone marrow failed to direct beta-chain synthesis in vivo and in vitro in a cell-free system. Molecular hybridization showed that the beta cDNA annealed to the RNAs at almost the same rate as the alpha and gamma cDNA. The beta cDNA-RNA hydrid formed efficiently and was thermally stable, whereas hybrids between gamma and beta sequences formed slowly and denatured at a significantly lower temperature. Thus, we conclude that the beta cDNA was annealing to beta-globin sequences in these two patients, and that nonfunctional beta-globin mRNA was present. Similar results were obtained in the reticulocyte RNA from an Italian patient with homozygous beta(0)-thalassemia.


A novel murine oncornavirus with dual eco- and xenotropic properties. Infection of Swiss mouse 3T3FL cells with a clonal isolate of Moloney leukemia virus (MLV-IC) resulted in virus progeny composed of at least three different murine helper oncornaviruses. Each entity was purified in appropriate cells by several sequential terminal dilution isolations and was grouwn to high titers. Besides ecotropic MLV-IC there was a pure xenotropic virus and a third novel virus with properties of both eco- and xenotropic viruses. The purified xenotropic virus had a wide host range, was restricted in mouse cells, and was inactivated by normal mouse sera like other xenotropic isolates. The purified virus with hybrid properties (HIX) could infect a wide range of mammalian cells, which included both N and B mouse cells. HIX gave single-hit titrations with equal titers on both mouse and cat indicator cells. Envelope properties of HIX were examined by virus preinfection interference, by interference involving viral glycoprotein, and by neutralization with specific antisera. Both xenotropic and MLV-IC type ecotropic determinants were found on the virus coat. The origins of HIX and the xenotropic virus were investigated in detail. The original MLV-IC stock had HIX type virus in low titer but no detectable pure xenotropic virus. Infection of mouse cells with a single infectious unit of the ecotropic virus from the MLV-IC virus stocks could at times give rise to HIX type virus. HIX type virus, passed once through heterologous rat cells, was subjected to long-term passage either in infected mouse or cat cells. After several months HIX type virus disappeared from some mouse and cat cell systems. The possible hybrid nature of HIX and the origins of newly appearing xenotropic viruses are discussed.


Observations on impulse conduction along central axons. This note calls attention to the facts that (i) the conduction velocities of central axons may not be predicted on the basis of diameter alone, (ii) that such structure-function relations as do exist may not be invariant. Data are presented which indicate that conduction velocities of rabbit callosal axons vary with the history of impulse conduction along the fiber. Increases and decreases of conduction velocity occur. Constant latency does not, therefore, constitute a necessary condition for identification of antidromically activated neurons, and variable latency does not constitute a sufficient condition for identification of synaptically activated neurons. The results are further discussed in terms of temporal coding of information in the central nervous system.


Rapid-flow resonance Raman spectroscopy of photolabile molecules: rhodopsin and isorhodopsin. We have devised a method for obtaining the resonance Raman spectrum of a photolabile molecule before it is modified by light. The essence of this technique is that the sample is flowed through the light beam at a sufficiently high velocity so that the fraction of photoisomerized (or photodestroyed) molecules in the illuminated volume is very low. This rapid-flow technique has enabled us to measure the resonance Raman spectrum of unphotolyzed bovine rhodopsin in Ammonyx LO detergent solution and in sonicated retinal disc membranes. The major features of these spectra, which are very similar to one another, are the protonated Schiff base line near 1660 cm-1, the ethylenic line at 1545 cm-1, lines due to skeletal modes at 1216, 1240, and 1270 cm-1, and a line due to C-H bending at 971 cm-1. The resonance Raman spectrum of unphotolyzed isorhodopsin formed by the addition of 9-cis-retinal to opsin was also measured. The spectrum of isorhodopsin is more complex and differs markedly from that of rhodopsin. In isorhodopsin, the ethylenic line is shifted to 1550 cm-1, and there are six lines between 1153 and 1318 cm-1. The rapid-flow technique described here makes it feasible to control the extent of interaction between light and any photolabile molecule. We present a theory for predicting the effective sample composition in the illuminated volume as a function of the flow rate, light intensity, and spectral characteristics of the photolabile species.


Spike-forming model of neural membrane: computer simulation of additional perfused axon experiments. In this paper a physical model of the neural membrane that I developed is tested for its ability to account for the details of the action potential spikes observed in squid giant axons, when perfused with sodium- and potassium-free fluids and surrounded by a calcium chloride solution. The near-zero resting potential of these perfused axons is accounted for by the model. The sizes, shapes, and conductances of the spikes observed with a number of different perfusates are also accounted for, when suitable values are assigned to the so-far unmeasured membrane constants that control the model properties. The three papers comprising the series are summarized at the end of this paper.


A normal level of adenosine deaminase activity in the red cell lysates of carriers and patients with severe combined immunodeficiency disease. The red cell lysates of two children with severe combined immunodeficiency disease (SCID) exhibited a virtually total absence of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) when standard volumes were assayed. Under these conditions the parents exhibited depressed specific activity except for one mother, whose lysate showed a normal value for activity. Upon storage of the lysate at 4 degrees, a significant amount of activity appeared in one of the SCID children, and the activity of the heterozygous carriers was stimulated. With the use of a sensitive spectrophotometric assay based on conversion of inosine to uric acid, it was shown that the specific enzymatic activity in each of the SCID patients increased progressively as the volume of lysate assayed was lowered. With the smallest amount of lysate this specific activity was in the normal range. Similarly, the specific activity of each of the parents' lysates increased to the level of normal (or, in one case, about twice normal) as smaller volumes were assayed. The activity in the SCID patient was inhibitable by 2-fluoroadenosine and N6-methyladenosine, known competitive inhibitors of human red cell adenosine deaminase. The lysate from the SCID patient was also shown to inhibit adenosine deaminase partially purified from a normal individual. The results are interpreted in terms of a genetically programmed production of an adenosine deaminase inhibitor in at least one variant of the severe combined immunodeficiency disease.


Synapse formation between clonal neuroblastoma X glioma hybrid cells and striated muscle cells. Clonal neuroblastoma X glioma hybrid cells were shown to form synapses with cultured, striated muscle cells. The properties of the synapses between hybrid and muscle cells were similar to those of the normal, neuromuscular synapse at an early stage of development. The number of synapses formed and the efficiency of transmission across synapses were found to be regulated, apparently independently, by components in the culture medium. Under appropriate conditions synapses were found with 20% of the hybrid-muscle cell pairs examined; thus, the hybrid cells form synapses with relatively high frequency.


Applications of synchrotron radiation to protein crystallography: preliminary results. X-ray diffraction photographs of protein single crystals have been obtained using synchrotron radiation produced by an electron-positron storage ring. The diffracted intensities observed with this unconventional source are a factor of at least 60 greater than those obtained with a sealed x-ray tube using the same crystal and instrumental parameters. Diffraction data have been collected by the precession method to higher resolution and using smaller protein crystals than would have been possible with a conventional source. The crystal decay rate in the synchrotron beam for several proteins appears to be substantially less than that observed with Ni-filtered Cu radiation. The tunable nature of the source (which allows selective optimization of anomalous contributions to the scattering factors) and the low angular divergence of the beam make the source very useful for single crystal protein diffraction studies.


Affinity of myosin S-1 for F-actin, measured by time-resolved fluorescence anisotropy. The association constant for myosin subfragment-1 (S-1) and actin was measured, using a new application of fluorescence depolarization which capitalizes on the fact that S-1 has high rotational mobility while F-actin does not. Uncoupling of the time dependences of the anisotropy decay and the association/dissociation phenomena allowed the experimentally determined anisotropy decay curve to be fitted by a sum of two terms weighted by the mole fractions of the free and bound S-1. At 4 degrees C, ionic strength 0.16 M, and pH 7.0, the association constant Ka is (1.73 +/- 0.35) X 10(6) M-1 at infinite dilution. This makes the -deltaG degrees of binding of F-actin to S-1 similar to the -deltaG degrees of binding of ATP to S-1, and the possible physiological relevance of the similarity to muscle contraction is discussed.


Cellulose microfibrils: visualization of biosynthetic and orienting complexes in association with the plasma membrane. Cellulose microfibril biosynthesis, assembly, and orientation in the unicellular green alga, Oocystis, is visualized in association with a linear enzyme complex embedded in the B face of the plasma membrane. Granule bands of the A face and complementary ridges of the B face are postulated to assist in the orientation of recently synthesized microfibrils. A model for microfibril synthesis and orientation is proposed and correlated with current hypotheses regarding cellulose biosynthesis in higher plants.


Translation of nonpolyadenylylated messenger RNA of sea urchin embryos. A large proportion of newly synthesized polyribosomal RNA of sea urchin blastulae is not polyadenylylated. The size distributions of the polyadenylylated and nonpolyadenylylated RNA are indistinguishable (mean size of 26 S). Upon translation of sea urchin polyribosomal RNA containing poly(A) and that without poly(A) in a wheat embryo cell-free protein-synthesizing system, where polypeptide synthesis is dependent on added messenger, both classes of RNA support peptide synthesis to the same extent. A preliminary analysis of the proteins synthesized in response to added mRNA (polyadenylylated and nonpolyadenylylated) indicates that these classes of mRNA molecules may code for different populations of proteins.


Prevention of viral-chemical co-carcinogenesis in vitro by type-specific anti-viral antibody. Low passage Fischer rat embryo cultures, which are normally very resistant to transformation by 3-methylcholanthrene but are highly susceptible when chronically infected with the Rauscher murine leukemia virus, were completely protected from transformation by methylcholanthrene when treated with neutralizing antibody specific for the leukemia virus prior to and during treatment with methylcholanthrene. Sister cultures were not protected by neutralizing antibody specific for the B-tropic radiation leukemia virus. This demonstrates clearly a definite type specific role for Rauscher murine leukemia virus in the 2-methylcholanthrene transformation system in rat cells.


Synthesis of acetylcholine receptors by cultured chick myotubes and denervated mouse extensor digitorum longus muscles. Mono-[125I]iodo- alpha - bungarotoxin - receptor complexes extracted from chick myotube cultures as well as from adult denervated extensor digitorum longus muscles of the mouse have been banded at their buoyant density in gradients of metrizamide-deuterium oxide. When cultures or denervated adult muscles are preincubated in media containing 2H- or 13C-substituted amino acids under conditions of active receptor accumulation, the mono-[125I]iodo-alpha-bungarotoxin-receptor complexes have an increased buoyant density and band in a position of higher density in the gradient relative to a marker of mono-[131I]iodo-alpha-bungarotoxin-receptor complexes extracted from cells preincubated in normal media. It is concluded that the accumulation of receptors on the surfaces of cultured chick myotubes and on the non-synaptic surfaces of extensor digitorum longus muscles following denervation are the result of de novo synthesis.


Polarity of actin filaments in Characean algae. Heavy meromyosin from rabbit muscle combines with oriented Nitella and Chara actin in vitro to form arrowhead structures directed opposite to the cytoplasmic flow in the living plant cell. All filaments and all bundles of filaments in the apically directed stream are similarly oriented; polarity with respect to the axis of the thallus is reversed in the downward stream. The actin filaments are attached to the chloroplasts at the ectoplasm-endoplasm interface, where the motive force for streaming is known to be generated.


Analysis of membrane halves: cholesterol. Membrane splitting by freeze-fracture has been used as a preparative tool for chemical analysis of outer and inner "half"-membranes. In a previous report I showed that monolayers of human erythrocytes, bound to cationized glass, fracture nonrandomly, producing membrane fractions substantially enriched in outer or inner "halves." For the present study cells were used in quantities compatible with microanalysis. For quantitation the total amount of membrane present and the fractional enrichment of outer and inner "half"-membranes were determined. Cholesterol was examined by quantitative thin-layer chromatography modified to assay nanogram amounts. Comparison of lipids extracted from intact membranes with lipids from fractured membranes indicated that cholesterol was asymmetrically distributed across the plane of the membrane, more being present on the exterior side than on the interior side.


Induction of human fibroblast proliferation by epidermal growth factor (EGF): enhancement by an EGF-binding arginine esterase and by ascorbate. The effect of mouse epidermal growth factor (mEGF) and an mEGF-binding arginine esterase on the growth of cultured human fibroblasts has been studied. Physiological concentrations (10(-9)-10(-10) M) of the growth factor were found to stimulate DNA replication and cell proliferation in quiescent cultures, and the arginine esterase, which is normally associated with mEGF in vivo, was shown to enhance this growth effect synergistically. The cellular response to mEGF was dependent upon a low, growth-limiting concentration of serum in the extracellular medium. Ascorbic acid, which alone exhibited no growth-promoting effect, could partially replace this requirement, and was found to elicit a rapid and marked increase in proline hydroxylation. Quiescent cultures in serum-free medium containing ascorbic acid were stimulated by the combination of mEGF and the esterase in a manner comparable to that achieved with serum shift-up. The possible requirement of a collagen-containing extracellular matrix for the growth response to mEGF is discussed.


Mutability of different genetic loci in mammalian cells by metabolically activated carcinogenic polycyclic hydrocarbons. The relationship between carcinogenesis and mutagenesis in mammalian cells has been determined with 10 polycyclic hydrocarbons with different degrees of carcinogenicity. Mutagenesis was determined in Chinese hamster cells with genetic markers that affect the surface membrane, nucleic-acid synthesis, and protein synthesis. The mutations were characterized by resistance to ouabain, 8-azaguanine, and temperature. Mutagenesis by the carcinogens required metabolic activation and this was provided by the presence of lethally irradiated metabolizing cells. The degree of carcinogenicity was related to the degree of mutagenicity for all three genetic markers. The most potent carcinogen, 7,12-dimethylbenz[a]anthracene, gave the highest mutagenicity and mutagenicity was obtained with 0.01 mug/ml. Treatment of the cells with aminophylline, which increases polycyclic hydrocarbon metabolism, increased mutagenesis by the carcinogens. It is suggested that such an experimental system with these and other mammalian cells should be useful as a sensitive assay for hazardous environmental chemicals.


Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate. Translation of rabbit globin mRNA in a wheat germ protein-synthesizing system is inhibited by the nucleotide 7-methylguanosine-5'-monophosphate (m7G5'p) but not by other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position. Translation of RNA of tobacco mosaic virus and poly(A) + HeLa RNA is also inhibited by m7G5'p. We show that m7G5'p prevents the association of mRNA with ribosomal subunits to form an initiation complex. We propose that m7G5'p interacts with a site on initiation factor(s) or ribosomes which is involved in mRNA recognition, presumably by binding to the 5'-terminal sequence m7G5'ppp. m7G5'p does not inhibit translation of poly(U) and RNA of satellite tobacco necrosis virus, which do not have the 5'-terminal sequence m7G5'ppp. In the case of RNA of satellite tobacco necrosis virus, some stimulation of its translation is consistently observed in the presence of m7G5'p; possible interpretations of this finding are discussed.


Genetics of somatic mammalian cells: genetic, immunologic, and biochemical analysis with Chinese hamster cell hybrids containing selected human chromosomes. Through hybridization of specific Chinese hamster cell auxotrophs with human cells and selection in media lacking the nutritional supplements required by the former cells, a series of stable hybrid clones can be prepared. These hybrids have genomes consisting of a common part--the complete or almost complete set of Chinese hamster chromosomes, plus a variable part--one or a few human chromosomes. The identity of the human chromosomes can be varied by utilizing different Chinese hamster auxotrophs and the appropriate selective media. The human chromosomes present can be determined by a combination of cytogenetic analysis with chromosome banding and testing for specific human marker genes. Hybrids containing single human chromosomes 11 and 12 and the combination of both 11 and 12 are described. The system appears to lend itself to various studies such as identification of human cell surface antigens, determination of their chromosomal loci, measurement of their distribution among cells of normal human tissues, study of interrelations among syntenic and asyntenic genes, and mutational analysis of the human genome.


Purkinje cell degeneration, a new neurological mutation in the mouse. A new autosomal recessive mouse mutation, Purkinje cell degeneration (pcd), is described. Mutants exhibit a moderate ataxia beginning at 3 to 4 weeks of age. The ataxia results from postnatal degeneration of virtually all cerebellar Purkinje cells beginning around 15 to 18 days of age and progressing rapidly over the next 2 weeks. In addition to the cerebellar disease there is slow progressive degeneration in the retina (photoreceptor cells) and olfactory bulb. Also, adult males have abnormal sperm.


Characterization of residual enzyme activity in fibroblasts from patients with adenosine deaminase deficiency and combined immunodeficiency: evidence for a mutant enzyme. A proportion of patients suffering from the autosomal recessive form of severe combined immunodeficiency have an inherited deficiency of adenosine deaminase (EC 3.5.4.4; adenosine aminohydrolase) (erythrocyte isoenzyme). We have, however, found residual adenosine deaminase activity in fibroblasts derived from four such patients. The enzyme responsible for this activity is biochemically homologous with the high-molecular-weight tissue isoenzyme of adenosine deaminase found in normal fibroblasts and tissues other than erythrocytes. The residual adenosine deaminase has an altered electrophoretic mobility, increased heat stability as compared to normals, and can be detected in fibroblasts of obligate heterozygotes. Our previous studies have indicated that the tissue and erythrocyte adenosine deaminase isoenzymes contain a common catalytic unit controlled by the gene affected in severe combined immunodeficiency with absent adenosine deaminase (erythrocyte isoenzyme). This residual adenosine deaminase therefore represents, most likely, a "mutant" enzyme in fibroblasts of patients with severe combined immunodeficiency. The data support the hypothesis that, in these patients, severe combined immunodeficiency is due to a mutation at the adenosine deaminase locus.


Chromatographic isolation of the hemagglutinin polypeptides from influenza virus vaccine and determination of their amino-terminal sequences. The influenza virus hemagglutinin polypeptides, HA1 and HA2, have been purified by gel filtration in the presence of sodium dodecyl sulfate from a vaccine preparation of the recombinant strain Heq1N2. Use of this technique for purification of the hemagglutinin polypeptides eliminated the need for proteolytic agents for removal of the hemagglutinin from the virus particles and 100-300 mg of virus yielded 10-30 mg of viral protein per chromatographic cycle. Because proteolysis is not required to remove the spikes from the viral envelope, the envelope-embedded HA2 polypeptide was purified in its entirety for structural analysis. Amino-terminal sequence analysis of the smaller polypeptide, HA2, revealed a cyclic repetition of glycyl residues through the first 24 residues at every third to fourth position. The sequence through the first 10 residues was identical to that presented by Skehel and Waterfield for other type A influenza viruses [(1975) Proc. Nat. Acad. Sci. USA 72, 93-97]. The HA1 (Heq/) polypeptide, on the other hand, had different amino acids at three or four out of the first 10 residues of the amino-terminal sequence when compared to HA1 from H0, H1, or H2 subtypes (Skehel and Waterfield). The present study has demonstrated the feasibility of the use of vaccine virus as a source of large quantities of viral protein for determination of primary structure.


Carcinogenicity of benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides on mouse skin. Benzo[a]pyrene and three arene oxides of benzo[a]pyrene (benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides) have been tested for carcinogenicity in mice by topical application of each compound (0.1 or 0.4 mumol) once every 2 weeks for 60 weeks. At the high dose, benzo[a]pyrene and the 7,8-oxide were highly carcinogenic, whereas the 4,5-oxide (K-region oxide) was weakly active and the 9,10-oxide was inactive. At the low dose, only benzo[a]pyrene was highly carcinogenic. The carcinogenic activities of the three arene oxides of benzo[a]pyrene were not correlated with their stabilities or mutagenic activities.


Model of solute and water movement in the kidney. Finite difference equations describing salt and water movement in a model of the mammalian kidney have been solved numerically by an extension of the Newton-Raphson method used for the medullary counterflow system. The method permits both steady-state and transient solutions. It has been possible to simulate behavior of the whole kidney as a function of hydrostatic pressures in renal artery, vein, and pelvis; protein and other solute concentrations in arterial blood; and phenomenological equations describing transport of solute and water across nephron and capillary walls. With the model it has been possible to compute concentrations, flows, and hydrostatic pressures in the various nephron segments and in cortical and medullary capillaries and interstitium. In a general way, calculations on the model have met intuitive expectations. In addition, they have reemphasized the critical dependence of renal function on the hydraulic and solute permeabilities of glomerular, postglomerular, and medullary capillaries. These studies provide additional support for our thesis that the functional unit of the kidney is not the single nephron, but a nephrovascular unit consisting of a group of nephrons and their tightly coupled vasculature.


Darwin's finches: population variation and natural selection. Van Valen's model, which relates morphological variation to ecological variation in an adaptive scheme, was investigated with individually marked and measured Darwin's finches on two adjacent Galápagos islands, Santa Cruz and Daphne Major. Results show that environmental heterogeneity is correlated with large continuous, morphological variation: variation in bill dimensions of Geospiza fortis is greater on Santa Cruz than on Daphne, as is environmental heterogeneity. Within populations of this species, different phenotypes distribute themselves in different habitat patches, select foods of different sizes and hardness, and exploit them with efficiencies that are phenotype- (bill size) dependent. These data constitute indirect evidence that natural selection has a controlling influence over the level of phenotypic variation exhibited by a population. Further evidence is that phenotypes did not survive equally well during the study period; on Daphne island G. fortis was apparently subjected to directional selection on bill tip length and G. scandens to normalizing selection on body weight and bill depth. Other factors which may have contributed to the establishment of a difference in variation between Santa Cruz and Daphne populations are the founder effect, genetic drift, and assortative mating. Annual climatic unpredictability is considered a source of environmental heterogeneity which, through its effect upon food supply, favors large morphological variation. It is predicted that species of large individual size are more influenced by this than are small species, and consequently exhibit greater size-corrected variation. The prediction is tested with data from six Geospiza species, and found to be correct.


Pregrowth hormone: product of the translation in vitro of messenger RNA coding for growth hormone. Membrane fraction RNA isolated from rat pituitary tumor (GC) cells has been translated in a wheat germ extract. A product was synthesized which was immunologically related to growth hormone, but which migrated more slowly than growth hormone upon sodium dodecyl sulfate-acrylamide gel electrophoresis. The mobility of the cell-free product on gels of this type was unchanged by treatment with either KOH or RNase. The mobilities during paper electrophoresis of the methionine-containing tryptic peptides obtained from the cell-free product were identical to those obtained from growth hormone synthesized and secreted by the GC cells. Molecular weights for growth hormone and the cell-free product of 19,500 and 24,000, respectively, were determined by gel electrophoresis of these proteins together with marker proteins of known molecular weights. No protein with the properties of the cell-free product was detected after a 2 min incubation of the GC cells with [35S]methionine. However, treatment of the GC cells, with a protease inhibitor, L-1-tosylamide-2-phenyl-ethylchloromethyl ketone (TPCK), led to the appearance of a new polypeptide, immunologically related to growth hormone, and with a mobility on gels identical to that of the cell-free product. These results strongly imply that the cell-free product represents a growth hormone precursor (pregrowth hormone) which is rapidly converted to growth hormone in pituitary cells.


Carbohydrate moieties of procollagen: incorporation of isotopically labeled mannose and glucosamine into propeptides of procollagen secreted by matrix-free chick embryo tendon cells. Cells obtained from chick embryo tendons incorporate isotopically labeled glucosamine and mannose into the pro-alpha1 and pro-alpha2 chains of procollagen as judged by sodium dodecyl sulfate-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The label was further localized to the propeptides of pro-alpha1 and pro-alpha2 by its chromatographic behavior after digestion with bacterial collagenase or alpha-chymotrypsin. Carbohydrate analysis of isolated pro-alpha chains showed the presence of labeled galactosamine in addition to mannose and glucosamine. Resistance to mild alkaline hydrolysis suggested that greater than 90% of the oligosaccharide units are not linked to the propeptide backbone by either serine or threonine.


Polypeptide chain initiation in eukaryotes: initiation factor requirements for translation of natural messengers. A protein-synthesizing system consisting of ribosomes and supernatant of undeveloped Artemia saline embryos is used for assay of mRNA translation and initiation factors. This system contains the components needed for chain elongation but has low levels of mRNA and initiation factors. Exogenous mRNA is readily translated upon addition of high-speed supernatant or ribosomal salt wash of developing embryos as a source of initiation factors (IF). This requirement can be largely satisfied by a mixture of the reticulocyte factors IF-MP, IF-M3, IF-M2A, and IF-M2B. A. salina IF-M1, which is present in undeveloped embryo supernatant, can be inactivated by A. salina IF-M1 antibody without affecting translation.


Magnetic circular dichroism of ferrous carbonyl adducts of cytochromes P-450 and P-420 and their synthetic models: further evidence for mercaptide as the fifth ligand to iron. Absorption and magnetic circular dichroism (MCD) spectra have been obtained for the ferrous carbonyl adducts of cytochromes P-450 and P-420 as well as synthetic model systems. Ferrous porphyrins with sodium methyl mercaptide and CO in benzene give MCD and absorption spectra which are almost identical to those of the natural enzyme, indicating that in P-450 a mercaptide serves as the fifth ligand in the ferrous carbonyl adduct. MCD spectra of models with either propyl mercaptan or N-methylimidazole as the axial ligand are identical with that of P-420. Thus, no unambiguous assignment of the axial ligand can be made in this case. The infrared stretching frequencies of ferrous porphyrin carbonyl complexes and the absorption spectrum of the CO adduct of Na[Fe1(meso-tetraphenylporphyrin dianion)] are consistent with the concept that in P-450 considerable electron density is transferred to the iron by the mercaptide ligand.


Amino-acid sequence of fragment A, an enzymically active fragment from diphtheria toxin. The amino-acid sequence of Fragment A from diphtheria toxin is reported. Fragment A (molecular weight, Mr, 21,145) is the major enzymically active fragment produced upon activation of the intact toxin (Mr about 60,000) by limited tryptic digestion and reduction. It, or a similar fragment, is believed responsible for the inhibition of protein synthesis in animal cells exposed to the toxin. Fragment A, which corresponds to the amino terminus of the toxin, is shown here to consist of three major forms (190, 192, and 193 residues) resulting from cleavage by trypsin adjacent to any of three closely spaced arginine residues. All three forms are enzymically active.


Transduction of chemical into electrical energy. The paper recalls some fundamental notions, developed by Otto Meyerhof, which were used in the analysis of the transduction of chemical into mechanical energy during muscular contraction. These notions formed the basis of the approach to the analysis of the transduction of chemical into electrical energy, i.e., the very principle underlying nerve and muscle excitability and bioelectricity. Instrumental for this purpose was the use, since 1937, of electric organs of fish, a tissue highly specialized for bioelectrogenesis.


Intramolecular energy transfer and molecular conformation. A discussion of the range of applicability of Forster long-range energy transfer in the determination of macromolecular dimensions and conformational dynamics is given. Emphasis is laid on the effect of restrictions in the orientational freedom of donor and acceptor and on the importance of the orientational averaging regime. The usefulness and limitations of polarized emission measurements in this regard are discussed.


Novel properties of a restriction endonuclease isolated from Haemophilus parahaemolyticus. The sequences in lambda DNA in and around six sites cut by Hph, a restriction enzyme isolated from Haemophilus parahaemolyticus, are compared. The enzyme produces a staggered cut around an AT or TA base pair, but the sequences immediately surroinding the cleavage sites bear no obvious relation to one another. Eight (in some cases nine) base pairs to one side of each cleavage site is the common sequence TCACC AGTGG. Two lines of evidence indicate that these bases constitute part or all of the Hph recognition site. First, mutations in this sequence prevent Hph cutting. Second, dimethylsulfate-mediated methylation of Gs and As in this site prevent cutting, whereas methylation of purines in the region between this sequence and the cleavage sites has no such effect. There is discernible 2-fold rotational symmetry neither in the common sequence nor around the cleavage sites.


Polypeptide chain initiation in eukaryotes: mechanism of formation of initiation complex. Artemia salina ribosomal subunits and highly purified reticulocyte initiation factors (IF) are used to study the mechanism of formation of the puromycin-sensitive initiation complex Met-tRNAi-80S ribosome-AUG. A complex with equimolar amounts of 40S subunit, GTP, and Met-tRNAi is formed at low Mg2+ concentration with a requirement for IF-MP (homogeneous) but not AUG or other factors. An 80S complex is formed only upon the further addition of AUG, IF-M2A, and IF-M2B, but not of either factor alone. This complex contains no GTP or GDP. A 40S complex, which cannot be converted to an 80S one, is formed when the nonhydrolyzable analog GMPPCP is substituted for GTP. IF-M2A has no effect on the formation of this complex, but IF-M2B enhances its formation.


Reciprocal reactivities of specific thiols when actin binds to myosin. We report measurements of the reactivity (degree of labeling, as mole of ligand per mole of protein, at constant exposure time) of the reactive thiol, "SH1", of a subfragment of myosin (S-1), and of Cys-10 of F-actin under various conditions, using N-iodo-[3H]acetyl-N-(1-sulfo-5-naphthyl)ethylenediamine, a fluorescent radioactive iodoacetamide analog. When either ADP or adenyloyl imidodiphosphate (simulating unhydrolyzed ATP) is bound to the enzymatic site of S-1, the reactivity of "SH1" is slightly enhanced, but when active ATPase is going on, reactivity is reduced by about a third, presumably due to the species, (S-1) ADP,Pi. The reactivity of Cys-10 alone is very low. When the complex, (S-1)-F-actin, is formed, the reactivity of SH1 is strongly decreased, and the reactivity of Cys-10 is strongly increased. The foregoing results explain our further observation (on glycerol-treated rabbit psoas fibers) that when fibers labeled in relaxation solution  are compared with fibers labeled in rigor solution, myosin is more reactive and actin is less reactive, in the former case; alpha-actinin and C-protein are also less reactive in the former case.


Studies on secondary structure of single-stranded RNA from bacteriophage MS2 by electron microscopy. A method allowing the demonstration and study by electron microscopy of secondary structure of viral RNA has been developed. Single-stranded RNA from the bacteriophage MS2 has been analyzed in the electron microscope in the presence of various concentrations of MgCl2. Depending on the salt concentration, the molecules display one to three large open loops which range in size from 10 to 20% of the total RNA length, and smaller closed loops which are approximately 3-5% of the total RNA length. Within one spreading, the conformation of the molecules is variable. However, the average complexity of the molecules increases with increasing salt, and individual loops which are infrequent at low salt increase in frequency with increasing salt. By analyzing the manner in which the individual loop appeared, it was possible to show that all molecules could be described by one basic pattern of secondary structure formation.


Detection of polycistronic and overlapping bacteriophage T7 late transcripts by in vitro translation. Bacteriphage T7 RNAs have been fractionated on preparative polyacrylamide gels. The in vitro coding capacities of the RNAs have been determined by translation of the RNAs in a cell-free system and analysis of the polypeptide products on sodium dodecyl sulfate polyacrylamide slab gels. The T7 early RNAs are fractionated according to their molecular weight and without intermolecular aggregation. Fractionation of the late T7 RNAs  gives rise to 10 major RNAs, ranging in size from 0.29 X 10(6) daltons to 2.05 X 10(6) daltons. Five of these RNAs are polycistronic and overlapping species are present for some T7 proteins. In particular, the gene 10 protein, the major capsid protein, is translated from at least three mRNAs. The smallest of these gene 10 mRNAs is monocistronic. A second gene 10 mRNA also codes for the gene 9 protein, and a third gene 10 mRNA codes for both gene 8 and gene 9 proteins. The T7 gene 3.5 protein, a T7 lytic enzyme, is also translated from several differently sized mRNAs. Comparison with published data on in vitro transcription by T7 RNA polymerase suggestes that transcription from multiple initiation sites and cleavage of larger precursors are both involved in generating the late T7 transcripts we observe. The overlapping mode of transcription could serve to amplify certain gene products.


Magnetic resonance studies of concanavalin A: location of the binding site of alpha-methyl-D-mannopyranoside. The longitudinal nuclear magnetic relaxation times of the methyl protons of alpha-methyl-D-mannopyranoside have been measured at 90 MHz and 270 MHz in solutions of concanavalin A complexed with: (i) Mn2+ and Ca2+; and (ii) Zn2+ and Ca2+. Zn2+ and Mn2+ are known to bind in site S1 and Ca2+ in site S2 of concanavalin A. Both sites must be occupied before monosaccharides will bind to the protein. In order to extract T1p, the paramagnetic contribution to the bound methyl relaxation time, from these observations the relaxation time of uncomplexed sugar was determined. Free Mn2+ contributed insignificantly to the latter value; however, outer sphere relaxation was found to be large at bound Mn2+ concentrations greater than 1 mM. Comparison of the results obtained at the two frequencies allowed the determination of the correlation time for the interaction between the methyl protons and the bound Mn2+ and the distance between them (21.5 +/- 1.2 A). In contrast to previous results from nuclear magnetic resonance studies, this distance is consistent with binding at the cavity proposed for the saccharide binding site by Becker et al. [J. Biol. Chem. 250, 1513 (1975)], although it does not preclude possible binding sites on the surface of the molecule.


A hepatic receptor of avian origin capable of binding specifically modified glycoproteins. Evidence is reported to support the following conclusions: (i) The presence of a specific hepatic receptor capable of recognizing and binding galactose-terminated glycoproteins is responsible for maintaining near zero levels of circulating asialoglycoproteins in mammalian serum. (ii) The absence of this galactose-specific binding protein in avian, and presumably reptilian, liver is correlated with high levels of circulating asialoglycoproteins in the serum of these species. (iii) The evolutionary significance of these observations is reinforced by the recognition of an avian hepatic binding protein specific for terminal, N-acetyl-glucosamine residues on glycoproteins.


Chemical reactivities of catalytic and noncatalytic zinc or cobalt atoms of horse liver alcohol dehydrogenase: differentiation by their thermodynamic and kinetic properties. Horse liver alcohol dehydrogenase (EC 1.1.1.1) contains one catalytic and one noncatalytic pair of zinc atoms that can be replaced selectively with cobalt and/or 65zince. We have now prepared "hybird" metalloenzymes by specifically replacing one or both pairs of zinc atoms with 65zinc and/or cobalt. Their differential chemical reactivities serve to characterize the metal atoms at either site. The spectral and kinetic properties of the resultant 65zinc, cobalt, and hybrid enzymes, as well as those of their complexes with 1,10-phenanthroline, identify the metal atoms that are at the catalytic sites and differentiate them from those at the noncatalytic sites. All data are in complete agreement with the results of the x-ray crystal structure analysis. Remarkably, under the conditions used, chemical reactivity, as gauged by thermodynamic methods under equilibrium conditions, identifies the catalytic metal atoms as those which are reactive to 1,10-phenanthroline, while this reagnet does not affect the noncatalytic pair. Under dynamic conditions the kinetics of the metal-metal exchange reveals the converse to be true: the chemical reactivity of the noncatalytic atoms is much higher and, hence, they exchange more rapidly. The results are examined in terms of thermodynamic and kinetic properties of metal complex ions which serve as the basis of possible mechanisms underlying these observations.


Structure of the iron complex in methemerythrin. The coordination of the ligands about the iron atoms in methemerythrin from Themiste dyscritum has been deduced from a 2.8 A resolution electron density map. The complex can be described in terms of two trigonal antiprisms about the pair of iron atoms in each subunit, the antiprisms having one face in common. Ligands at eight of the nine coordination positions are protein side chains, the ninth presumably being water. Comparison of the electron density map for T. dyscritum methemerythrin with the sequence of Phascolopsis gouldii hemerythrin suggests six aromatic side chain ligands (five histidine and one tyrosine) and two nonaromatic side chain ligands. The latter provide atoms at two of the three vertices of the face shared by the two antiprisms, and these along with the presumed water at the third vertex form bridges between the iron atoms of each pair.


Synthesis of Rauscher murine leukemia virus-specific polypeptides in vitro. The biosynthesis of specific polypeptides directed by purified viral messenger RNA from JLS-V9 cells infected with Rauscher leukemia virus has been studied in a rabbit reticulocyte lysate. The 35S viral mRNA gives rise to two major products of 65,000 and 72,000 molecular weight. The synthesis of specific polypeptides was also investigated in lysates derived from infected cells. The main products were polypeptides with molecular weights of 65,000, 76,000, and 82,000, and were preferentially made in association with membranes. The relative content of the virus-specific polypeptide of 65,000 molecular weight, synthesized in a cell-free system supplemented with purified polyribosomes, is considerably higher for membrane-bound polyribosomes.


Transbilayer distribution and movement of cholesterol and phospholipid in the membrane of influenza virus. The transfer of radioactive cholesterol from influenza virus to excess phosphatidylcholine-cholesterol vesicles has been studied. Viral cholesterol was found to exist in two pools, one rapidly exchangeable. Evidence is presented that the rapidly exchangeable pool corresponds to cholesterol present on the outer surface of the viral bilayer, while the slowly exchangeable pool corresponds to inner surface cholesterol. Approximately equal amounts are present in each pool, suggesting that cholesterol distribution is not markedly asymmetric in the viral bilayer. A half-time for the rate of equilibration between the two sides of the bilayer (flip-flop) was about 13 days at 37 degrees with a 90% confidence interval of 3.4- infinity days. Similar experiments were carried out which followed the time course of transfer of labeled phospholipids from the viral bilayer to phospholipid vesicles, catalyzed by phospholipid exchange protein from beef heart. From these experiments the half-times for the flip-flop of phosphatidyl-choline and spingomyelin were found to be indeterminately in excess of 10 and 30 days at 37 degrees, respectively. These results suggest that exchange of the major components of the viral bilayer between the two surfaces occurs very slowly relative to the turnover times of most membrane constituents, and provide a plausible mechanism for the maintenance of membrane asymmetry over biologically relevant time periods.


Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes. A protein fraction was obtained from human erythrocyte ghosts by solubilization with Triton X-100 or octylglucoside. Triton X-100 was removed from the protein by Bio-Beads SM-2 and octylglucoside, by diafiltration. The solubilized protein fraction catalyzed D-glucose uptake when reconstituted in sonicated liposomes. The uptake was time dependent and inhibited by mercuric ions or cytochalasin B. The results indicate that the uptake represents transport of the sugar into the liposomes rather than binding to the reconstituted liposomes.


Chromatin-like organization of the adenovirus chromosome. Staphylococcal nuclease (nucleate 3'-oligonucleotidohydrolase; EC 3.1.4.7) cleaved DNA within disrupted adenovirus particles into a regular series of fragments with a repeat unit of 200 base pairs. Since this pattern did not eppear when DNA alone was digested, we postulate that the orderly arrangement of core polypeptides protects discrete regions of DNA from nuclease attack. The 23 X 10(6) dalton adenovirus DNA molecule can accommodate 180 units of roughly 200 base pairs. Based on the stoichiometry of core polypeptides, we calculate that each repeat unit contains six copies of polypeptide VII and a single copy of polypeptide V. This model is bases on proposals for the structure of eukaryotic chromatin. Very brief nuclease digestion generated 1800 base pair fragments (1/20 of the adenovirus chromosome). This result is discussed in terms of a higher order folding of viral DNA within the virus particle.