Liposarcoma of the oropharyngeal region. Review of the literature and report of two cases.

Liposarcomas of the oropharyngeal regions are exceedingly rare. Their silent, slow growth, submucosal or deep location, circumscription, and firm but resilient texture often suggest a diagnosis of a cyst or benign soft-tissue neoplasm. Two cases of well-differentiated myxoid liposarcoma are presented. The literature of over half a century was reviewed, and all documented cases were critically analyzed. The clinical data correlated with the histopathology and biologic behavior of the tumors indicated that two thirds of all liposarcomas of the head and neck are of the myxoid type. The well-differentiated myxoid liposarcoma was the most common one and has an over-all favorable prognosis. Lipomas of the oropharyngeal regions are relatively common and small, generally not exceeding 2.5 cm. in greatest diamter. A lobulated firm tumor of fast tissue significantly exceeding this size should be strongly suspected of being a liposarcoma.


Description of a computerized tumor recall program for premalignant and aggressive oral lesions.

The rationale for establishing a computerized tumor recall program as an adjunct to a diagnostic biopsy service to assist practitioners in instituting periodic reexamination of patients with oral lesions that have been diagnosed histologically as either premalignant or locally aggressive is given. The necessary forms, with a description of pertinent information, are described in conjunction with the proper sequential procedure for completing the forms before and after information has been submitted to a computer center.


Focal odontoblastic dysplasia: dentin dysplasia type III?

A new, nonsyndromic dentin defect, focal odontoblastic dysplasia, is described on the basis of clinical, radiographic, histologic, and scanning electron microscopic criteria. A provisional classification is proposed for this disease entity according to the nosology of Shields and associates. Four additional cases in the literature which possibly represent this new entity are presented, and a possible genetic etiology is discussed.


Endodontic morphology. IV. A comparative study.

One hundred sixty-four extracted human teeth of all denominations were subjected to radiographic study after their canal systems were prepared with endodontic instruments and perfused with a water-soluble, radiopaque contrast medium. The same specimens had been studied prior to endodontic instrumentation by means of the same technique, and a comparison of the two sets of radiographs was made. Results of this comparative study were combined with information gained during the instrumentation procedures, and an analysis of the success rate for each group of specimens has been given. The over-all rate of success was high (92 per cent), being highest in the case of maxillary incisors and canines (100 per cent) and lowest in mandibular incisors and canines (81.8 per cent). The belief that the canals of molars present more difficulties than those of other teeth was not substantiated in this experiment, with the success rate for maxillary and mandibular molars being 92.1 per cent. Discussed were the potential for the packing of debris into the apical ends of root canals during instrumentation, the presence of residual traces of alloy within canals after canal preparation, and the known and unknown fracturing of endodontic instruments.


Extraradicular communicating dens invaginatus.

The morphologic occurrence of dens invaginatus often results in pathologic involvement of the pulp and associated periodontal tissues. Treatment must include endodontic therapy to reduce the organic and infectious content of the pulp and invaginated tract. Concervative therapy, however, is frequently insufficient to eliminate the irritant force to a level that is biologically acceptable to the host. Therefore, surgical intervention has been considered by most authors to be the most acceptable approach for correction of the deformity and associated alveolar defects. Both cases reviewed here represent the dens invaginatus type of malformations that have primary zones of irritation within the anomalous tract. A report similar to our Case 2 appeared in the literature 2 weeks after initial therapy of the nonpupally involved lateral incisor. These cases are published to document the occurrence of these forms of dens invaginatus and their associated periradicular pathosis, to recommend treatment, and to note the apparent favorable response to the involved tissues.


The lack of pulpal pathosis in rice rats with the periodontal syndrome.

The purpose of this study was to determine in the rice rat model whether a relationship exists between the severity of periodontal disease and the presence of severity of pulpal lesions. The results indicated that, even in the presence of severe loss of alveolar bone, extending close to the root apices, there were no detectable changes in the pulpal tissues as compared to the pulps of teeth in which there was no loss of alveolar bone. The scarcity of accessory pulp canals in the rice rat may be related to our findings.


The radiologic features of fibrous dysplasia of the craniofacial bones.

The radiographs of twenty-five patients (eight males, seventeen females) with histologically proven craniofacial fibrous dysplasia were analyzed. The lesions could be classified into six radiologic types: (1) peau d'orange, (2) whorled plaquelike, (3) diffuse sclerotic, (4) cystlike (multilocular or unilocular), (5) pagetoid, and (6) chalky types. The peau d'orange type was by far the most common (40 per cent) followed by the plaquelike (20 per cent, cystlike (16 per cent), and sclerotic (12 per cent). Most of the patients were young, with 60 per cent being underthe age of 20 years and 96 per cent under the age of 40 years.


Treatment of acute non-lymphoid leukemias: comparison of two protocols.

The results of treatment of 57 patients suffering from acute non-lymphoid leukemia by two protocols are compared. The more aggressive Coap protocol rendered a higher remission rate (57.1%), than the mild Guyer protocol where the remission rate has been 25%. The best results have been achieved in the former group in the younger population; in the latter group there has been no age-effect relationship. Although the remission rate differed in both protocols there has been no statistically significant difference in survival.


Recent results in cancer research related to non-solid tumors in children.

The frequency of acute leukaemia, and the recent results of therapy are described. The requirements to achieve these relatively good results (early and exact diagnosis, intensive therapy, prophylaxis and treatment of side effects, mental and physical rehabilitation) are also discussed.


[How can the largest possible number of people be reached by a prophylaxis program in a country with a scattered population?].

Two prophylactic programs are described suited to Icelandic conditions: a basic program for the majority of children and youth, and an additional program for individuals who have unusually high caries activities, -called the risk-group. The basic program consists of a) information distributed to groups on diets, including a restricted use of carbohydrates, b) instructions in dental hygiene, c) regular use of fluoride in the form of mouth-wash or tooth-brushing with a fluoride-solution. The additional program aims at individual motivation, explanation of cause and effect and intensive fluorideutilization.


On subthreshold solutions of the Hodgkin-Huxley equations.

Subthreshold solutions of the Hodgkin-Huxley equations are considered here by means of the linearized forms of these equations. An asymptotic theory is obtained, based on dimensional analysis and scaling arguments. Explicit expressions for the crest speed are obtained and are shown to be in good agreement with experiment, with computation, and with an exact asymptotic value which is also obtained here.


Netropsin-poly(dA-dT) complex in solution: structure and dynamics of antibiotic-free base pair regions and those centered on bound netropsin.

The biphasic duplex-to-strand transition for the netropsin.poly(dA-dT) complex, phosphate/drug mole ratio (P/D) = 50, has been investigated by high-resolution proton nuclear magnetic resonance (NMR) spectroscopy at the nonexchangeable base and sugar protons in 0.1 M cacodylate solution. The NMR spectral parameters monitor the structure and dynamics of the opening of antibiotic-free base pair regions (55 degrees-65 degrees) and the opening of base regions centered on bound netropsin (90 degrees-100 degrees). The gradual addition of netropsin to poly(dA-dT) results in structural perturbations extending into the antibiotic-free base pair regions that begin to level off above 0.02 antibiotic molecules per polynucleotide phosphate (P/D = 50). The NMR chemical shift parameters at the antibiotic-free base pair regions in the P/D = 50 complex suggest changes in the glycosidic torsion angles of the deoxyadenosine and thymidine residues and less pronounced changes in the base pair overlap geometries. The dissociation rates of the antibiotic-free base pair regions are at least an order of magnitude slower in the P/D = 50 netropsin.poly(dA-dT) complex compared to related parameters for poly(dA-dT) and the P/D = 50 ethidium bromide-poly(dA-dT) complex. There is decreased segmental mobility at the antibiotic-free strand regions in the temperature range (65 degrees-90 degrees) between the two transitions in the biphasic melting curve of the P/D = 50 netropsin-poly(dA-dT) complex. Netropsin stabilizes at least five base pairs, with their center at its binding site.


Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale.

A simple technique is described that uses a continuous wave laser with electromechanical modulation to obtain time-resolved Raman spectra of transient species on the millisecond timescale. The time behavior of the vibrational bands of the intermediates involved in the proton pumping of bacteriorhodopsin is determined. From these results, along with resonance enhancement and power dependence studies, the bands that appear in the continuous wave Raman spectrum of bacteriorhodopsin can be assigned to three intermediates in the photochemical cycle of bacteriorhodopsin, bR570, bL550, and bM412. The Raman spectra of bR570 and bM412 are compared with published spectra of model Schiff bases of all-trans and 13-cis retinal.


Light-regulated permeability of rhodopsin:egg phosphatidylcholine recombinant membranes.

Purified rhodopsin was incorporated into phospholipid bilayers of egg phosphatidylcholine to give recombinant membrane vesicles, which were examined by proton and phosphorus nuclear magnetic resonance spectroscopy. Increased rhodopsin content in the membranes appears to progressively inhibit the molecular motions of the methyl, methylene, and phosphate groups of the phospholipid molecules. This indicates that regions of the rhodopsin molecule interact in a manner that affects the phospholipids from the aqueous interface to the bilayer midline. In the dark, the recombinant vesicles were sealed to europium, manganese, or cobalt ions. Light exposure allowed rapid equilibration of Mn2+ and Co2+, and somewhat slower equilibration of Eu3+ across the membrane. Light changed the membrane permeability, and the gradient in chemical potential resulted in a net ion movement across the rhodopsin:phospholipid recombinant membrane. The results suggest rhodopsin is a transmembrane protein.


"Viral" expansion of enzyme flux and use of quasi-chemical approximation for two-state enzymes with enzyme-enzyme interactions.

Two examples of enzyme systems with interactions, at steady state, are treated here. In both cases, the enzyme cycle has two states and quasi-equilibrium in spatial distributions obtains at steady state (because f alpha + f beta = 1). The first example is a dilute solution of enzyme molecules in a solvent. The flux (turnover) per molecule is expanded in powers of the enzyme concentration (a "viral" expansion). Aggregation of the enzyme molecules in solution is considered as a special case. In the second example, we treat an arbitrary lattice of enzyme molecules, with nearest-neighbor interactions, using the well-known quasi-chemical approximation. The flux per molecule is obtained. Critical behavior and hysteresis are illustrated.


Transition state structures of a dipeptide related to the mode of action of beta-lactam antibiotics.

The tetrahedral adducts formed during nucleophilic attack by a hydroxyl ion on the carbonyl carbon of a model dipeptide, glycylglycine, were studied by modified-intermediate-neglect-of-differential-overlap molecular orbital calculations. This dipeptide is taken to represent the D-alanyl-D-alanine terminus of the polypeptides involved in the cross-linking transpeptidation reaction of peptidoglycan in bacterial cell walls. It was found that nucleophilic attack on one face of the carbonyl carbon leads to a transition intermediate species structurally similar to that afforded by the bicyclic nucleus of penicillins and cephalosporin antibiotics. The results support the concept that the beta-lactam antibiotics, which are known to inhibit various bacterial cell wall enzymes, may act as transition state analogs. Also, the structure formed from nucleophilic attack on the so-called alpha face of the dipeptide is more similar to the antibiotic structures than is that from attack on the opposite face. In agreement with other types of experiments, the results suggest that the alpha face may be the one approached by a nucleophile in the receptor site(s) of the appropriate cell wall enzymes.


Hydrophobicity, hydrophilicity, and the radial and orientational distributions of residues in native proteins.

The radial distributions of the Calpha and side-chain atoms in a sample of 13 native proteins have been examined. It is found that there are substantial differences in behavior between different atoms of the same amino acid. In particular, the Calpha atoms of polar residues show no particular preference for being far from the center of mass. In light of these results, a new criterion for hydrophobicity and hydrophilicity is proposed--namely, the orientational preference of the side chain. The distribution of this property is shown, and it is suggested that this provides a basis for incorporating hydrophobic interactions into a protein folding algorithm.


Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation.

A study was carried out on mechanisms, independent of activated Factor XI, capable of activating Factor IX. The reaction product of tissue factor and Factor VII functioned as a potent Factor IX activator in the assay system used. Activated Factor IX itself activated Factor X; thrombin failed to activate Factor IX. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis confirmed that the reaction product of tissue factor and Factor VII activated Factor IX, with replacement of the band corresponding to native factor IX [molecular weight (Mr) 55,000] by bands corresponding to the heavy chain (Mr 27,000) and light chain (Mr 17,000) of activated Factor IX. When either Factor VII or calcium ions were left out of incubation mixtures, the band of native Factor IX persisted unchanged. Contact of blood with tissue factor represents a second mechanism, bypassing activated Factor XI, for the activation of Factor IX during hemostasis. It may help to explain the discrepancy between the mild bleeding of hereditary Factor XI deficiency and the severe bleeding of hereditary Factor IX deficiency.


Origin of replication of pBR345 plasmid DNA.

A small (approximately 1100 base pairs) ColE1-type plasmid, pBR345, was constructed from plasmid pMB1 by a series of in vitro recombinant manipulations. Approximately 9% of the supercoiled pBR345 DNA obtained from cultures amplified with chloramphenicol appears to be replicative intermediates with replicating "eye" structures of uniform size. Results obtained from electron microscopy and biochemical analyses have enabled us to localize the origin of replication at the same position as that reported for ColE1. A sequence of 420 nucleotides surrounding this origin has been determined. A comparison between this sequence and the one determined for the origin of replication of ColE1 is presented.


Identification of the major adducts formed by reaction of benzo(a)pyrene diol epoxide with DNA in vitro.

Covalent binding of the benzo[a]pyrene metabolite (+/-)7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to calf thymus DNA was investigated. Enzymatic hydrolysis of the carcinogen-modified DNA and subsequent separation via reversed-phase high-pressure liquid chromatography resulted in the detection and isolation of seven distinct products. High-resolution mass spectrometry indicates that these products are covalent adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine. The deoxyguanosine and deoxyadenosine adducts involve binding between the activated hydrocarbon (benzo[a]pyrene diol epoxide) and exocyclic amino groups of the respective purines.


Phage T4-modified RNA polymerase transcribes T4 late genes in vitro.

Initiation of T4 late RNA synthesis has been achieved in an in vitro system prepared from Escherichia coli cells infected with wild-type or maturation-defective mutant T4 phage. The system uses a cellophane membrane as a mechanical support for concentrated cell lysates and for added streptolydigin-resistant RNA polymerases. Transcriptional activity and selectivity of added RNA polymerases are tested while endogenous RNA polymerase activity is inhibited by streptolydigin. T4-modified RNA polymerase is required for substantial stimulation of T4 late RNA synthesis.


Spatial requirement for coupling of iodotyrosine residues to form thyroid hormones.

A linear random copolymer of tyrosine and lysine and two synthetic oligopeptides containing two tyrosine residues in addition to lysine residues give thyroid hormone (thyroxine and triodothyronine) residues in good yield upon enzymatic iodination with thyroid peroxidase. These synthetic peptides may serve as simple models for thyroglobulin, the protein in which biosynthesis of the thyroid hormone takes place. For the formation of significant amounts of hormone, such model compounds must contain at least two properly spaced tyrosine residues.


An internal deletion mutant of a myosin heavy chain in Caenorhabditis elegans.

Unc-54 I is the structural gene for a myosin heavy chain present in a major fraction of the total myosin of Caenorhabditis elegans. The allele e675, which possesses a normal amount of myosin but fails to assemble thick filaments, has been shown previously to contain a novel heavy chain of molecular weight 2 X 10(5), shorter by 10(4) than the wild-type (N2) unc-54 gene product. The structural alteration of the E675 heavy chain is an internal deletion of 10(4) molecular weight near the COOH terminus of the molecule. This has been determined by mapping the partial digestion products of heavy chain fragments labeled specifically at their NH2 termini.


Lipoprotein lipase cofactor activity of a carboxyl-terminal peptide of apolipoprotein C-II.

Apolipoprotein C-II (apoC-II) is a small protein found associated with the plasma lipoproteins. It serves a unique function in the activation of the enzyme lipoprotein lipase (triacylglycerol acyl-hydrolase, EC 3.1.1.3). ApoC-II contains a single arginine residue, permitting tryptic cleavage into two peptides after succinylation of the native protein. The succinylated amino-terminal peptide, approximately 50 residues, did not activate lipoprotein lipase. The succinylated carboxyl-terminal peptide, about 29 residues, had significant cofactor activity. Relative to native apoC-II, the maximal activation observed with the succinylated carboxyl-terminal peptide was 50% lower and the concentration required for half-maximal activity was approximately 10 times higher. Mixtures of the carboxyl- and amino-terminal peptides had no more activity than the carboxyl-terminal peptide alone. Localization of functional properties to the carboxyl region is a feature also common to apolipoproteins C-III, A-II, and A-I.


Regulation of proline oxidase activity by lactate.

We found that proline oxidase, the first enzyme of the proline degradative pathway, is inhibited by lactate. The Km of the enzyme for proline increases with increasing concentrations of lactate. Since proline can be a source for gluconeogenesis, regulation of proline degradation by lactate may serve as a mechanism for allocation of metabolic fuel sources. The marked inhibition of proline oxidase at levels of lactate that commonly occur in both genetic and acquired lactic acidosis may cause the previously unexplained hyperprolinemia seen in these metabolic disorders.


Retinoic acid (vitamin A acid) induced transcriptional control of interferon production.

The production of interferon was used to study the site and mechanism of action of retinoic acid (vitamin A). The data are consistent with a site of action at the gene level, because it appears that interferon production is blocked at the transcriptional step by a retinoic acid-induced protein. (i) The effect of retinoic acid is probably on an early cellular function associated with interferon production rather than an effect on the inducer [virus or poly(I).poly(C)]. (ii) The suppression of interferon production by retinoic acid is blocked by cycloheximide, indicating that a newly synthesized protein (repressor) mediates the suppression. (iii) When allowances are made for the time required for the synthesis of the retinoic acid-induced protein, the time course of retinoic acid suppression of interferon production is superimposable on the time course of actinomycin D suppression because the slopes are parallel. These data provide evidence for transcriptional control of a specific protein (interferon) by retinoic acid. Additionally, they support the existence of transcriptional control of interferon production after addition of inducer.


Developmental control of messenger RNA for hepatic tryptophan 2,3-dioxygenase.

The enzyme tryptophan 2,3-dioxygenase [EC 1.13.11.11; L-tryptophan:oxygen 2,3-oxidoreductase (decyclizing)] first appears in the livers of young rats around the 15th postnatal day, and increases to the adult level by the 22nd day. Studies have shown that the appearance and subsequent development of the enzyme activity result from an increase in the rate of its synthesis and thus in the amount present in the liver. In this study, we have attempted to ascertain whether the appearance and development of tryptophan 2,3-dioxygenase mRNA coincided with, and thus led to, the development of enzyme activity, or whether the biosynthesis of this enzyme was due to a developmental event enabling translation of a preexisting, sequestered, reservoir of its mRNA. Using a cell-free protein-synthesizing system based on a wheat germ S30 supernatant, we measured the level of tryptophan 2,3-dioxygenase mRNA in the livers of rats between 0 and 22 days of age. We found that functional tryptophan 2,3-dioxygenase mRNA is not detectable in rat liver until the 15th postnatal day. It increases to the adult level by the 22nd postnatal day, in parallel with the enzyme. The appearance and development of tryptophan 2,3-dioxygenase are the direct consequence of the parallel appearance and development of its mRNA. It has been shown that glucocorticoids, which induce tryptophan 2,3-dioxygenase activity in adult rats, are capable of inducing the appearance of this enzyme precociously in 8- and 10-day-old rats. We have found that it is also possible to induce tryptophan 2,3-dioxygenase catalytic activity with hydrocortisone precociously in 4-day-old rats. Moreover, precocious induction of enzyme activity and the induction that occurs during the enzyme's normal developmental rise to the adult level between 15 and 22 days, are mediated through parallel increases in the level of tryptophan 2,3-dioxygenase mRNA. The present findings indicate that glucocorticoids are developmental hormones that act upon the postnatal hepatocyte to evoke elevated levels of the mRNA species coding for tryptophan 2,3-dioxygenase; the findings are compatible with the hypothesis that such hormones act by initiating and accelerating transcription of the structural genes coding for the alpha and beta protomers of this enzyme.


Distribution of rat liver albumin mRNA membrane-bound and free in polyribosomes as determined by molecular hybridization.

Recently, we purified rat liver albumin mRNA and prepared albumin [(3)H]cDNA. Using albumin [(3)H]cDNA in molecular hybridization experiments, we have now determined the distribution of albumin mRNA sequences in membrane-bound and free liver polyribosomes prepared by techniques in which there is high recovery of polyribosomes without evidence of degradation. By using molecular hybridization to measure specific mRNA sequence content, numerous problems could be avoided in interpretation of results as obtained by cell-free protein synthesis or immunological methods. Under these conditions, 98% of albumin mRNA sequences in polyribosomes are found in the membrane-bound fraction.


Sites of transcription initiation in vivo on Xenopus laevis ribosomal DNA.

We report the results of a novel method for locating sites of transcription initiation using a complex of capping enzymes from vaccinia virions that catalyze the reaction pppG + S-adenosylmethionine + (p)ppXpYpZp..... -->(7m)GpppXpYpZp..... [Ensinger, M. J., Martin, S. A., Paoletti, E. and Moss, B. (1975) Proc. Natl. Acad. Sci. USA 72, 2525-2529]. This enzyme complex will cap di- or triphosphate termini but will not cap monophosphate or hydroxyl termini. Xenopus laevis 40S precursor rRNA from oocytes is capped by these enzymes, and we conclude that it has 5'-polyphosphate termini. Therefore, 40S RNA must represent the primary transcript of amplified X. laevis ribosomal DNA. The majority of 40S molecules with polyphosphate termini begin with the sequence (p)ppAAG. There is evidence, however, that the 5' terminus may be heterogeneous. The majority of all detectable initiation events were localized close to the region coding for the 5' end of the 40S RNA. No initiation sites were detected in the nontranscribed spacer, but an apparent initiation site in the middle of the transcribed region was also observed.


Defects of glycosyltransferase activities in human fibroblasts of Pk and p blood group phenotypes.

We demonstrate that human fibroblasts of the rare Pk phenotype lack globoside, which was identified as the blood group P antigen, and that p cells possess neither globoside nor trihexosyl ceramide, which was identified as Pk antigen. Our investigations indicate also that these glycosphingolipid patterns are most likely caused by inherited preferential biosynthetic pathways in the abnormal phenotypes rather than by excess catabolism of the antigens. Evidence is presented that the fibroblasts of Pk phenotype lack beta-N-acetylgalactosaminyltransferase (globoside synthetase; UDP-N-acetylgalactosamine:trihexosylceramide beta-N-acetylgalactosaminyltransferase; EC 2.4.1.79) activity, and those of p are deficient in alpha-galactosyltransferase (trihexosylceramide synthetase; UDP galactose:lactosylceramide alpha-galactosyltransferase) and possibly also in globoside synthetase. The diminished globoside synthetase activity in p cells, however, is not caused by the defect in the gene coding for this enzyme. It appears, rather, to be caused by a failure in gene expression because one-third of Pk X p hybrids became able to express P antigenicity with a time lag of 3-4 days after cell fusion [Fellous, M., Gerbal, A., Nobillot, G. & Weils, J. (1977) Vox Sang. 32, 262-268].


High resolution two-dimensional electrophoresis of human plasma proteins.

The two-dimensional electrophoretic technique of O'Farrell has been adapted to the analysis of human plasma proteins, and 30 polypeptides have been identified in the pattern produced. Genetic variants involving charge (isoelectric point) or size (molecular weight in the presence of sodium dodecyl sulfate) changes should be routinely detectable in at least 20 proteins at once, facilitating studies of human mutation rates.


Orientation of bacteriorhodopsin in Halobacterium halobium as studied by selective proteolysis.

The orientation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by proteolytic degradation of purple membrane sheets, reconstituted vesicles, and whole cells, with the following results: (i) Bacteriorhodopsin in purple membrane sheets is cleaved at a single site by Pronase or trypsin; a polypeptide segment of about 15 amino acids is lost from the carboxyl end. Carboxypeptidase A sequentially releases amino acids from the carboxyl end; the tetrapeptide sequence -Ala-Ala-Thr-Ser(COOH) was tentatively deduced for this terminus. (ii) The apomembrane, which lacks retinal, undergoes a second cleavage with trypsin releasing a fragment of approximately 6300 molecular weight from the amino terminus. (iii) Vesicles reconstituted from the purple membrane sheets and synthetic lecithins, in which the direction of proton pumping is opposite to that in the whole cells, have the carboxyl terminus of bacteriorhodopsin accessible to proteolysis. (iv) In envelope vesicles, which largely pump protons in the same direction as the whole cells, the carboxyl terminus is largely protected against proteolysis. (v) Treatment of whole cells with proteinase K hydrolyzes the cell wall proteins but has no effect on acteriorhodopsin. However, the same treatment after lysis of the cells results in degradation of the hydrophilic region at the carboxyl terminus. The results show that the carboxyl terminus as well as the additional cleavage site near the amino terminus observed in apomembrane are on the cytoplasmic side of the purple membrane.


Oligopeptides as potential antiaggregation agents for deoxyhemoglobin S.

Oligopeptides that mimic segments of the amino acid sequence of hemoglobin S at potential contact sites can be used to inhibit aggregation. These oligopeptide inhibitors raise the minimum gelling concentration of deoxyhemoglobin S so that chemical modification does not have to be used. The hexapeptide amides of both betaS 1-6 which is believed to be one of the contact areas among aggregates, and betaA 1-6 of hemoglobin A increase the minimum gelling concentration by more than 70%. The hexapeptide amide beta79-84 behaves like beta1-6 (beta being betaS or betaA). Shorter oligopeptides, such as betaS3-6, are less effective as an inhibitor but longer ones, such as betaS1-8, are no more effective than beta1-6. Permutations of the sequence, such as betaS125634, do not alter the percent increase in the minimum gelling concentration. Leu- and Met-enkephalin increase the minimum gelling concentration just as beta1-6 does, but (Pro)6 is not very effective. Thus, the use of complementary oligopeptides as inhibitors is extended to include certain "flexible" peptides, which can adapt themselves to interfere with the molecular contacts and thereby gelation of deoxyhemoglobin S.


The bicistronic nature of lens alpha-crystallin 14S mRNA.

The A2 and B2 polypeptide chains of calf lens alpha-crystallin are synthesized on a 14S, 1500-nucleotide mRNA and a 10S, 735-nucleotide mRNA, respectively. The 10S mRNA is theoretically compatible with the size of the B2 chain, but the 14S mRNA contains approximately twice the required number of nucleotides necessary for A2 chain synthesis. This fact raises the question of the function of the additional nucleotide sequence in the 14S mRNA. The following observations on 14S mRNA suggest that it may contain an additional cistron. (i) Under a number of denaturing conditions, 14S mRNA continues to retain its initial size characteristics. (ii) In addition to synthesis of the A2 chain, 14S mRNA directs the synthesis of another polypeptide with the same electrophoretic mobility as that of the B2 chain. (iii) Molecular hybridization of the 14S mRNA with the cDNA produced from the 10S mRNA suggests that 2 mol of the cDNA bind to 1 mol of the 14S mRNA. (iv) Examination of the nucleotide sequences of the 10S and 14S mRNAs by two-dimensional maps of RNase A and T1 digests indicates marked similarity. The overall data suggest that the additional cistronic component may carry coding information for an alpha-crystallin polypeptide or a closely related polypeptide species.


DNA sequencing with chain-terminating inhibitors.

A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.


Hepatic mitochondrial cytochrome P-450: isolation and functional characterization.

A CO-binding heme protein was solubilized and partially purified from the inner membrane fraction of rat liver mitochondria by a modification of a method [Imai, Y. & Sato, R. (1974) Biochem. Biophys. Res. Commun. 60, 8-14] developed to purify cytochrome P-450 from liver microsomes. The partially purified preparation contained protoheme and its spectral properties are characteristic of the heme proteins of the cytochrome P-450 family. The isolated cytochrome P-450 preparation could reconstitute a CO-sensitive, NADPH-dependent 26-hydroxylation activity for 5beta-cholestane-3alpha,7alpha,-12alpha-triol when supplemented with NADPH-adrenodoxin reductase and adrenodoxin, both purified from bovine adrenocortical mitochondria. Unlike a cytochrome P-450 purified from liver microsomes of drug-untreated rats, however, the liver mitochondrial cytochrome P-450 could not catalyze NADPH-dependent benzphetamine N-demethylation in the presence of adrenodoxin reductase and adrenodoxin or function with the purified microsomal NADPH-cytochrome c reductase plus Emulgen 913 as an electron-donating system. It is concluded that the rat liver inner mitochondrial membrane houses a species of cytochrome P-450 functional in 5beta-cholestane-3alpha,7alpha,12alpha-triol 26-hydroxylation.


Two distinct adenosine-sensitive sites on adenylate cyclase.

The effects of adenosine and adenosine analogs on adenylate cyclases from several tissues have been examined. Two adenosine-reactive sites have been identified: (i) the "R" site, occupancy of which usually leads to activation of cyclase and which requires integrity of the ribose ring for activity, and (ii) the "P" site, which mediates inhibition and requires integrity of the purine ring for activity. Biphasic effects of adenosine are explained by the presence of both sites on a single adenylate cyclase. Comparison of these data with those in the literature indicates that adenosine-reactive "P" and "R" sites are present generally.


Presence of protein A24 in rat liver nucleosomes.

Two-dimensional gel profiles of the 0.2 M H2SO4-soluble proteins of monomer nucleosomal fractions were found to contain protein A24. Protein A24 is of interest because it is composed of histone 2A and "ubiquitin", apparently joined by an isopeptide linkage [Goldknopf, I.L. & Busch, H. (1977) Proc. Natl. Acad. Sci. USA 74, 864-868; Hunt, L.T. & Dayhoff, M.O. (1977) Biochem. Biophys. Res. Commun. 74, 650-655]. Monomer nucleosomal fractions were obtained by sucrose density gradient centrifugation of micrococcal nuclease digests of rat liver nuclei. As shown by their DNA size, the monomer fractions were highly purified. Proteins A24 and Bu, another protein of unknown characteristics, were found along with histones 1, 2A, 2B, 3, and 4 in the monomer fractions in relative amounts similar to those found in extracts from whole nuclei and chromatin. Other acid-soluble proteins found in the nuclear and chromatin extracts were essentially absent from the monomer fraction. Inasmuch as protein A24 and Bu were found in lesser amounts than the histones, it is suggested that they are associated with specialized subsets of nucleosomes.


Alternative aspirins as antisickling agents: acetyl-3,5-dibromosalicylic acid.

Acetyl-3,5-dibromosalicylic acid (dibromoaspirin) is shown to be a potent acylating agent of intracellular hemoglobin in vitro. Transfer of the actyl group of dibromoaspirin to amino groups of hemoglobins A and S seems to occur predominantly at just two or three sites on these proteins. This acetylation produces moderate increases in the oxygen affinities of normal and sickle erythrocytes. Furthermore, treatment of intracellular hemoglobin S with dibromoaspirin directly inhibits erythrocyte sickling. This antisickling effect is paralleled by an increase in the minimum gelling concentration of deoxy hemoglobin S extracted from sickle erythrocytes that had been exposed to low concentrations of dibromoaspirin. These observations suggest that dibromoaspirin might be an effective antisickling agent in vivo.


Nucleotide sequence of 5' terminus of alfalfa mosaic virus RNA 4 leading into coat protein cistron.

The sequence of the 5'-terminal 74 nucleotides of alfalfa mosaic virus RNA 4, the mRNA for the viral coat protein, has been deduced by using various new techniques for labeling the RNA at the 5' end with 32P and for sequencing the 5'-32P-labeled RNA. The sequence is NpppGUUUUUAUUUUUAAUUUUCUUUCAAAUACUUCCAUCAUGAGUUCUUCACAAAAGAAAGCUGGUGGGAAAGCUGG. The AUG initiator codon is located 36 nucleotides in from the 5' end; the nucleotide sequence beyond corresponds to the amino acid sequence of the coat protein. This 5' noncoding region is rich in U (58% U); except for the 5'-terminal G, the next G in is part of the initiator AUG codon.


New method for localizing proteins in periodic structures: Fab fragment labeling combined with image processing of electron micrographs.

Fab fragments prepared from antisera directed against purified bacteriophage T4 structural proteins and head-related structures were used to label proteins on the surface of T-even giant phage capsids. Optically filtered electron micrographs of the Fab-labeled capsids reveal both the location of specific proteins within the capsomeres and differing conformational states of the protein subunits. We describe parameters affecting the utility of this technique for the study of molecular organization and protein conformation in periodic biological structures.


Histone H3 disulfide dimers and nucleosome structure.

The arginine-rich histone, H3, isolated from avian erythrocytes, can dimerize by forming a disulfide linkage between the single cysteine sulfhydryl residues at position 110 of the H3 polypeptide chain. The H3 dimer can be substituted for undimerized H3 in experiments in which the nucleosome is reconstituted from DNA and mixtures of the four "core" histones, H2A, H2B, H3, and H4. We report here that reconstituted nucleosomes containing H3 dimer are indistinguishable, by a number of criteria, either from native nucleosomes or from reconstitutes containing H3 monomer. The criteria include the pattern of susceptibility of the complex to nucleases, the amount of DNA supercoiling induced by histone binding, and the hydrodynamic properties of reconstituted nucleosome "core" preparations. The results suggest that the residues in the neighborhood of position 110 on each H3 molecule are in close contact in the nucleosome. If, as has been proposed, the nucleosome has a dyad axis, then the disulfide bridge between H3 molecules must lie on this axis.


Muscarinic acetylcholine receptors of the developing retina.

Six- and 13-day chicken embryo retinas contain 10 and 320 fmol per mg of protein of specific binding sites for 3-[3H]quinuclidinyl benzilate, a ligand of muscarinic acetylcholine receptors. Most of the receptors of 13-day embryo retina were found, by autoradiography, to be localized in two sharp bands within the inner synaptic layer of the retina. In the adult, the receptors were found almost exclusively in three bands in the inner synaptic layer of the retina. A possible mechanism for generating sets of stratified or columnar neurons and relating one set to another is proposed.


Reversible and irreversible inhibition by anesthetics of the calcium-induced luminescence of aequorin.

The actions of various kinds of so-called membrane-stabilizing drugs on Ca2+-induced flash intensity of purified aequorin, a photoprotein obtained from Aequorea aequorea, were examined. All drugs used in this experiment, including inhalational anesthetics, tetracaine, chlorpromazine, and morphine, depressed flash intensity, and the inhibition increased with time. The inhibition followed a mixture of reversible and irreversible kinetics. A method of analyzing irreversible kinetics is presented. The dissociation constants for reversible inhibition and the rate constants for irreversible inhibition are presented for each agent.


Electron microscopy of fibers and discs of hemoglobin S having sixfold symmetry.

Aggregated forms of deoxyhemoglobin S were examined with a field emission transmission electron microscope. Images of isolated helical fibers were obtained from sickled cell lysates stained directly on the electron microscope grid. Optical and digital analyses of the electron micrographs showed that the fibers are similar to those characterized by J. T. Finch, M. F. Perutz, J. F. Bertles, and J. Döbler [(1973) Proc. Natl. Acad. Sci. USA 70, 718-722] in that they consist of stacked discs each composed of six hemoglobin molecules. The fibers exhibit an outer diameter of 160-170 A and an inner diameter of about 60 A with an axial spacing of 58 A per disc. The fiber can be described as a helix consisting of 56 discs per helical turn. We observed discs of six hemoglobin molecules, which may be stable substructural components of the fibers. They were observed in preparations of hemoglobin fibers and exhibited 6-fold symmetry by power spectrum analysis. A reconstructed image of a disc digitally filtered for 6-fold symmetry has a maximum external diameter of approximately 170 A and a central hole of 60 A diameter and is similar to the axial projection of a single disc from a low-resolution, three-dimensional reconstructed model of a fiber.


Random walk analysis of potassium fluxes associated with nerve impulses.

We present a novel recursion method for obtaining theoretical expressions for unidirectional single-file fluxes of ions through narrow membrane channels containing an arbitrary number of ion sites. The theory is applied to experimental tracer fluxes associated with nerve impulses from cephalopod giant axon membranes at various temperatures between 7 degrees and 27 degrees. The comparison between the theoretical and experimental one-way fluxes suggests that the potassium channel in nerve membrane contains three ion sites, which is consistent with the deduction by Hodgkin and Keynes that the potassium channel contains two or three sites on the basis of the ratio of tracer influx to tracer efflux. The analytical results in this paper provide a further test of the single-file model for nerve and other membrane preparations.


Genetic dissection of the control of normal differentiation in myeloid leukemic cells.

Normal myeloid precursors and MGI(+)D(+) myeloid leukemic cells can be induced to differentiate to mature cells by the normal protein inducer MGI. The sequence of differentiation is the induction of C3 and Fc rosettes, C3 and Fc immune phagocytosis (IP), synthesis and secretion of lysozyme, and formation of mature macrophages and granulocytes. Mutant clones of myeloid leukemic cells have been isolated with differences in the time of induction of C3 and Fc rosettes and C3 and Fc IP, in which lysozyme was induced without going through the stage of Fc or C3 IP, and with differences in inducibility by MGI to mature macrophages or granulocytes. Only one out of five MGI(-)D(-) clones gave rise to MGI(+)D(+) mutants. The ability to obtain mutants from this clone was associated with its special chromosome constitution, and these mutants showed a change in their ability for cap formation by concanavalin A. The steroid inducer dexamethasone can induce in MGI(+)D(+) clones differentiation to macrophages but not to granulocytes. Differentiation by steroid inducer in different clones occurred either with or without induction of Fc rosettes and Fc IP, and induction of C3 rosettes was not always associated with induction of C3 IP. The use of mutants that differ in their competence to be induced by MGI or steroid inducer has shown that there are separate controls for the induction of C3 and Fc rosettes, C3 and Fc IP, lysozyme, macrophages, and granulocytes.


Nerve growth factor prevents vinblastine destructive effects on sympathetic ganglia in newborn mice.

Vinblastine injections in newborn mice produce severe atrophy of sympathetic ganglia; after a 7-day treatment the ganglia are 80% reduced in volume. Histological examinations show that this effect is due to a marked decrease in the neuronal cell population. The most precocious ultra-structural alterations are localized in the nuclear compartment, followed by axonal swelling and microtubule disappearance. Simultaneous injections of nerve growth factor entirely prevent the noxious effects of the vinca alkaloid, and result in partial appearance of the growth effects of nerve growth factor. Such protective action is not due to inhibition of vinblastine uptake which is the same in control mice and in mice pretreated with nerve growth factor. It is suggested that nerve growth factor prevents the vinca alkaloid action by favoring the assembly or organization, or both, of microtubules, which, from in vitro studies, have been proved to be inaccessible to vinblastine.


Mosaic mice with teratocarcinoma-derived mutant cells deficient in hypoxanthine phosphoribosyltransferase.

Mutagenized stem cells of a cultured mouse teratocarcinoma cell line were selected for resistance to the purine base analog 6-thioguanine. Cells of a resistant clone were completely deficient in activity of the enzyme hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), the same X-linked lesion as occurs in human Lesch-Nyhan disease. After microinjection into blastocysts of another genetic strain, the previously malignant cells successfully participated in normal embryogenesis and tumor-free, viable mosaic mice were obtained. Cells of tumor lineage were identified by strain markers in virtually all tissues of some individuals. Mature function of those cells was evident from their tissue-specific products (e.g., melanins, liver proteins). These mutagenized teratocarcinoma cells are therefore developmentally totipotent. Retention of the severe HPRT deficiency in the differentiated state was documented in extracts of mosaic tissues by depressed specific activity of the enzyme, and also by presence of unlabeled clones in autoradiographs of explanted cells incubated in [(3)H]hypoxanthine. Some mosaic individuals had mutant-strain cells in only one or a few tissues. Such animals may provide unique opportunities to identify the tissue sources of particular aspects of the complex disease syndrome. The tissue distribution of HPRT-deficient cells suggests that selection against them is particularly strong in blood of the mosaic mice, as is already known to be the case in human heterozygotes. This phenotypic parallelism supports the expectation that afflicted F(1) male mice that might be obtained from mutant germ cells can serve as a model of the human disease.


Angiotensin stimulation of bovine adrenocortical cell growth.

Factors controlling proliferation of adrenocortical cells have been studied in monolayer cultures of bovine adrenocortical cells. Angiotensin II stimulated cell proliferation and [3H]thymidine incorporation into DNA with a half-maximal effective concentration of 0.96 +/- 0.27 nM. Similar sensitivity to angiotensin III with reduced sensitivity to angiotensin I and tetradecapeptide renin substrate was observed. Although sensitivity to angiotensin II was equivalent to that for fibroblast growth factor (1.5 nM half-maximal effective concentration), maximal effects of angiotensin were less than for fibroblast growth factor and serum. High concentrations of insulin (1-10 micrometer) also stimulated [3H]thymidine incorporation into DNA and cell proliferation. [Sar1,Ile5,Ile8]Angiotensin II, a competitive antagonist of angiotensin II, blocked angiotensin II stimulation of DNA synthesis but did not affect fibroblast growth factor and insulin stimulation of DNA synthesis. Corticotropin (ACTH) blocked the stimulatory effects of both angiotensin II and fibroblast growth factor. The dose-response curves for angiotensin II stimulation of steroidogenesis were similar to those for stimulation of [3H]thymidine incorporation into DNA. Among the seven cell types examined, only adrenocortical cells responded to angiotension II with stimulation of DNA synthesis.


Evidence that pyrimidine dimers in DNA can give rise to tumors.

The Amazon molly, Poecilia formosa, is a small fish that grows in clones. Hence, cells from one animal may be transplanted to another without danger of rejection. Cells from thyroid and adjacent tissue were irradiated with UV light in vitro and injected into the abdominal cavity of isogeneic recipients. At appropriate UV doses and numbers of cells injected, all recipients showed exuberant thyroid proliferation. We give arguments and data indicating that the proliferation is a tumor, not a goitrogenic response. If the UV irradiation is followed, but not preceded, by photoreactivating illumination, the yield of thyroid growths is markedly decreased. Because other investigations have shown that photoreactivation monomerizes UV-induced cyclobutylpyrimidine dimers in DNA and does not affect other photoproducts, our data indicate that pyrimidine dimers in DNA can give rise to tumors.


DNA degradation in terminally differentiating lens fiber cells from chick embryos.

During the terminal differentiation of lens fiber cells, nuclear DNA is known to accumulate free 3'-OH ends and is progressively lost from the nucleus. Toward the end of this process, nuclei undergo pycnosis and disappear. The size of the DNA in the epithelia and in early and late stages of fiber cell development was examined by electrophoresis on nondenaturing agarose/polyacrylamide gels. Low molecular weight DNA of discrete sizes appears only at the final stages of nuclear degeneration in central fiber cells and persists after the disappearance of the nuclei. These low molecular weight DNA fragments appear as multiples of a monomeric unit and are similar to the fragments produced by the digestion of epithelial cell nuclei by micrococcal nuclease. The data indicate that in lens fiber nuclei the double-strand breaks in vivo affect the chromatin during nuclear degeneration, and the data suggest that the DNA of these cells is organized into chromatin composed of discrete subunits.


Transitions in collagen types during matrix-induced cartilage, bone, and bone marrow formation.

The localization of types I, II, and III collagens during bone matrix-induced sequential differentiation of cartilage, bone, and bone marrow was studied by specific immunofluorescence. Subcutaneous transplantation of coarse powders of demineralized rat bone matrix into allogeneic recipients resulted in new bone formation. After a transient appearance of polymorphonuclear leukocytes in the implant, fibroblasts appeared in close continguity to the matrix on day 3. Type III collagen was then localized as a fine network around the invading fibroblasts. On days 4--6 smaller amounts of type I were also detected around these proliferating cells. With the onset of chondrogenesis, type II collagen was detected in the cartilage matrix on day 6 and persisted until the early stages of bone formation. Vascular invasion of the implant was accompanied by osteogenesis on day 10. Type I collagen was demonstrated in the newly deposited bone matrix coating the surfaces of cartilage spicules and particles of implanted bone powder. On day 17 and thereafter, type III collagen was localized as a fibrous array around nests of hematopoietic cells.


Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity.

The protease(s) responsible for removing the amino-terminal extension of nascent presecretory proteins (signal peptidase) has been extracted from rough microsomes of dog pancreas with the detergent sodium deoxycholate. Preprolactin and pre-growth-hormone, prepared by in vitro translation of bovine pituitary RNA in the wheat germ system, were used to assay signal peptidase in the extract. When added to the wheat germ system during translation, the extract reduced the size of preprolactin and pre-growth-hormone to that of prolactin and growth hormone, respectively. Post-translational addition of the extract also reduced the size of preprolactin and pre-growth-hormone to that of the authentic hormones. The prolactin produced by post-translational cleavage of radiolabeled preprolactin has been shown, by partial amino-terminal sequence analysis, to have the correct amino terminus. This post-translational assay has permitted the investigation of the subcellular localization of the enzyme. Sodium deoxycholate extracts of rough microsomes were active, whereas extracts of smooth microsomes were inactive. However, without detergent treatment, neither rough nor smooth microsomes were capable of cleaving preprolactin in the post-translational assay. From this we conclude that the signal peptidase activity is confined to the rough endoplasmic reticulum and is latent. Finally, we have detected two small peptides which we believe could be the signal peptides generated by the endoproteolytic cleavage of preprolactin and pre-growth-hormone by signal peptidase.


Liposome--lymphocyte interaction: saturable sites for transfer and intracellular release of liposome contents.

The water-soluble dye 6-carboxyfluorescein was trapped in the internal aqueous compartments of small sonicated dioleoyl lecithin vesicles and used to assess the kinetics of transfer of vesicle contents to human lymphocytes. By using flow microfluorometry, the initial rate of dye transfer to the cells was measured as a function of the concentration of vesicles in the external medium. The rate of transfer consists of at least two components, one of which saturates at high vesicle concentration and the other of which does not saturate in the range of concentrations explored. The saturable component was competitively inhibited by vesicles not containing dye. Both the saturable and nonsaturable components of transfer were inhibited by fetal calf serum or bovine serum albumin but neither component was affected by bovine IgG, choline chloride, or heparin. Pretreatment of the lymphocytes with trypsin or Pronase had no effect on either component. The saturable component can be interpreted in terms of a two-step process in which vesicles bind reversely to sites on the cell surface, and dye is then transferred into the cell from the vesicle-site complex.


Molecular evolution of human and rabbit beta-globin mRNAs.

The primary structures of human and rabbit beta-globin mRNAs are compared. Using as a standard the extent of nucleotide substitutions inferred from the hypervariable amino acid residues of fibrinopeptides A and B, which are thought to change largely by neutral evolution, we show that not all silent mutations in globin mRNA are neutral. The divergence of the sequences is limited in part by the selective usage of synonymous codons. The divergent nucleotides tend to be distributed nonrandomly: in the coding region silent substitutions are most rare in segments that are also deficient in substitutions leading to replacements.


Assignment of the human gene for galactose-1-phosphate uridyltransferase to chromosome 9: studies with Chinese hamster-human somatic cell hybrids.

Chinese hamster-human somatic cell hybrids were analyzed for the expression of human galactose-1-phosphate uridyltransferase (GALT; UDPglucose:alpha-D-galactose-1-phosphate uridyltransferase, EC 2.7.7.12) by electrophoresis and for the presence of human chromosomes cytogenetically with the aid of Q-banding. Three of the 10 randomly chosen independently derived primary hybrid lines showed the presence of human GALT. Human chromosome 9 was consistently present in the hybrid lines expressing human GALT and consistently absent in the lines not expressing it. Biochemical analysis alone of 11 independently derived hybrid lines showed human GALT to be syntenic with known chromosome 9 markers (soluble aconitase, adenylate kinase 1, and adenylate kinase 3). Previous studies on chromosome assignment of this locus, utilizing somatic cell hybrids, have yielded inconsistent results; one group assigned GALT to chromosome 2, and another assigned it to chromosome 3. However, we believe that, based on our results and other published evidence, the correct assignment of the human GALT locus is to chromosome 9.


Increased dexamethasone resistance of cystic fibrosis fibroblasts.

We demonstrate previously that fibroblasts from cystic fibrosis (CF) patients are significantly more resistant to the toxic effects of ouabain than normal human fibroblasts are. Ouabain is generally assumed to act primarily at the level of the cell membrane and to cause killing of cells by inhibiting ion transport. We have therefore examined the ability of normal and CF cells to survive exposure to ethacrynic acid, another inhibitor of ion transport, and to survive colchicine and aminopterin, resistance to which has been associated with membrane alterations in other cells. The effect of dexamethasone, which has a sterol nucleus similar to that of ouabain but is thought to have a different site of cellular action, was also tested. Exposure of the cells to ethacrynic acid, colchicine, or aminopterin did not reveal any differences in survival between normal and CF fibroblasts. However, CF cells survived exposure to dexamethasone significantly better than normal cells did. These results suggest that normal and CF cells do not differ in terms of a generalized resistance to ion transport inhibitors or to drugs that must pass through the membrane to be active. Instead, the results raise the possibility that CF cells have an enhanced resistance to drugs that have the sterol nucleus found in ouabain and dexamethasone.


Design of molecular control mechanisms and the demand for gene expression.

Regulation by a repressor protein is the mechanism selected when, in the organism's natural environment, there is low demand for expression of the regulated structural genes. Regulation by an activator protein is selected when there is high demand for expression of the regulated structural genes. These general conclusions are useful in relating physiological function to underlying molecular determinants in a wide variety of systems that includes repressible biosynthetic pathways, inducible biosynthetic enzymes, inducible drug resistance, and prophage induction, as well as inducible catabolic pathways, for which a special case of this prediction previously was reported [Savageau, M. A. (1974) Proc. Natl. Acad. Sci. USA 71, 2453-2455].


Microsurgically produced homozygous-diploid uniparental mice.

Shortly after fertilization, either the male or the female pronucleus was microsurgically removed from 202 F(1) hybrid eggs derived from crosses of two inbred strains. Subsequent incubation of these haploid eggs in medium containing cytochalasin B, which inhibits cytokinesis but not nuclear division, enabled the remaining pronucleus to become diploid. After nuclear diploidization and transfer to regular culture medium, cleavage commenced normally, and a total of 135 successfully manipulated eggs continued in development and yielded 93 morulae and blastocysts. These embryos were surgically transferred to the uteri of pseudopregnant foster mothers who gave birth to seven live female offspring. Five of the females were derived from the maternal genome (gynogenesis) and the remaining two mice inherited only the paternal genes (androgenesis), depending on whether the female or male pronucleus had been retained in the egg, respectively. Homozygosity for a number of genetic loci positioned on different chromosomes and effecting the coat color phenotype and strain-specific allelic variants of several enzymes, urinary and plasma proteins, and hemoglobins could be demonstrated unequivocally in all instances. Chromosomal analysis revealed a normal diploid karyotype including two X chromosomes. Thus far, six of the seven homozygous-diploid (isogeneic) females have proved to be fertile and have given birth to progeny corresponding only to the pronuclear genotype of the mother.


Interactions of C-reactive protein and complement with liposomes.

Interactions between C-reactive protein (CRP) and liposomal model membranes containing phosphatidylcholine were investigated. These interactions, in the presence of human serum, resulted in consumption of each of the components of the classical complement pathway (C1-C9) and also resulted in complement-dependent damage and release of trapped glucose from certain types of liposomes. CRP-initiated lysis of liposomes was strongly dependent upon membrane lipid composition. Optimal activity occurred with positively charged liposomes containing galactosylceramide (galactocerebroside); positively charged liposomes lacking galactocerebroside released much less glucose, while negatively charged liposomes, either with or without galactocerebroside, did not release glucose at all. Glucose release was inhibited by free phosphocholine. Lesser, but significant, "background" glucose release independent of the presence of CRP also was observed with positively charged liposomes containing galactocerebroside, and this was associated with marked preferential consumption of the later-acting complement components (C3-C9). C2-deficient human serum failed to support CRP-dependent glucose release, but glucose release was observed upon reconstitution of the serum with C2. Guinea pig complement also did not support CRP-mediated glucose release, but upon addition of human C1q substantial glucose release was observed. We conclude that (i) CRP can sensitize appropriate liposomes for complement-dependent damage via the primary complement pathway starting at the level of C1q; (ii) of those studied, liposomes that are most susceptible to membrane damage contain phosphatidylcholine, have a positive charge, and contain a ceramide glycolipid; and (iii) such liposomes also are sensitive, although to a much lesser degree, to complement-dependent lysis initiated in the absence of CRP and involving consumption of terminal in excess of early acting complement components.


Antigen-binding mutants of mouse myeloma cells.

A cultured mouse myeloma cell line, S107, that secretes an IgA phosphocholine-binding immunoglobulin has been cloned in soft agar and overlaid with phosphocholine-hemocyanin. Spontaneous mutants that secrete immunoglobulin with a decreased ability to precipitate antigen were detected with this plate assay and occur at a very high frequency. From one such mutant, phenotypic revertants arise spontaneously with a frequency of 0.28-2.8%. This mutant and one of its revertants were studied, and they were found to differ from the parent and from each other serologically and in antigen binding. While it is not yet clear whether these findings bear any relationship to the normal generation of antibody diversity, they do indicate that it is possible to generate antigen binding diversity in somatic cells.


Evidence for ovarian "inhibin": suppression of the secondary rise in serum follicle stimulating hormone levels in proestrous rats by injection of porcine follicular fluid.

The present studies were carried out to see if porcine follicular fluid could inhibit increases in serum follicle stimulating hormone (FSH) levels when injected into the rat. For these studies the pentobarbital-treated proestrous rat was chosen as the major test animal model. If an artificial surge of luteinizing hormone (LH) is administered to these rats, it can induce a synchronized secondary rise in FSH secretion rate. Normal saline-treated rats were also used as test animals. They exhibit preovulatory endogenous "surges" of LH and FSH, and also a secondary FSH rise. Porcine follicular fluid was harvested from medium-sized and large (3- to 10-mm diameter) follicles and treated with charcoal to remove endogenous steroids. Charcoal-treated porcine serum served as a control solution. The fluid was injected intraperitoneally in two 0.5-ml doses into pentobarbital-treated proestrous rats immediately and 3 hr after LH injection. Follicular fluid, but not the serum, suppressed the secondary, LH-induced FSH rise (P < 0.01) in a dose-dependent manner, without altering the effects of LH upon serum ovarian steroid levels or follicular rupture. It was effective down to a total dose of 200 mul. Porcine follicular fluid also blocked the secondary FSH surge in normal proestrous rats exhibiting endogenous LH/FSH primary surges. Thus, it would appear that porcine follicular fluid contains a non-steroidal substance(s) that can block the secretion of FSH that is secondary to a natural or artificial LH surge.


Demonstration of different contractile mechanisms for angiotensin II and des-Asp1-angiotensin II in rabbit aortic strips.

Evidence of selective inhibition, differences in dose-response relationships, and cross-tachyphylaxis studies suggest that separate receptors and/or mechanisms may be involved in responses to angiotensin (Ang), [Sar1]Ang II, and Ang III (= des-Asp1-Ang II). The extracellular Ca2+ requirement for contractile responses induced by angiotensin peptides and norepinephrine was determined in rabbit aortic strips. Responses to K+ and [Sar1]Ang II were attenuated markedly by treatment with SKF-525A, verapamil, or Ca2+-free buffer. The response to Ang II was not impaired by verapamil, was blocked partially by SKF-525A, and was reduced markedly in Ca2+-free medium. Norepinephrine- and Ang III-induced contractions were not dependent on extracellular Ca2+. K+, Ang II, and [Sar1]Ang II required extracellular Ca2+ to induce contraction of the rabbit aorta. The data indicate that Ang III may have a mechanism of action that differs from that of [Sar1]Ang II and Ang II.


Mapping of human chromosomal regions related to neoplasia: evidence from chromosomes 1 and 17.

In clonal aberrations leading to an excess or partial excess of chromosome 1, trisomy for bands 1q25-1q32 was noted in the myeloid cells from all of 34 patients who had various disorders such as acute leukemia, polycythemia vera, and myelofibrosis. This was not the result of a particularly fragile site in that region of the chromosome because the break points in reciprocal translocations that involve it occurred almost exclusively in the short arm. Two consistent rearrangements that have been observed in chromosome 17 produced either duplication of the entire long arm or a translocation of the distal portion of the long arm to chromosome 15. The nonrandom chromosomal changes found in hematologic disorders can now be correlated with the gene loci on these chromosomes or chromosomal segments. Seventy-five genes related to various metabolic enzymes have been mapped; it may be significant that chromosomes carrying gene loci related to nucleic acid metabolism are more frequently involved in hematologic disorders (and other malignancies as well) than are gene loci related to intermediary or carbohydrate metabolism. Furthermore, the known virus-human chromosome associations are closely correlated with the chromosomes affected in hematologic disorders. If one of the effects of carcinogens (including viruses) is to activate genes that regulate host cell DNA synthesis, and if translocations or duplications of specific chromosomal segments produce the same effect, then either of these mechanisms might provide the affected cell with a proliferative advantage.


Molecular basis of reovirus virulence: role of the S1 gene.

A genetic approach has been used to define the molecular basis for the different patterns of virulence and central nervous system cell tropism exhibited by reovirus types 1 and 3. Intracerebral inoculation of reovirus type 3 into newborn mice causes a necrotizing encephalitis (without ependymal damage) that is uniformly fatal. Animal inoculated with reovirus type 1 generally survive and may develop epedymal cell damage (without neuronal necrosis) and hydrocephalus. Using recombinant clones derived from crosses between reovirus types 1 and 3, we have been able to determine that the S1 genome segment is responsible for the differing cell tropism of reovirus serotypes and is the major determinant of neurovirulence. The type 1 S1 genome segment is responsible for ependymal damage with subsequent hydrocephalus; the type 3 S1 genome segment is responsible for neuronal necrosis and neurovirulence. We postulate that these differences are due to the specific interaction of the sigma1 outer capsid polypeptide (the protein coded for by the S1 genome segment) with receptors on the surface of either ependymal cells or neuronal cells.


Histrionicotoxin enhances agonist-induced desensitization of acetylcholine receptor.

Dihydroisohistrionicotoxin inhibits acetylcholine receptor-dependent 22Na+ uptake of cultured chick muscle cells with a KI of 0.2 micrometer. The inhibition is noncompetitive with respect to agonists. The toxin enhances desensitization of the receptor by agonists which is accompanied by a 10-fold increase in receptor affinity for agonists. Dihydroisohistrionicotoxin increases the affinity of the desensitized form of the receptor for agonists but not antagonists. The results suggest that dihydroisohistrionicotoxin inhibits the acetylcholine receptor by causing an increase in the affinity of the desensitized form of the receptor for agonists and thereby stabilizing the desensitized state.


Effect of increased potassium concentrations on particle motion within a neurosecretory structure.

Intensity fluctuation spectroscopy was used to detect particle motion within the pericardial organ of the crab Carcinus while it was perfused with media with increased potassium concentrations. Replacement of some or all of the sodium ions of the normal medium with potassium ions caused a graded increase in motion. Evidence is presented which indicates that this effect is not a consequence of membrane depolarization or calcium ion influx, but is due to the flux of potassium chloride and water into the cytoplasm of the cells.


Determination of transmitter function by neuronal activity.

The role of neuronal activity in the determination of transmitter function was studied in cultures of dissociated sympathetic neurons from newborn rat superior cervical ganglia. Cholinergic and adrenergic differentiation were assayed by incubating the cultures with radioactive choline and tyrosine and determining the rate of synthesis and accumulation of labelled acetylcholine and catecholamines. As in previous studies, pure neuronal cultures grown in control medium displayed much lower ratios of acetylcholine synthesis to catecholamine synthesis than did sister cultures grown in medium previously conditioned by incubation on appropriate nonneuronal cells (conditioned medium). However, here we report that neurons treated with the depolarizing agents elevated K(+) or veratridine, or stimulated directly with electrical current, either before or during application of conditioned medium, displayed up to 300-fold lower acetylcholine/catecholamine ratios than they would have without depolarization, and thus remained primarily adrenergic. Elevated K(+) and veratridine produced this effect on cholinergic differentiation without significantly altering neuronal survival. Because depolarization causes Ca(2+) entry in a number of cell types, the effects of several Ca(2+) agonists and antagonists were investigated. In the presence of the Ca(2+) antagonists D600 or Mg(2+), K(+) did not prevent the induction of cholinergic properties by conditioned medium. Thus depolarization, either steady or accompanying activity, is one of the factors determining whether cultured sympathetic neurons become adrenergic or cholinergic, and this effect may be mediated by Ca(2+).


Distribution of 59Fe-labelled iron-poly(soribitol-gluconic acid) complex in normal and anaemic rats.

An iron carbohydrate complex, iron-poly(sorbitol-gluconic acid), Ferastral, was labelled with 59Fe, and its distribution in rats was studied. The animals were intramuscularly treated with a dose of 10 mg of iron/kg. Three groups of animals were used: group A: non-anaemic and group B: anaemic rats, both kept on iron-deficient diet, and group C: non-anaemic rats kept on iron-supplemented diet. Urinary and faecal excretion, distribution in the body and incorporation in blood of the 59Fe was followed up to 28 days. The total excretion after that time was 15%. There was a rapid initial phase followed by a slower continuous one. After 28 days group A had 25, group B 13 and group C 40% of the given dose remaining at the site of injection. The corresponding values in liver after 28 days were 7, 4 and 17% of the given dose, respectively. In blood a continuous increase was observed. At 28 days after administration 26, 43 and 17% of the given dose had been incorporated in the red blood corpuscles of the respective groups. These results show that the iron complex is absorbed from the site of injection and is utilized for haemoglobin synthesis. They also show that the disposition of the complex is influenced by the iron content of the diet.


The absorption and availability of iron from iron-poly(sorbitol-gluconic acid) complex in rats as measured by phlebotomy.

The local and systemic availability of the iron in an iron-poly(sorbitol-gluconic acid) complex (Ferastral) has been studied in rats. The animals were treated intramuscularly with the complex in a dose corresponding to 10 mg of iron per animal. The technique of repeated phlebotomy was used to induce and maintain constant levels of anaemia. The phlebotomies were started immediately after injection and at 14 and 28 days after dosing. At the end of the appropriate interval, the remaining amount of iron at the injection sites as well as the degree of utilization of the iron retained in the animals were estimated. It was shown that the amount of iron at the site of injection of these anaemic animals was less than 3% regardless of the time when the series of phlebotomies were started. It was also shown that the degree of utilization of the iron from the complex was 90% of the retained amount of iron when the phlebotomies immediately followed dosing, and 86% and 79% when initiated 14 days and 28 days respectively. The significance of the decrease in utilization is discussed.


Disposition in rats of the carbohydrate polymer in Ferastral--an iron-poly (sorbitol-gluconic acid) complex.

The disposition and pharmacokinetics in rats of 14C-sorbitol-Ferastral has been studied as an initial step towards elucidating the metabolic fate of the polymeric organic moiety. The period of time studied was 24 h. Following i.v. administration of a dose corresponding to 10 mg/kg of iron approximately 60% of the isotope was found in the urine after this period of time. A further 3% was demonstrated in the faeces whilst the expired air accounted for about 10% in the form of 14CO2. Thus, a relatively large proportion of the radiolabel was promptly excreted via the kidneys whilst the expired air and probably bile constituted secondary excretion routes. The physico-chemical nature of the urinary product was similar to the parent carbohydrate compound. The residual 14C radioactivity was distributed mainly between the liver, which contained approximately 45% of the retained label, carcass with 36% and large intestine with 7%. The plasma disappearance pattern was biphasic. Similar experiments with the 14C-sorbitol polymer starting material were performed for purposes of comparison.


Distribution of the carbohydrate polymer in 14C-labelled iron-poly (sorbitol-gluconic acid) complex after intramuscular administration in normal and anaemic rats.

The carbohydrate polymer in an iron-poly (sorbitol-gluconic acid) complex was prepared by using uniformly labelled 14C-sorbitol or 14C-gluconic acid. The distribution of these complexes 7, 14 and 28 days after administration has been followed in non-anaemic and anaemic rats. Most of the 14C from the labelled complexes was found in the expired CO2, faeces and urine. Twenty-eight days after injection of 14C-labelled Ferastral, less than 13% of the given dose of either 14C-sorbitol or 14C-gluconic acid labelled preparation could be found in the body. There was no specific accumulation of 14C in any organ except in the bones, where 3% of the given dose could be seen 28 days after 14C-gluconic acid labelled Ferastral to both non-anaemic and anaemic animals.


Studies on the placental transfer in rats of 59Fe- and 14C-labelled iron-poly (sorbitol-gluconic acid) complex.

The placental transfer of an iron-poly (sorbitol-gluconic acid) complex, Ferastral, has been studied. By use of the 59Fe-, 14C-sorbitol, and 14C-gluconic acid labelled complexes it was shown that the passage of the iron moiety is related to foetal need and that only negligible amounts of 14C from either 14C-sorbitol or 14C-gluconic acid labelled iron complexes were found in the foetus. This implies that the intact complex does not pass the placental barrier.


Metabolic studies of iron-poly (sorbitol-gluconic acid) complex, Ferastral.

The metabolism of iron-poly (sorbitol-gluconic acid) complex (Ferastral) was studied in 6 blood donors with latent iron deficiency and 13 patients with manifest iron deficiency anaemia. Between 125 and 1 000 mg of iron were injected. In latent deficiency an average of 19% of the dose given was excreted during the first two weeks. The corresponding figure for patients with manifest iron deficiency was 11%. An average of 8% of the dose was retained at the site of injection after 40 days. In the two groups, respectively, 61% (range 47%-70%) and 58% (range 24%-86%) of the iron retained was incorporated into red cells after 40 days, which is judged to be quite satisfactory. Some local side effects were observed.


Absorption, excretion, and utilization of an intramuscular iron-poly (sorbitol-gluconic acid) complex, Ferastral.

An iron-poly (sorbitol-gluconic acid) complex (Ferastral) labelled with 59Fe was administered by intramuscular injection to clinically and haematologically normal male volunteers. Urinary excretion of radioactivity was measured for the first 48 hours following injection, and was 2%-10% of the administered dose. Disappearance of radioactivity at the site of injection and accumulation of radioactivity in the liver, spleen, and sacrum were measured by external scintillation counting. Plasma radioactivity was measured at intervals for four days and red cell incorporation of 59Fe reached levels of 29%-57.5%. An unexpected finding was that the highest levels of urinary excretion of radioactivity did not coincide with the highest levels of plasma radioactivity.


Studies on iron stores built up by an iron-poly (sorbitol-gluconic acid) complex, Ferastral, in man. Preliminary report.

The characteristics of iron stores built up by a new parenteral iron-poly (sorbitol-gluconic acid) complex, Ferastral, have been studied in iron depleted non-anemic blood donors. The results from studies on the first three subjects are presented. The availability of this storage iron for the chelator, desferrioxamine, was found to be in the same range as normal iron stores. The pattern of distribution in reticulo-endothelial cells of the bone marrow could not be differentiated from natural storage iron. No visible iron could be detected in liver parenchymal cells 40 days after iron administration. The results from these preliminary studies suggests normal bioavailability of this material for Hb-synthesis. The absence of iron in liver parenchymal cells might be explained by the short time interval between the iron administration and the fine needle aspiration biopsy.


Interaction between Ferastral and plasma factors.

Ferastral, iron-poly (sorbitol-gluconic acid) complex, in aqueous solution is completely adsorbed by magnesium carbonate; Ferastral in serum solution is only partially adsorbed by magnesium carbonate. Adsorption by magnesium carbonate removes the fraction of Ferastral in serum solution which donates iron more readily to transferrin. It is suggested that this fraction comprises the smaller molecular species of the Ferastral complex and that these species have a faster rate of absorption from an intramuscular injection site than the larger molecular species of the iron complex preparation. These findings shed some light on the patterns of serum transferrin bound iron compared with serum Ferastral concentrations which occur with time after intramuscular Ferastral injection.


The assay of iron-poly (sorbitol-gluconic acid) complex (Ferastral) and its separation from transferrin in serum.

A new iron-poly (sorbitol-gluconic acid) complex (Ferastral) has been studied. A method of assay is described. The iron complex may be separated from serum transferrin using a Sephadex DEAE A50 column. This binds the iron complex and elutes iron-transferrin which can then be assayed. It is shown that the assay of serum transferrin unsaturated binding capacity using excess 59FeCl2 and MgCO3 adsorption, is valid in the presence of Ferastral. Serum unsaturated iron binding capacity may therefore be used to follow the binding of Ferastral iron by transferrin. These methods may be used to follow the distribution of iron in plasma after an intramuscular injection of Ferastral.


Studies on an iron-poly(sorbitol-gluconic acid) complex for parenteral treatment of iron deficiency anaemia.

A preparation containing an iron-poly(sorbitol-gluconic acid) complex for parenteral treatment of iron deficiency anaemia is described. The physical and chemical properties of the iron complex have been studied by using electrophoresis and gel permeation chromatography. A rapid absorption from the injection site after intramuscular administration to rabbits takes place, 70% of the iron being absorbed after 24-48 hours. Thereafter, the absorption rate is slower, and 32 days after the injection 94% has been absorbed from the injection site. In rabbits the maximum level of iron in serum is reached after 12-24 hours; in dogs after 1-3 hours. Disappearance from the serum takes place slowly. The complex is exclusively absorbed via the lymphatic route. Nine to ten per cent of the given dose is excreted by the kidney within 72 hours in rats and 24 hours in rabbits after intramuscular administration. On administration of the preparation to rats, made anaemic by phlebotomy, a rapid increase of haemoglobin values is observed as well as a very high utilization of the retained amount of the given dose.


Transfer of iron from Ferastral and other organic complexes to transferrin as measured by reticulocyte uptake.

The transfer of iron from the iron carbohydrate complexes, Ferastral, Imferon, and Jectofer, and from ferric chloride has been studied by the effect of such transfer in reducing reticulocyte uptake of 59Fe from labelled transferrin. There are plasma factors which augment the transfer of iron from complex to transferrin. The pattern of transfer from Ferastral and from Imferon are similar: at concentration of 5000 microgram/100 ml and 1250 microgram/100 ml in plasma these complexes transfer about 0.8% and 1.7%, respectively. Jectofer transfers about four times these amounts under similar conditions. In the case of Ferastral there is evidence of an equilibrium between transferrin-bound and Ferastral-bound iron. The characteristics of Ferastral assessed in this way suggest that it may prove suitable for therapeutic use as a total dose infusion.


Kinetic studies on an intramuscular iron-poly (sorbitol-gluconic acid) complex.

Levels of iron and unsaturated iron binding capacity in serum and the renal excretion of iron were studied in 15 anaemic patients receiving Ferastral. Various dose schedules were used, 100 mg Fe daily for 10 days, 200 mg Fe daily for 5 days, and 500 mg Fe twice at various intervals from 2 to 7 days. A continuous increase of iron in serum was seen after the doses 100 mg and 200 mg daily as well as after 500 mg given at intervals of 2 and 3 days. Longer 500 mg dose intervals resulted in a fluctuation in the iron concentration. Peak levels varied between 740 and 5 260 microgram/100 ml (132 and 942 mumol/1). A transient decrease in the UIBC was seen. Urinary excretion of iron varied between 8.9 and 26.9% of the dose given. No local or general side effects occurred.


Patterns of plasma iron fractions after intramuscular Ferastral.

The distribution of plasma iron fractions after intramuscular Ferastral is described for times up to eight days after injection. Transferrin-bound iron reaches a peak value in 4-8 hours and falls towards normal values while circulating iron-complex concentration remains high. The peak concentration of iron-complex occurs 8-24 hours after injection, and is virtually cleared from the circulation in 6-8 days. An approximate T 1/2 of 30 hours for plasma Ferastral is deduced from the rate of fall from peak values. UIBC values fell as transferrin-bound iron increased, but did not reach zero in any subject studied.


Compensation for blood loss in plastic surgery with ferastral and autologous blood transfusion.

A study has been made to assess the effect on haematological status of pre-operative parenteral iron in a operation accompanied by conisderable blood loss. Forty-two patients who were to undergo breast reducing operations were selected for the trial. Patients were divided into three groups: group 1 which received no iron treatment, group 2 which received Ferastral, 250 mg Fe, 21 days before operation and group 3 which received Ferastral, 250 mg Fe, 21 days before operation plus an autologous blood transfusion during operation. The blood loss at operation which was estimated to be between 500 and 1 000 ml in 86% of the cases resulted in a decrease in haemoglobin in all groups. On the 14th postoperative day the decrease in Hb was significantly lower in both group 2 and 3 compared to group 1 (P less 0.05 and P less than 0.01, respectively). Forty per cent of the patients were anaemic in group 1, 10% in group 2 and 15% in group 3. It is concluded that the need for blood transfusion may be avoided in operations where the blood loss is less than 1 000 ml, provided sufficient iron is available for spontaneous blood replacement to take place.


Iron-deficiency in the tropics.

Different populations in the tropics show wide variation in their iron status, even when living in the same area. These differences range from the most profound iron-deficiency anaemia to nutritional haemosiderosis. The extremes of dietary iron content so far reported are 5 mg per day in Mauritius to 180 mg per day in Ethiopia. Contamination with inorganic iron is an important source in some communities. Generally, the iron in food is in excess of requirement, but lumenal factors are all important in deciding how much is available for absorption. The iron of some diets is readily absorbed, but in other only a small percentage is utilizable. Hookworm infection is the commonest cause of severe iron-deficiency in many parts of the world. Its prevalence and its density depends on social habits, and may vary considerably even within tribes. The development of iron-deficiency anaemia depends on the hookworm load being sufficient to cause iron loss in excess of available intake. Anaemia impairs work capacity, reproductive ability of women and the development of children, and has wide social and economic repercussions. An intramuscular preparation by which 500 mg of iron may be administered in one injection has most useful application in areas where medical care is minimal.


Pharmacological studies on an iron-poly-(sorbitol-gluconic acid) complex for parenteral treatment of iron deficiency anaemia.

Intravenous injection of the iron-poly(sorbitol-gluconic acid) complex (IPSG) to cats anaesthetized with sodium pentobarbitone caused a transient decrease in mean arterial blood pressure and a temporary increase in central venous pressure, heart rate and femoral blood flow at large doses (cumulative doses up to 744 mg/kg). Tachyphylaxis developed upon repeated administration. A temporary reduction in the magnitude of the blood pressure responses to noradrenaline and isoprenaline was obtained after large doses of IPSG. The blood pressure effects of acetylcholine, histamine and bilateral carotid occlusion were not affected. No definite effects were seen on the electrocardiograms. The transient cardiovascular effects were interpreted as being due to the presence of small amounts of ferrous iron in the preparation. IPSG did not significantly affect blood coagulation or platelet aggregation as judged from results of in vitro and in vivo experiments. Immunogenicity studies in rabbits and antibody analyses of sera from patients treated with IPSG failed to demonstrate any effect of IPSG on the immune system.


Oncological study in rats of Ferastral, an iron-poly-(sorbitol-gluconic acid) complex, after intramuscular administration.

Four groups of Sprague-Dawley rats were given Ferastral, an iron-poly(sorbitol-gluconic acid) complex (IPSG) or Imferon, an iron-dextran complex, intramuscularly twice a week for 17 weeks. The experiment lasted for 95 weeks. Each compound was given to two groups, one low dose group and one high dose group. Depending on the body mass, the dose levels varied between 2.5 and 10 mg (25-50 mg/kg body mass) and between 5 and 20 mg (50-100 mg/kg body mass) of iron per rat, respectively. The mean total dose of iron per rat was calculated to be 235 and 495 mg, respectively. Another group of animals served as a control. From about the 30th experimental week onwards tumours developed at the intramuscular injection sites in the groups given Ferastral and Imferon. The tumours appeared to be sarcomas. In almost all the treated animals that lived longer than 30 weeks after the start of the experiment, sarcomas were present at the intramuscular injection sites. The sarcomas appeared earlier in the high dose groups than in the low dose groups and slightly earlier in the rats given Ferastral than in those given Imferon. No other pathological changes, including neoplasms, were considered to be related to the treatment.


[Oral health for everyone--an unattainable goal? Analysis of a questionnaire among 1200 Swiss recruits].

The attitude of 1085 Swiss military recruits towards the dentist and dental care, their knowledge of periodontal disease and its prevention, the incidence of smoking and the frequency of eating sweets as well as interest in their own teeth in general was determined by use of a questionnaire. The results showed a positive attitude on the part of the participants towards their own teeth and a definite motivation for good oral health. The oral hygiene habits of only a minority could be classified as sufficient, the knowledge of periodontal disease and its prevention were limited. Smoking and eating sweets were found to be widespread. It can be stated, therfore, that hygiene instruction for improvement of oral health by the dental profession as well as motivational efforts towards general health education at home and at school were unfortunately unsatisfactory.


[Transplantation of oral mucosa using a mucotome].

"Free gingival grafts--a technique for obtaining transplant tissue using a newly developed Mucotom": A new instrument for obtaining palatal soft tissue grafts was designed. It has a mechanically oscillating blade. The Mucotom is driven by the dental unit like a handpiece. The attached gingiva in the mandibular anterior region was widened in over 100 cases through a simplified surgical procedure and by taking the grafts with the Mucotom. Intermediate split thickness grafts resulted in good esthetics and function. The superior cutting ability of the instrument produced smooth grafts of uniform thickness.


[Surface analysis of 2 composite filling materials after various methods of finishing and sealing].

The surface quality of 2 composite filling materials (Adaptic with coarse hard quartz fillers and Cosmic with fine softer glass fillers) was evaluated using profilometry, scanning electron microscopy and light reflectometry after finishing with experimental diamond discs, 3M discs, corundum discs, polishing paste and also after sealing. Plaque formation on standardized surfaces of both composites was photographically recorded in 20 subjects. The sealed surfaces and the surfaces finished by the matrix alone were approximately 10 times less rough than after finishing procedures. Under in vivo conditions they reflected the most light, for both composites. The sealer failed to cover the whole composite surface. The unfinished and sealed surfaces lost their shine 3-7 days after placement in the mouth. Decrease in reflectance was independent of mechanical oral hygiene procedures. There were no significant differences in the roughness produced by the various discs on Adaptic surfaces. Diamond discs with particle size 1-3 micrometer resulted in least surface roughness and were the only instruments not destroying the filler particles of either composite. 3M discs and the rough corundum discs caused significantly more surface roughness of the Cosmic surface. Reflectometry showed that the finishing methods unfavourably affected composite surface smoothness. Among all finishing methods polishing paste produced the highest reflection value on the Cosmic surface. Adaptic surfaces were always associated with more plaque formation than Cosmic surface after all finishing methods.


[Adpatation and marginal fit of thixotropic composites and syringe system composites. In vitro findings].

In a dye penetration test and a scanning electron microscopic evaluation MOD-restorations made of a thixotropic material (ZKP-A-5(2)) and of two syringe system composites (Concise-Capsule-Composite3, Compocap-S4) were investigated with respect to microleakage and marginal adaptation. These materials did not reveal better marginal micromorphology than a comparable two-paste composite system (Concise3); Compocap-S showed markedly less microleakage. The quality of marginal adaptation, however, was improved to 52% when a low viscosity sealant (ZKP-A-2(2), Concise Enamel Bond3) was applied before insertion of the restorative. A sealant pretreatment is therefore compulsory in thixotropic and syringe packed composites as well. The perfection reached with these composite brands is equal to that of adhesive restorations made of Concise or Adaptic5 [6,7]. Restorations performed with new, complete composite systems (Nimetic6, GC's Epolite 100/Epobond7) revealed moderate results when compared to standard materials like Concise or Adaptic [6,7].


[Evaluation of 16 months old variously fashioned incisor fillings].

The influence of the cavity preparation form, enamel conditioning, and application of a low viscosity sealer upon marginal adaptation and microleakage of anterior teeth proximal composite restorations was investigated in vivo. Restorations inserted using the principles of Adhesive Restoration showed the best results. Experimental adhesive fillings placed into minimally prepared saucer-shaped cavities with no macroscopic dentin retention demonstrated likewise satisfactory results because of their small volume. Definitely inferior marginal adaptation and increased microleakage were obtained with other restorative techniques. Trace areas of etched enamel could be observed after 16 months in those cases where enamel conditioning was not followed by a sealer application. The utilization of a sealer, therefore, improves not only the marginal adaptation of the composite material but in addition offers a protective seal to the previously conditioned enamel surface. Once the adhesive restoration is completed, it is recommended that a fluoride solution be applied to all enamel surfaces in the operational field. This fluoride treatment results in the formation of extremely high concentrations of fluorapatite in enamel areas which might have been inadvertently exposed to etching solutions during the procedure and not sealed or covered by composite material.


[Core build-ups from composites--evaluation of the technics of application].

The advantages and disadvantages of composite materials when used in direct core restoration of endodontically treated teeth is discussed. With the aid of dye penetration tests and scanning electron microscopy, in vitro fabrication of gold and amalgam cores were compared to those of 3 different composite core techniques. A compression technique used in the application of composite to the Radix -anchor post was judged successful. Neither modification of preparation nor application of a sealer before composite insertion improved margin adaptation or reduced microleakage of restoration margins. Acid etching of dentin definitely decreased marginal quality; therefore, for core construction using composites, the acid etch technique is not indicated. The proper use of composites is superior to gold and comparable to amalgam in core fabrication of endodontically treated teeth, when evaluating marginal adaptation and the reduction of microleakage at the composite-dentin interface.