HeLa cell variants that differ in sensitivity to monofunctional alkylating agents, with independence of cytotoxic and mutagenic responses.

Different strains of the established human cell line HeLa differ substantially in sensitivity to ethyl methanesulfonate (EtMes). The EtMes doses effective for either cytotoxicity or mutation induction in a line of HeLa S3 cells are about 1/10th those required in the CCL2 HeLa line of the American Type Culture Collection. By plating the sensitive HeLa S3 line in the presence of highly cytotoxic doses of EtMes, we obtained a clone (designated A6) that displays about 7-fold greater resistance to EtMes toxicity. This A6 isolate is also cross resistant to other simple monofunctional alkylating agents-exhibiting about 4-fold increased resistance to methyl methanesulfonate and 10- to 15-fold increased resistance to N-methyl-N'-nitro-N-nitrosoguanidine but is similar to the S3 parent in sensitivity to mitomycin C, UV radiation, and gamma-rays. In contrast to the results for cytotoxicity, the A6 variant and the S3 parent showed the same high susceptibility to EtMes induction of ouabain-resistant mutations. This is direct biological evidence that different alkylation lesions are normally responsible for mutagenic and cytotoxic effects. The S3 and A6 cell lines may differ in DNA repair capability specific to certain potentially lethal alkylation products. The comparative sensitivity of the A6 cells to alkylation mutagenesis may also prove useful in cell genetic studies by facilitating the generation of multiple mutants for recessive alleles and permitting exceptionally sensitive detection of specific mutagenic effects.


Mathematical model for studying genetic variation in terms of restriction endonucleases.

A mathematical model for the evolutionary change of restriction sites in mitochondrial DNA is developed. Formulas based on this model are presented for estimating the number of nucleotide substitutions between two populations or species. To express the degree of polymorphism in a population at the nucleotide level, a measure called "nucleotide diversity" is proposed.


Successful treatment of autoimmune disease in (NZB/NZW)F1 female mice by using fractionated total lymphoid irradiation.

Adult female (NZB/NZW)F1 hybrid mice with documented autoimmune glomerulonephritis resembling systemic lupus erythematosus were treated with fractionated total lymphoid irradiation (TLI), a modification of the radiotherapeutic regimen currently used for the treatment of Hodgkin disease. After TLI, proteinuria subsided in all mice that had undergone radiotherapy, and the mean survival increased from 359 days in untreated controls to 545 days. It is suggested that TLI should be further investigated as a new approach for immunoregulation of autoimmune disorders.


Tumor cell variants obtained by mutagenesis of a Lewis lung carcinoma cell line: immune rejection by syngeneic mice.

It has been reported that, by mutagenesis of a malignant mouse teratocarcinoma cell line, it is possible to obtain cell variants that are incapable of forming progressive tumors in syngeneic mice. These variants, which were called "tum-," are eliminated from the host by an immune rejection process. We report here that similar variant cell clones can be obtained at high frequency from a Lewis lung carcinoma cell line treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Syngeneic C57BL/6 mice reject these tum- clones and acquire a strong radioresistant immune protection against the immunizing clone. When the challenging tum- clone differs from the immunizing clone, a weaker radioresistant immune protection can be demonstrated with some, but not all, combinations. All the tum- clones induce a significant protection against the original Lewis lung malignant cells. These results imply that each Lewis lung tum- variant carries on its surface a singular antigen in addition to one or more weak antigens already present on the original tumor cell line. This antigenic pattern is similar to that found on teratocarcinoma tum- variants. Our results suggest that the procedure of using a mutagen in order to generate tum- variants carrying new transplantation antigens may be generally applicable to cancer cells.


Change in specificity of antibodies to a random synthetic branched polypeptide in mice tolerant to its ordered analogs.

The crossreactivity between the random synthetic polypeptide antigen, (Tyr,Glu)-poly(DLAla)- -poly(Lys), and its ordered sequence analogs, (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(Lys) and (Tyr-Glu-Tyr-Glu)-poly(DLAla)- -poly(Lys), has been studied on the level of tolerance induction. Induction of tolerance to the random (Tyr,Glu)-poly(DLAla)- -poly(Lys) affected the response of the tolerant mice to the homologous antigen as well as to (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(Lys), which was shown previously to represent the major determinant of (Tyr,Glu)-poly(DLAla)- -poly(Lys). In contrast, these mice responded with high antibody titers to the hardly crossreacting (Tyr-Glu-Tyr-Glu)-poly(DLAla)- -poly(Lys). Mice tolerant to the ordered peptide antigen (Tyr-Glu-Tyr-Glu)-poly(DLAla)- -poly(Lys) did not respond to the homologous polypeptide; however, their immune response to either (Tyr-Glu)-poly(DLAla)- -poly(Lys) or (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(Lys) was not affected. Mice that were tolerant to (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(Lys) responded well to (Tyr-Glu-Tyr-Glu)-poly(DLAla)- -poly(Lys). Furthermore, these mice produced high antibody titers after immunization with the random (Tyr,Glu)-poly(DLAla)- -poly(Lys). However, the antibodies produced were not specific to the major determinant of (Tyr,Glu)-poly(DLAla)- -poly(Lys), namely, Tyr-Tyr-Glu-Glu, but were directed to minor determinants of the random polypeptide, including Tyr-Glu-Tyr-Glu, which are not immunopotent when nontolerant mice are immunized with (Tyr,Glu)-poly(DLAla)- -poly(Lys). Thus, whereas antigenic specificity reflects itself also at the level of tolerance induction, the animals that had been made tolerant are capable of responding to previously silent antigenic determinants.


C4a: the third anaphylatoxin of the human complement system.

The activation peptide C4a was isolated from C1s-cleaved C4, the fourth component of complement. The peptide appeared to be homogeneous by electrophoresis on cellulose acetate and by polyacrylamide gel electrophoresis. C4a has a molecular weight of 8650 and an electrophoretic mobility at pH 8.6 of +2.1 x 10(-5) cm2V-1 sec-1. Carboxypeptidase B released approximately 1 mol of arginine per mol of C4a. The partial COOH-terminal sequence was determined to be Leu-Gln-Arg-COOH. The isolated C4a was spasmogenic for guinea pig ileum at a concentration of 1 microM and it desensitized the muscle (i.e., produced tachyphylaxis) with respect to human C3a anaphylatoxin (at 0.33 microM) but not with respect to human C5a anaphylatoxin. Increased vascular permeability was observed in human skin after intradermal injection of 1 nmol of C4a, as evidenced by immediate erythema and edema formation. The spasmogenic, tachyphylactic, and vascular activities of C4a were abrogated by removal of the COOH-terminal arginine, a property that is characteristic also of the C3a and C5a anaphylatoxins. Contamination of C4a with either C3a or C5a has been ruled out by using radioimmunoassays for these peptides. Although C4a is considerably less active than are C3a and C5a, the present observations suggest that C4a constitutes a heretofore unrecognized anaphylatoxin that is related biologically and chemically to the activation peptides of C3 and C5.


Electrophoretically pure mouse interferon exerts multiple biologic effects.

Electrophoretically pure mouse interferon was examined for a number of biologic effects previously ascribed to crude or partially purified interferon preparations. These effects include: inhibition of the growth of a transplantable tumor in mice; inhibition of cell multiplication of mouse tumor cells in vitro; enhancement of the expression of histocompatibility antigens on mouse tumor cells in vitro; inhibition of antibody formation in vitro; inhibition of sensitization to sheep erythrocytes and the expression of delayed type hypersensitivity in mice; enhancement of natural killer cell activity in vivo and in vitro; enhancement of cell sensitivity to the toxicity of poly(I)-poly(C); and enhanced production ("priming") of interferon production in vitro. Our results establish that the molecules responsible for the antiviral action of interferon are also responsible for these varied biologic effects.


Uroporphyrinogen I synthase induction in normal human bone marrow cultures: an early and quantitative response of erythroid differentiation.

Uroporphyrinogen I (URO) synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] activity increased when cultures of normal human bone marrow cells were incubated with erythropoietin. The increase of URO synthase activity was a linear function of erythropoietin concentration in the culture medium and was proportional to the extent of heme synthesis as determined by 55Fe incorporation into heme. The onset of the increase in URO synthase activity, which occurred on day 4 of incubation with erythropoietin, preceded by 3 days the appearance of hemoglobin in colonies derived from erythroid progenitor cells. These results indicate that induction of URO synthase in normal human bone marrow cells incubated with erythropoietin is an early event and that URO synthase activity is a useful quantitative index of erythroid differentiation.


Manganese and zinc blockade of enzyme induction: studies with microsomal heme oxygenase.

Heme oxygenase (decyclizing) [heme,hydrogen-donor:oxygen oxidoreductase (alpha-methene-oxidizing, hydroxylating), EC 1.14.99.3] is greatly induced in the kidney by the administration of nickel or tin. Manganese, when administered simultaneously with nickel or tin in an equimolar amount, substantially inhibited the induction of heme oxygenase. The extent of inhibition was 80% and 98%, respectively. In rats pretreated up to 8 hr with manganese, the level of induction of heme oxygenase by nickel or tin was markedly reduced in a time-dependent fashion. Manganese treatment after the inducing metal was relatively ineffective in preventing the induction of heme oxygenase. Manganese in vitro did not inhibit heme oxygenase in the microsomes isolated from either control or tin-induced rats and in vivo did not increase the rate of catabolism of the induced enzyme. Magnesium was unable to block nickel or tin induction of heme oxygenase. Zinc in equimolar amounts could also substantially reduce the extent of induction of renal heme oxygenase when administered simultaneously with nickel or tin. In addition, simultaneous zinc administration blocked to a considerable extent the induction of hepatic heme oxygenase by nickel, cobalt, or cadmium. These findings indicate the existence of metal-metal interactions that can greatly influence the regulatory mechanism for the induced synthesis of heme oxygenase, the rate-limiting enzyme in heme degradation.


Human epidermal cell cultures: growth and differentiation in the absence of differentiation in the absence of dermal components or medium supplements.

Human epidermal cells grew and differentiated in vitro, provided that the pH of the culture medium was at 5.6-5.8, the seeding density was optimal (approximately 2.5 x 10(5) cells per cm2), and the incubation temperature was maintained at 35-37 degrees C. Under these conditions, epidermal cells from many different skin locations grew to confluency within 15-20 days and formed multi-layered sheets whose differentiated structure resembled that of the full depth of skin epidermis. Cell proliferation and differentiation did not require a feeder layer, a collagen substrate, a high concentration of fetal bovine serum, or added hormones. The sheets of differentiated epidermal cells could be dissociated from the plastic surfaces of the tissue culture flasks. The use of such cultured cells for wound dressing is proposed.


Alterations in nociception and body temperature after intracisternal administration of neurotensin, beta-endorphin, other endogenous peptides, and morphine.

The antinociceptive and hypothermic effects of intracisternal administration of 11 endogenous neuropeptides and morphine were evaluated in mice. Of the substances tested, only neurotensin (NT) and beta-endorphin exerted significant antinociceptive and hypothermic effects; NT was the most potent in inducing hypothermia whereas beta-endorphin was the most potent antinociceptive agent via this route of administration. Both NT, and beta-endorphin were, on a molar basis, considerably more potent antinociceptive agents than morphine, [Met]enkephalin, or [Leu]enkephalin. NT-induced analgesia and hypothermia both were significantly dose-dependent. Substance P was found to produce significant hyperalgesia and hyperthermia. Bombesin produced a significant hypothermic effect, whereas somatostatin and luteinizing hormone-releasing hormone (luliberin) produced hyperthermia. None of the other peptides studies [bradykinin, thyrotropin-releasing factor (thyroliberin), melanocyte-stimulating hormone release-inhibiting factor (melanostatin), somatostatin, [Met]enkephalin, and [Leu]enkephalin] produced any significant alterations in colonic temperature or response to a noxious stimulus with the doses tested. These data demonstrate that NT and beta-endorphin, two endogenous brain peptides, are potent in inducing hypothermia and in producing an antinociceptive state.


Specific [3H]phencyclidine binding in rat central nervous system.

[3H]Phencyclidine (PCP) bound specifically and with high affinity (Kd = 0.15 microM at pH 7.4) to a single saturable class of binding sites in rat brain membrane preparations. Specific binding constituted approximately 70% of total binding at 0 degrees C and 33% of total binding at 37 degrees C (at 10 nM [3H]PCP). Bound [3H]PCP could be displaced by nonradioactive PCP, a series of its derivatives, and the psychotomimetic opiate N-allylnorcyclazocine (SKF 10,047) with relative potencies that closely paralleled those determined in animal behavioral tests. Muscarinic cholinergic ligands inhibited [3H]PCP binding, but only at 0.1 mM and in rank order at variance with that for binding to muscarinic sites or for pharmacological potencies. Other drugs, including opiates other than SKF 10,047, were unable to displace specifically bound [3H]PCP at 0.1 mM. [3H]PCP binding was most enriched in crude synaptosomal subcellular fractions, and was about three times higher in hippocampus (region of highest density) than in cervical spinal cord (region of lowest density). Trypsin and Pronase reduced specific [3H]PCP binding. Thus, PCP may exert its effects on the central nervous system via binding to specific brain receptor sites.


beta-Endorphin: analgesic and hormonal effects in humans.

The pharmacokinetics and the hormonal, analgesic, and behavioral effects of several doses of human beta-endorphin were evaluated after intravenous administration to three patients and intracerebroventricular administration to one patient with pain caused by cancer. These effects were compared to the hormonal effects of intravenously administered morphine sulfate in two patients and an enkephalin analog in two baboons. The mean terminal half-life after intravenous administration of 5 or 10 mg of human beta-endorphin to three patients was 37 min; the mean volume of distribution was 178 ml/kg, and the metabolic clearance rate was 3.2 (ml/min)/kg. The half-life of beta-endorphin in cerebrospinal fluid after intracerebroventricular administration was 93 min, and the volume of distribution was 0.74 ml/kg. A rapid rise in plasma prolactin followed both intravenous and intracerebroventricular beta-endorphin. Intravenous administration did not affect plasma growth hormone, but intracerebroventricular administration suppressed plasma growth hormone. No significant change in plasma growth hormone was noted after intravenous administration of morphine to humans, but plasma growth hormone decreased in one baboon after administration of the enkephalin analog. beta-Endorphin-stimulated release of prolactin occurred at doses lower than those required to produce analgesic and other behavioral effects. When both hormonal and analgesic effects were observed (after 7.5 mg were given intracerebroventricularly), the onset of the hormonal response slightly preceded the analgesic and behavioral responses. These studies suggest that the hormonal effects of beta-endorphin are species dependent and are similar to those of morphine. Hormonal and analgesic effects of beta-endorphin appear to result from the activation of opiate receptors that differ in their locations and characteristics.


In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in presence of their striatal target cells.

Long-term survival of mesencephalic and striatal cells from mouse embryos in dissociated primary cultures is described. Catecholaminergic neurons in mesencephalic culutres were identified histochemically and by measuring [3H]dopamine uptake and synthesis from [3H]tyrosine. According to experiments using specific inhibitors of catecholamine uptake, at least two-thirds of the catecholaminergic neurons are dopaminergic. These neurons differentiated whether or not striatal target cells were present, but striatal cells stimulated the development of the dopaminergic neurons. [3H]Dopamine uptake was increased by at least 2-fold regardless of the age of the cocultures (4-15 days). Enhanced [3H]dopamine synthesis was also observed (at least 2-fold) at later times (12-15 days).


Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord.

Extracts of chicken brain and spinal cord increase the total number of acetylcholine receptors and the number of acetylcholine receptors and the number of receptor clusters on uninnervated skeletal myotubes in culture. The active component in these extracts may be unique to neural tissue. Spinal cord cells grown in culture contain the active factor and they secrete it into the medium. Most of the activity is associated with a small molecule possibly a peptide. Such a factor may be responsible for the clustering of receptors at newly formed nerve-muscle synapses.


Theory of the dependence of population levels on environmental history for semelparous species with short reproductive seasons.

A population that is strongly self-regulating through density-dependent effects is expected to be such that, if many generations have elapsed since its establishment, its present size should not be sensitive to its initial size but should instead be determined by the history of the variables that describe the influence of the environment on fecundity, mortality, and dispersal. Here we discuss the dependence of abundance on environmental history for a semelparous population in which reproduction is synchronous. It is assumed that at each instant t: (i) the rate of loss of members of age a by mortality and dispersal is given by a function rho of t, a, and the present number x = x(a,t) of such members; and (ii) the number x(0,t) of members born in the population is given by a function F of t and the number of x(a(f),t) at a specified age a(f) of fecundity. It is further assumed that the functions rho and F have the forms rho(x,a,t) = pi(1)(a,t)x + pi(2)(a,t)x(2) and F(x(a(f),t),t) = nu(t)x(a(f),t). For such a population, a change in the environment is significant only if it results in a change in nu(t) pi(1)(a,t), or pi(2)(a,t), and, hence, the history of the environment up to time t is described by giving nu(tau), pi(1)(a,tau), and pi(2)(a,tau) for each tau </= t and all a in [0, a(f)]. We show that the dependence of x on the history of the environment can be calculated explicitly and has certain properties of "fading memory"; i.e., environmental events that occurred in the remote past have less effect upon the present abundance than comparable events in the recent past.


1H nuclear magnetic resonance investigation of flexibility in DNA.

In this paper we report successful observations of proton NMR spectra of native double helical salmon sperm and calf thymus DNA of various lengths. Measurements of the linewidths arising from proton--proton dipolar interactions are used to obtain information about the dynamic behavior of DNA helices in solution. Depending upon which protons are used to monitor the local internal motions of the DNA, different results are obtained. The lowfield resonances from hydrogen-bonded imino protons in the base pairs indicate that the correlation time for reorientation of base pairs is less than 3 x 10(-7) sec, whereas correlation times for motion of neighboring sugar protons relative to the aromatic protons are less than 5 x 10(-8) sec for DNA that is over 200 base pairs long. These observations indicate that there is considerably more internal flexibility in the DNA molecues, especially in the backbone, than is indicated by classic hydrodynamic studies of DNA.


Antibodies bound to lipid haptens in model membranes diffuse as rapidly as the lipids themselves.

A pattern photobleaching method has been used to measure the rates of lateral diffusion of fluorescent-labeled specific anti-nitroxide IgG bound with both combining sites to nitroxide-containing phospholipids in liposomal membranes composed of dimyristoyl phosphatidylcholine at 28 degrees C ("fluid"), dipalmitoyl phosphatidylcholine at 32 degrees C ("Solid"), and dipalmitoyl phosphatidylcholine containing 15 or 25 mol% cholesterol ("solid" or "fluid," respectively, at 32 degrees C). The diffusion coefficients of the bound immunoglobulin were found to be the same as those of fluorescent-labeled phospholipids in each case even though these diffusion coefficients range from 10(-11) to 10(-8) cm2/sec. Hapten-containing liposomal membranes of the type studied here have previously been shown to elicit a number of antibody-dependent immune responses. Therefore, this work indicates that membrane-bound but otherwise freely diffusing antibodies are sufficient for these reponses.


Characterization of an endopeptidase involved in pre-protein processing.

Proteolytic removal of the pre-segment from growing nascent chains of pre-human placental lactogen (hPL) occurred during in vitro translation of placental mRNA if crude membranes derived from ascites lysates, dog pancreas, or rat liver rough endoplasmic reticulum were added to the translation mixtures. The cotranslational proteolytic event was inhibited by the peptide protease inhibitor, chymostatin, but not by leupeptin, antipain, or elastatinal. The proteases involved in cleavage were solubilized with detergent and converted completed pre-hPL to hPL (post-translational processing). Direct assay of the solubilized membranes, with synthetic fluorogenic aminocoumarin peptide substrates, revealed no significant tryptic or elastase-like activity, but activity against a chymotrypsin substrate [(succinyl-Ala-Ala-Phe)-7-amino-4-methyl-coumarin] was found. This activity was dependent upon both an endopeptidase and an aminopeptidase. Although bestatin inhibited the aminopeptidase activity, it had no effect on the endopeptidase or on post-translational cleavage. Although this endopeptidase cleaved on the COOH side of an alanine residue, it was not inhibited by elastatinal. However, it was inhibited by high levels of chymostatin and by some serine protease inhibitors.


Enhanced nonenzymatic glucosylation of human serum albumin in diabetes mellitus.

Use of an ion exchange chromatographic method and a colorimetric method with thiobarbituric acid showed that levels of nonenzymatically glucosylated serum albumin were increased in patients with poorly controlled diabetes mellitus compared to controls. The two methods correlated well (r = 0.99) and clearly discriminated between normal and poorly controlled diabetic populations. The levels of glycosylated hemoglobin were also measured in both populations. Several patients apparently in good control based on glycosylated hemoglobin measurements were found to have increased levels of glycosylated albumin. Because albumin has a shorter circulating half-life than does the human erythrocyte, the plasma concentration of glucosylated albumin should be expected to reflect short-term control of hyperglycemia in diabetes. The studies reported here suggest that the level of glucosylated albumin may indeed be a sensitive indicator of moderate hyperglycemia and of early glucose intolerance.


Synthesis of fluorescent nucleotide analogues: 5'-mono-, di-, and triphosphates of linear-benzoguanosine, linear-benzoinosine, and linear-benzoxanthosine.

The fluorescent nucleotide analogues (the 5'-mono-, di-, and triphosphates of lin-benzoguanosine, lin-benzoxanthosine, and lin-benzoinosine) have been prepared for use as dimensional probes of enzyme binding sites. They have quantum yields in aqueous solution of 0.39, 0.55, and 0.04 and fluorescent lifetimes of 6, 9, and approximately equal to 1.5 nsec, respectively. lin-Benzoinosine 5'-monophosphate is a substrate for xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2), providing lin-benzoxanthosine 5'-monophosphate, and lin-benzoinosine 5'-diphosphate is a substrate for polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase. EC 2.7.7.8), giving poly(lin-benzoinosinic acid). The benzologues of the purine diphosphates are substrates for pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40), which is used to prepare the triphosphates.


Differential activities of glycolipid glycosyltransferases in Tay-Sachs disease: studies in cultured cells from cerebrum.

Four different glycolipid:glycosyltransferase activities involved in the biosynthesis in vitro of gangliosides and blood group-related glycosphingolipids have been tested in a simian virus 40-transformed glial cell culture derived from the cerebrum of a fetus with Tay-Sachs disease (TSD). The TSD cultured brain cells contained little activity of either UDP-Gal:GM2(beta 1-3)galactosyltransferase (GalT-3; EC 2.4.1.62), which catalyzes the formation of GM1a from GM2 (tay-Sachs) ganglioside, or GDP-Fuc:nLcOse4Cer (alpha 1-2)fucosyltransferase (FucT-2; EC 2.4.1.89), which catalyzes the formation of H1 glycolipid from nLcOse4Cer. These cells contained a potent inhibitor of the second reaction (catalyzed by a Golgi-rich membrane fraction from bovine spleen), whereas no inhibition of the first reaction (catalyzed by a membrane fraction from 14-day-old embryonic chicken brain) was observed. The activity of UDP-Gal:LcOse3Cer(beta 1-4)galactosyltransferase (GalT-4; EC 2.4.1.86) was 30- to 80-fold higher than the activity of GalT-3. The presence of CMP-AcNeu:nLcOse4Cer sialyltransferase activity and the absence of either GalT-3 or FucT-2 suggested a probable pathway for the synthesis of sialylneolactotetraosylceramide [GM1b(GlcNAc)] in addition to a specific blockage of GM1a ganglioside synthesis from GM2 in these TSD transformed cells.


Mechanism for circularization of linear DNAs: circular parvovirus MVM DNA is formed by a "noose" sliding in a "lasso"-like DNA structure.

During an electron-microscopic survey with the aim of identifying the parvovirus MVM transcription template, we observed previously unidentified structures of MVM DNA in lysates of virus-infected cells. These included double-stranded "lasso"-like structures and relaxed circles. Both structures were of unit length MVM DNA, indicating that they were not intermediates formed during replication; they each represented about 5% of the total nuclear MVM DNA. The proportion of these structures was unchanged after digestion with sodium dodecyl sulfate/Pronase and RNase and after mild denaturation treatment. Cleavage of the "lasso" structures with EcoRI restriction endonuclease indicated that the "noose" part of the "lasso" structure is located on the 5' side of the genomic single-stranded MVM DNA. A model is presented for the molecular nature of the circularization process of MVM DNA in which the "lasso" structures are identified as intermediates during circle formation. This model proposes a mechanism for circularization of linear DNAs.


Structure of genes for human growth hormone and chorionic somatomammotropin.

A 2.6-kilobase (kb) EcoRI restriction endonuclease fragment containing human growth hormone (hGH; somatotropin) gene sequences and a 2.8-kb EcoRI fragment containing human chorionic somatomammotropin (hCS; choriomammotropin) gene sequences have been identified by hybridization to cloned cDNA. Human DNA was cleaved with EcoRI and fractionated by preparative agarose gel electrophoresis; DNA in the size range 2--3 kb was ligated to lambda gt WES.lambda B DNA and viable recombinant bacteriophage were recovered by in vitro packaging. After infection of Escherichia coli and screening of phage plaques, single isolates of hGH and hCS gene sequences were obtained. Restriction endonuclease mapping showed that the hGH gene contains three intervening sequences interrupting the coding sequence. Partial DNA sequence analysis of the hGH gene, obtained by the chain termination method, confirmed the location of the intervening sequences and the identity of the fragment.


Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts.

Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is subject to receptor-mediated endocytosis by fibroblasts. Prior work demonstrated charge heterogeneity in beta-glucuronidase and showed that high-uptake forms are more acidic than slowly internalized forms. Considerable indirect evidence implicated mannose 6-phosphate as an essential part of the recognition marker on high-uptake enzyme forms. Here we report the purification of beta-glucuronidase from human spleen and demonstrate enzymatically that mannose 6-phosphate is released on acid hydrolysis of pure enzyme varies directly with its susceptibility to pinocytosis by fibroblasts. Enzyme forms resolved by CM-Sephadex chromatography differed over an 18-fold range in uptake rate and in mannose 6-phosphate content. The most acidic forms had 4.4 mol of mannose 6-phosphate per mol of enzyme. The mannose 6-phosphate was released from the enzyme by treatment with endoglycosidase H with concomitant loss of susceptibility to adsorptive endocytosis. Thus, these studies provide direct evidence that mannose 6-phosphate is present on high-uptake enzyme forms, that it is present in the recognition marker for uptake, and that it is present on oligosaccharide that is released by endoglycosidase H.


Identification of gamma-glutamyl phosphate in the alpha 2 chains of chicken bone collagen.

Purified components of chicken bone collagen contain approximately 4 atoms of organic phosphorus per mol of collagen, located principally in the alpha 2 chains. Previous analyses have demonstrated the absence of O-phosphoserine, O-phosphothreonine, and other phosphorylated hydroxy amino acids, phosphoamidated amino acids, and phosphorylated sugars. In the present report we establish that chicken bone collagen contains gamma-glutamyl phosphate. This was accomplished by the isolation of tritiated alpha-amino-delta-hydroxyvaleric acid after reductive cleavage with NaB[3H]H4 of the gamma components, the alpha 2 chains, and peptides enriched in organic phosphorus that were derived from the alpha 2 chains. Tritiated alpha-amino-delta-hydroxyvaleric acid was not detected in any of the following unphosphorylated proteins after cleavage with NaB[3H]H4:albumin and lysozyme, the alpha 2 chains of several unmineralized tissues, and, most importantly, dephosphorylated alpha 2 chains of chicken bone collagen. The alpha 2 chain of chicken bone collagen is the first structural protein found to contain an acyl phosphate.


Comparison of adult, embryonic, and dystrophic myosin heavy chains from chicken muscle by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and peptide mapping.

Chicken myosin heavy chains from adult fast white muscle fibers (both normal and dystrophic), adult slow red fibers, and embryonic presumptive fast white fibers were compared by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by peptide mapping. The heavy chain of slow red myosin migrated electrophoretically more slowly than the heavy chains of the other myosins and differed markedly from them in its peptide maps. The heavy chain of dystrophic fast white myosin was similar to its normal counterpart by peptide mapping but showed slight differences. The peptide map of the heavy chain of embryonic presumptive fast white myosin had the general features of that of the heavy chain of fast white, not slow red, fibers but contained definite differences from the former. The results are consistent with the existence of a separate gene for the heavy chain of embryonic presumptive fast white myosin.


Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.

A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.


Construction and cloning of rat albumin structural gene sequences.

A recombinant plasmid containing a DNA segment complementary to rat liver albumin mRNA has been constructed, cloned, and used to examine the organization of albumin gene. The 18S fraction of total liver poly(A)-containing RNA was copied into a double-stranded cDNA by avian myeloblastosis virus reverse transcriptase and Escherichia coli DNA polymerase I. The cDNA was inserted into the HindIII site of the plasmid pBR322 via the addition of specific oligonucleotide linkers. Recombinant plasmids were screened by hybrid arrest of mRNA translation and hybridization with specific cDNAs. Thereby, a plasmid was identified that contained a 1200-nucleotide insert corresponding to a segment adjacent to the 5'-terminal region of albumin mRNA. The inserted sequence was used as a hybridization probe to detect five EcoRI fragments of genomic DNA which encode albumin mRNA. These were compared to eight EcoRI fragments identified within the rat genome by albumin cDNA. We conclude that the albumin gene (or genes) is interrupted at more than one site in the coding DNA by intervening sequences. Furthermore, we were able to distinguish those fragments that encode the 5' and 3' ends of the mRNA.


Characterization and regulation of reductase kinase, a protein kinase that modulates the enzymic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

The activity of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA reductase; mevalonate:NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34] can be modulated in vitro by a phosphorylation-dephosphorylation reaction sequence. A microsomal reductase kinase catalyzes the phosphorylation of HMG-CoA reductase and histones. Histone phosphorylation was enhanced 2- to 3-fold by cyclic AMP. Reductase kinase exists in interconvertible active and inactive forms. Incubation of reductase kinase with phosphoprotein phosphatase resulted in a time-dependent decrease in the ability of reductase kinase to catalyze the phosphorylation of histones and to inactivate HMG-CoA reductase. Incubation of phosphoprotein phosphatase-inactivated reductase kinase with [gamma-(32)P]ATP plus Mg(2+) and a partially purified protein kinase designated reductase kinase kinase resulted in parallel increases in protein-bound (32)P radioactivity and ability to inactivate HMG-CoA reductase. Incubation of (32)P-labeled reductase kinase with phosphoprotein phosphatase resulted in a time-dependent loss of protein-bound (32)P radioactivity and a decrease in the ability to inactivate HMG-CoA reductase. Polyacrylamide gel electrophoresis of purified reductase kinase incubated with reductase kinase kinase and [gamma-(32)P]ATP plus Mg(2+) revealed that the (32)P radioactivity and reductase kinase enzymic activity were located in a single electrophoretic position. Dephosphorylation of (32)P-labeled purified reductase kinase with phosphoprotein phosphatase was associated with significant loss of radioactivity and enzymic activity in the protein band ascribed to reductase kinase. These results provide evidence that the activity of reductase kinase, like HMG-CoA reductase, is modulated by a reversible phosphorylation-dephosphorylation reaction sequence.


Orientation of rhodopsin alpha-helices in in retinal rod outer segment membranes studied by infrared linear dichroism.

Frog retinal rod outer segments, oriented by a magentic field, were shown to contain rhodopsin alpha-helical segments preferentially aligned perpendicular to the plane of the disc membrane, by the technique of infrared linear dichroism. Infrared absorption parallel and perpendicular to the rod axes by peptide C parallel to O groups, whose absorption band contains alpha-helical and random coil components at slightly different frequencies, showed positive dichroism centered on the alpha-helix frequence. We conclude that the alpha-helical portion of the protein has an average orientation in the transmembrane direction. Furthermore, infrared spectra of rods in 2H2O Ringer's solution exhibit two distinct peptide amino group absorption bands: the unexchanged N-2H band, which is nondichroic. This implies that the oriented part of the protein is in the lipid bilayer, supporting a model for rhodopsin with a hydrophobic core containing partially oriented alpha-helices and hydrophilic ends consisting of unoriented polypeptide.


Protein-heme interaction in hemoglobin: evidence from Raman difference spectroscopy.

Raman difference spectroscopy measurements on native and chemically modified human deoxyhemoglobins stabilized in either the R or the T quaternary structure revealed frequency differences in the oxidation state marker lines. The differences indicate that the R structure has an effective increase in the electron density of the antibonding pi* orbitals of the porphyrin rings. This increase is explained by a charge transfer interaction between donor orbitals and the pi* orbitals of the porphyrins. The relative amount of charge transferred, which is inferred from the Raman difference measurements, correlates with some but not all factors that influence the energetics of the quaternary structure equilibrium. In addition, the free energy of cooperativity for a variety of ligated proteins follows the same order as that of the degree of charge depletion of the pi* orbitals upon ligation as determined from the frequency of a Raman mode. The proposed electronic interaction between the protein and heme could result in energies large enough to provide a significant contribution to the energetics of hemoglobin cooperativity.


Filament interaction in intact muscle fibers monitored by light scattering.

Measurement of changes in optical properties of intact muscle fibers during contraction has proven to be difficult or, in some cases, impossible due to movement of the muscle relative to the incident beam. In this paper we describe a technique for immobilizing single fibers in clear gelatin, which permits measurement of light scattering signals undistorted by movement artifacts. We also describe the phase and amplitude relationship between changes in intensity of light scattering (at 90 degrees to incident beam) and tensions induced by electrically activating single fibers. With tensions that range up to 50% Po (Po = maximal tension measured by exposure of fibers to 200 mM K+), the maximal increase in light scattering is about 25% of that for resting fibers. The scattering increase precedes tension, and at low temperatures the interval between the two peaks can be 50--100 msec. We interpret these data on intact fibers, as we did our earlier data from studied on skinned fibers, as indicating that increases in light scattering power of muscle are primarily due to attachment of myosin cross-bridges to actin filaments.


Synthesis of labile, serum-dependent protein in early G1 controls animal cell growth.

We present a model to account for several major observations on growth control of animal cells in culture. This model is tested by means of kinetic experiments which show that exponentially growing animal cells whose ability to synthesize total protein has been inhibited with cycloheximide (by up to 70%) grow at rates approximately proportional to their rates of protein synthesis. However, virtually the entire elongation of the cell cycle occurs in the part of the G(1) phase that depends on a high concentration of serum in the medium. This part of the cycle has earlier been suggested to lie prior to the restriction point-i.e., the point beyond the main regulatory processes of G(1). The remainder of the cycle, from restriction point to mitosis, is markedly insensitive to these concentrations of cycloheximide as well as to growth regulation. We quantitatively account for the specific lengthening of that part of the cycle involved in growth regulation by assuming that cells must accumulate a specific protein in a critical amount before they can proceed beyond the restriction point. The lability of this protein (half-life about 2 hr) makes its accumulation unusually sensitive to inhibition of total protein synthesis by cycloheximide. Its production appears to depend on growth factors provided by serum. The model can also account for greater variations of G(1) durations as the growth of cell populations is made slower. It also predicts two sorts of quiescence: one of cells slowly traversing G(1), in slightly suboptimal conditions; the other of cells that enter G(0) under inadequate conditions. Transformation of different sorts could create cells with altered variables for initiation, synthesis, or inactivation of the regulatory protein or could altogether eliminate the need for the protein.


Presence and expression of Friend erythroleukemia virus-related sequences in normal and leukemic mouse tissues.

The nature and distribution of sequences related to the murine erythroleukemia virus, Friend spleen focus-forming virus (SFFV), have been analyzed by using a radioactive cDNA probe specific for the SFFV genome (cDNA(sff)). From the proportion of high molecular weight viral [(32)P]RNA which hybridized to cDNA(sff), it was estimated that these sequences represent about 50% of the SFFV genome, indicating a genetic complexity of about 3300 nucleotides. cDNA(sff) hybridized extensively (80-95%) to SFFV virion RNA and to cellular RNA from murine and rat cells productively or nonproductively infected with SFFV. Only background homology was detected between cDNA(sff) and viral RNA from a number of murine [Friend murine leukemia virus (MuLV), Moloney-MuLV, and Kirsten sarcoma virus] and nonmurine (Rous sarcoma virus, feline leukemia virus, baboon endogenous virus, and Mason-Pfizer mammary tumor virus) retroviruses. Limited homology was also detected to a number of murine xenotropic and mink cell focus-inducing viruses (20-35%) as well as Rauscher leukemia virus (50%). Nucleotide sequences homologous to cDNA(sff) were also detected in the DNA of normal cells of several mouse strains as single or a few copies per cell. Thermal denaturation analysis indicated that duplexes formed between cDNA(sff) and normal DBA/2J cellular DNA have a reduction in melting temperature of 2 degrees C when compared with the dissociation of hybrids between cDNA(sff) and homologous sequences in SFFV-infected mouse spleen cell DNA. Examination of cellular RNA from uninfected mouse cells indicated that SFFV-related RNA sequences were also expressed in varying degrees in different tissues of adult DBA/2J mice. The highest amounts were observed in cells from bone marrow and spleen, whereas considerably lower amounts were found in cells from the thymus and kidney. No SFFV-related sequences could be detected in RNA extracted from liver, muscle, or fibroblasts. The presence of these SFFV-related sequences in normal, uninfected mouse cell DNA and their differential expression in hematopoietic tissues suggest that these sequences may be an integral part of the program of both normal and leukemic hematopoietic cell differentiation.


Acute stimulation of aromatization in Leydig cells by human chorionic gonadotropin in vitro.

Aromatization of testosterone in Leydig cells purified from mature rat testes was assessed. Leydig cells incubated for 4 hr with increasing concentrations of [3H]testosterone. At saturating concentrations of testosterone, human chorionic gonadotropin (hCG) acutely stimulated aromatization. This stimulation was first observed at 1 hr, an 8-fold increase being found during a 4-hr incubation. The maximal amount of estradiol produced at saturating concentrations of testosterone and hCG was 1.8 ng per 10(6) cells. These results demonstrate that purified Leydig cells have a high capacity for aromatization and that hCG can acutely stimulate aromatization independently of stimulating testosterone synthesis in vitro.


Regulation of translational initiation ina temperature-sensitive mutant of BHK cells.

The role of various essential components in mammalian cell protein chain initiation, such as the 40S ribosomal subunit and the initiation factors, have been studied largely in fractionated systems and little is known about the mechanisms of their regulation. The process was studied in an unfractionated system derived from temperature-sensitive (ts) 422E cells. The mutant cells fail to assemble 60S ribosomal subunits at the nonpermissive temperature with little effect on the production of 40S ribosomal subunits. Although the resulting imbalance in ribosomal subunits has little immediate effect on the overall rate of protein synthesis, the nascent 40S subunits in mutant cells appear to be markedly unstable at the nonpermissive temperature. The initial step in protein synthesis, initiation factor elF-2-promoted binding of [35S]Met-tRNAf Met to 40S ribosomal subunits, remains unimparied in ts 422E cells at the nonpermissive temperature. However, most of the nascent 40S ribosomal subunits in ts 422E cells band at a higher density, suggesting their failure to bind initiation factor elF-3. These newly synthesized 40S subunits in the mutant cells are unstable, and may be degraded before their appearance in the polysomes.


Stimulation of phospholipid metabolism in embryonic muscle cells treated with phospholipase C.

Phospholipid metabolism is dramatically stimulated in cultured myogenic cells in which cell fusion was inhibited with phospholipase C (phosphatidylcholine choline-phosphohydrolase; EC 3.1.4.3). Phospholipase C was active under the culture conditions as shown by the degradation of exogenous phosphatidylcholine. Rates of incorporation of 32Pi and [methyl-3H]choline into lipids were about 5-fold greater in phospholipase-treated cells than in either untreated fusing cells or untreated cells prevented from fusing by calcium deprivation. The greatest stimulation in the phospholipase C-treated cultures occurred with synthesis of phosphatidylcholine and sphingomyelin; synthesis of phosphatidylinositol and cardiolipin was not stimulated. Degradation of cellular [32P]phosphatidylcholine and appearance in the culture medium of the degradation product [32P]phosphocholine were both increased. Levels of total cellular phospholipids and of individual phospholipid classes were similar in control and phospholipase-treated cells. The results suggest that the membrane phospholipid composition in myogenic cells is controlled by a regulatory mechanism which increases the synthesis of phospholipids that are degraded in the presence of the phospholipase.


Isolation of a high molecular weight actin-binding protein from baby hamster kidney (BHK-21) cells.

A high molecular weight protein (HMWP) with properties similar to those of both actin-binding protein (ABP) and filamin has been isolated from cultured baby hamster kidney (BHK-21) cells. The protein was present in an actomyosin-depleted sucrose extract of the cells and was eluted, upon gel chromatography on Sepharose 4B, near the void volume. The subunit migration on sodium dodecyl sulfate/polyacrylamide gels and the amino acid composition of HMWP were similar to those of ABP and filamin. HMWP bound to and crosslinked F-actin from rabbit muscle, as shown by the formation of a gel that was sedimented with low-speed centrifugation. This interaction was insensitive to temperature and low concentrations of calcium ions, although it may depend on the presence of myosin. Observation of thin sections of the actin-HMWP gel revealed crosslinked complexes of laterally aggregated actin filaments. The axial period of the dense crosslinks was 34 nm. The HMWP may be involved in regulation of microfilament organization.


Fluorescent phallotoxin, a tool for the visualization of cellular actin.

A fluorescent derivative of phalloidin has been synthesized possessing high affinity to filamentous actin. This compound was used for visualization of actin-containing structures in eukaryotic nonmuscle cells. Due to its low molecular weight (1250), fixation for formaldehyde was sufficient to render the membrane permeable for the labeled peptide. Bundles of microfilaments are the predominant pattern in the flat rat kangaroo PtK1 cells, whereas a net of concentric fibers characterizes the more spherical bovine kidney MDBK cells. Specificity of staining was confirmed by competition experiments with unlabeled phalloidin.


Biological detection of specific mRNA molecules by microinjection.

We have used direct microinjection of messenger RNA into individual mouse and human cells to assay for specific translation products. We have been able to detect the synthesis of human fibroblast interferon, thymidine, kinase, hypoxanthine phosphoribosyltransferase, adenine phosphoribosyltransferase, and propionyl-CoA carboxylase in response to injected mRNA. Using the interferon system as a model, we have quantitated interferon synthesis and followed partial purification of interferon mRNA sequences on sucrose density gradients. The methods we have utilized should be applicable to other systems in which sensitive assays exist for gene products and should provide a screening procedure for isolating specific mRNA sequences.


Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S.

Murine erythroleukemia cells (MELC) are induced to erythroid differentiation by culture with 4 mM hexamethylenebisacetamide. The relationship between onset of accumulation of globin mRNA and cell cycle phase has been examined in MELC fractionated with respect to cell cycle (G1, early S, mid S, and late S/G2) by the technique of centrifugal elutriation. This technique provides synchronized populations of cells without use of chemicals that block cell function (e.g., DNA synthesis). It is shown that the initial onset of accumulation of globin mRNA occurs during the first G1 phase after a complete S traversed in culture with inducer. Once globin mRNA synthesis is initiated, it continues throughout the cell cycle. Studies of synchronized MELC populations exposed to hexamethylenebisacetamide starting in G1, mid S, or late S/G2, provide evidence that an effect of the inducer during G1 or early S is critical to initiation of accumulation of newly synthesized globin mRNA and to the prolongation of G1, both of which are associated with terminal erythroid differentiation.


Calculation of evolutionary trees from sequence data.

Evolutionary trees are usually calculated from comparisons of protein or nucleic acid sequences from present-day organisms by use of algorithms that use only the difference matrix, where the difference matrix is constructed from the sequence differences between pairs of sequences from the organisms. The difference matrix alone cannot define uniquely the correct position of the ancestor of the present-day organisms (root of the tree). Furthermore, methods using the difference matrix alone often fail to give the correct pattern of tree branching (topology) when the different sequences evolve at different rates. Only for equal rates of evolution can the difference matrix (when used with the so-called matrix method) yield exactly the correct topology and root. In this paper we present a method for calculating evolutionary trees from sequence data that uses, along with the difference matrix, the rate of evolution of the various sequences from their common ancestor. It is proven analytically that this method uniquely determines both the correct topology and root in theory for unequal rates of sequence evolution. How one would estimate an ancestral sequence to be used in the method is discussed in particular for the 5S RNA sequences from prokaryotes and eukaryotes and for ferredoxin sequences.


Polymorphism and loss of duplicate gene expression: a theoretical study with application of tetraploid fish.

We studied the fixation of null alleles at independent duplicate loci, assuming that wild-type active alleles mutate irreversibly to nonfunctional null alleles and that the population is finite and panmictic. Solving the two-dimensional Kolmogorov backward equation numerically, we obtained the rate at which one of the active genes is lost and the amount of heterozygosity at specified times. Previously harmful genes, including recessive lethals, can be fixed at one of the duplicate loci, which would not happen with a single locus. Examination of data from several fish families showed that the rate of fixation of null alleles is too slow and the amount of heterozygosity too small to be compatible with complete recessivity at all loci. Our conclusion differs in this regard from that of Bailey et al. [Bailey, G.S., Poulter, R. T. M. & Stockwell, P. A. (1978) Proc. Natl. Acad. Sci. USA 75, 5575--5579]. They also reported that the time taken for 50% of the loci to be fixed for null alleles is approximately 15N + v-3/4, in which N and v are the effective population sizgote is lethal. We found that the fixation rate depends not only on N, but also on Nv.


Cytoplasmic inheritance of erythromycin resistance in human cells.

An erythromycin-resistant mutant, ERY2301, was isolated from ethidium bromide-treated HeLa cells in the presence of erythromycin at 300 micrograms/ml. ERY2301 cells were enucleated and the anucleate cytoplasts were fused with D98/AH-2, a hypoxanthine phosphoribosyltransferase-deficient variant of HeLa cells. The resultant cybrids were isolated in a double selective medium containing erythromycin and 6-thioguanine. Cybrid formation occurred at a frequency of 10(-3) to 10(-4). In vitro protein synthesis by intact and Triton X-100 treated mitochondria isolated from ERY2301 was resistant to the macrolide antibiotics erythromycin and carbomycin, but was sensitive to chloramphenicol. These results suggest that the site of erythromycin resistance in ERY2301 may be at the level of mitochondrial protein synthesis and indicate that this trait is cytoplasmically inherited and, therefore, presumably encoded in the mitochondrial genome.


Regulation of the tissue specificity of an enzyme by a cis-acting genetic element: evidence from interspecific Drosophila hybrids.

Homologous genes for alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) are expressed in qualitatively different patterns during the development of two closely related species of Hawaiian Drosophila. In interspecific hybrids, each parental structural allele is expressed according to the developmental program characteristic of the species from which it is derived. This provides strong evidence for a cis acting control element.


DNA restriction endonuclease analysis for localization of human beta- and delta-globin genes on chromosome 11.

DNA from a clone of a mouse-human hybrid that retained a human chromosome consisting of the major part of chromosome 11 and region q25-26-qter of the X chromosome was digested with various restriction endonucleases, subjected to electrophoresis in agarose gels, and transferred to nitrocellulose filters. The restriction digest pattern of the clone, when hybridized with a 32P-labeled plasmid fragment containing human beta-globin gene sequences, was a composite of the normal human and mouse (A9) patterns. When back-selected in 6-thioguanine to eliminate the 11 translocation chromosome, the hybrid cells showed only the A9 restriction pattern. These results substantiate the localization of beta- and delta-globin genes to human chromosome 11 and exclude the region 11q23-qter as the site.


Telomere replication, kinetochore organizers, and satellite DNA evolution.

Robertsonian rearrangements demonstrate one-break chromosome rearrangement and the reversible appearance and disappearance of telomeres and centromeres. Such events are quite discordant with classical cytogenetic theories, which assume all chromosome rearrangements to require at least two breaks and consider centromeres and telomeres as immutable structures rather than structures determined by mutable DNA sequences. Cytogenetic data from spontaneous and induced telomere-telomere fusions in mammals support a molecular model of terminal DNA synthesis in which all telomeres are similar and recombine before replication and subsequent separation. This, along with evidence for a hypothetical DNA sequence, the kinetochore organizer, readily explains latent telomeres, latent centromeres, and reversible (one-break) Robertsonian rearrangements. A second model, involving simply recombination between like satellite DNA sequences on different chromosomes, explains not only how one satellite can simultaneously evolve on different chromosomes, but also why satellite DNA is usually located near centromeres or telomeres and why it maintains a preferred orientation with respect to the centromere.


Cell cytotoxicity due to specific influenza antibody production in vitro after recent influenza antigen stimulation.

Peripheral blood leukocytes, obtained from volunteers after vaccination or natural illness with influenza, were assayed for cytotoxicity against influenza virus-infected cells. Approximately 7 days after vaccination or the onset of respiratory illness, peak cytotoxicity was demonstrated in a chromium-release assay. Secretion of specific antibody against hemagglutinin from the leukocytes during in vitro incubation was demonstrated in quantities that would mediate the cell cytotoxicity observed. Antibody secretion was inhibited by exposure to cycloheximide but not by exposure to trypsin. The secretion of antibody against hemagglutinin from peripheral blood leukocytes occurred only at the time of maximal cytotoxicity. We thus demonstrate secretion of specific antibody in vitro after recent viral antigen stimulation. Moreover, this antibody is capable of conveying cytotoxic capacity to peripheral blood leukocytes that may be important in the recovery process from acute viral infection.


Nicotinic acetylcholine receptor: evidence for a functionally distinct receptor on human lymphocytes.

The presence of three distinct cholinergic receptors on human lymphocytes was suggested by the effects of carbamoylcholine on lymphocyte proliferation in vitro. The cells responded to both 0.1 nM and 1 microM carbamoylcholine by increased proliferation which was blocked by the muscarinic antagonist atropine. This effect occurred in both mitogen-stimulated cells (maximum effect at 24 hr) and nonstimulated cells (maximum effect at 72 hr). In contrast, 1--10 nM carbamoylcholine produced diminished in vitro proliferation, an effect which was blocked by the nicotinic antagonists alpha-bungarotoxin and d-tubocurarine.


Studies on murine Ss protein: demonstration that S region encodes structural gene for fourth component of complement.

Although genes controlling the expression of certain complement components have been shown to be linked to the major histocompatibility complex of several species, the structural genes that encode these molecules have been more difficult to map. In this study, the three constitutive polypeptide chains of the fourth component of murine complement (C4) (alpha, beta, and gamma) were isolated from 14 different inbred strains and compared by peptide mapping on analytical sodium dodecyl sulfate gels. The peptide patterns of the alpha and gamma subunits appeared to be nearly identical, but two distinctly different patterns were observed for the C4 beta chain. This structural variant was mapped to the S or G region and, as such, provides direct evidence that a structural gene for a complement component is encoded within a major histocompatibility complex.


Apolipoproteins in human cerebrospinal fluid.

The presence of apolipoproteins A-I, E, C-II, and C-III and the absence of apolipoprotein B was demonstrated in human cerebrospinal fluid. The concentration of apolipoproteins was measured by electroimmunoassay. Apolipoproteins E, C-II, and C-III were present in cerebrospinal fluid at 3--5% of their concentration in plasma; the cerebrospinal fluid level of apolipoprotein A-I was 0.4%. Most of the cerebrospinal fluid apolipoproteins were present in the rho less than 1.21 g/ml lipoprotein fraction. The major apolipoporteins of cerebrospinal fluid are E and A-I. The possible mechanism of transfer and the physiological and pathophysiological role of apolipoproteins in cerebrospinal fluid are postulated.


Cholesterol homeostasis in the rat with a portacaval anastomosis.

Studies were undertaken to determine the effect of portacaval anastomosis on cholesterol homeostasis in rats fed sucrose/lard under conditions of normal body growth. Four to 6 weeks after portacaval shunt surgery, we found decreases in plasma cholesterol and triglyceride concentrations, total liver weight, and hepatic microsomal protein concentration. Measurements oof hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) activity showed decreases in specific activity and total liver activity in portacaval shunt rats, but the enzyme diurnal rhythm remained. Decreased reductase activity in shunted rats was not due to an altered Km for D-HMG-COA, nor was an enzyme inhibitor found in the livers of the portacaval shunt animals. Sterol balance measurements in rats with shunts showed a 22% decrease in whole body cholesterol synthesis rate compared to controls. These metabolic studies, coupled with postmortem data, showed diminished bile acid synthesis, unchanged fecal neutral steroid excretion, and decreased net tissue accumulation of cholesterol during growth. The decreased whole body cholesterol synthesis rate ultimately led to a diminished total carcass cholesterol concentration in the rats with shunts.


Restriction endonuclease cleavage map and location of unique features of the DNA of hepatitis B virus, subtype adw2.

DNA of hepatitis B virus (HBV) of hepatitis B surface antigen (HBsAg) subtype adw2 made fully double stranded by the virion DNA polymerase and radiolabeled either by the virion DNA polymerase reaction or by nick-translation with 32P-labeled deoxynucleoside triphosphates was used to establish a map of restriction endonuclease cleavage sites by the method double and triple enzyme digestion and to determine the relative positions of several unique physical features of this DNA. The five restriction sites for enzyme HincII, the two sites each for BamHI, Ava I, and Bgl II, and the single sites for EcoRI, Pst I, Hpa I, and Taq I were positioned relative to each other. Within this map, the single-stranded region in HBV DNA has been localized and the locations of nicks in each strand (a and b) have been determined with respect to restriction sites on the circular map. Comparison of restriction endonuclease cleavage patterns of DNAs of HBV of HBsAg subtypes adw2, ayw3, and adrq+ revealed consistent differences among subtypes and occasional differences within subtypes.


Regulation of the state of phosphorylation of specific neuronal proteins in mouse brain by in vivo administration of anesthetic and convulsant agents.

The effect of drug treatment in vivo on the state of phosphorylation of two specific neuronal proteins, proteins Ia and Ib, has been studied in mouse brain. For this purpose, animals were killed by immersion into liquid nitrogen, and proteins Ia and Ib were extracted by a procedure designed to prevent alterations in their state of phosphorylation. Several anesthetic agents (pentobarbital, chloral hydrate, and urethane) each caused a decrease in the state of phosphorylation of these proteins. Conversely, the convulsant agents pentylenetetrazol and picrotoxin each caused an increase in the state of phosphorylation of these proteins. Neither the anesthetic nor the convulsant agents affected the total amount of these proteins. The results are compatible with a role for proteins Ia and Ib in neuronal function.


Target recognition in neurogenesis: formation of the Mauthner axon cap.

To learn whether presence of a specific neuron, the Mauthner (M) cell, is required for the organization, during embryogenesis, of an associated synaptic neuropil, the M-axon cap, M-cell precursors were experimentally deleted in embryos of the zebra fish (Brachydanio rerio) and the axolotl (Ambystoma mexicanum). Examination of early larvae revealed that in about half of the cases M-axon caps were present in the absence of the corresponding M-cells. The locations and structures of such caps were approximately normal. We conclude that the M-cell is not a necessary target for normal development of the axon cap.


Speech reception by the hearing-impaired: methods of testing and the development of new tests.

The development of tests for measuring speech reception in the hearing impaired is reviewed. The rationale for a new closed-response-set nonsensesyllable test is presented followed by a description of the test. Preliminary normative and comparative data for this Nonsense Syllable Test are reported. Current work on the development of related tests for young, profoundly deaf children, and measuring reception of the prosodic features of speech is also described.


Some aspects of auditory space, speech perception and the use of hearing aids.

The auditory space of an individual is operationally defined as that perceptual area that lies between the upper tolerance level for loud sounds and the threshold of hearing for a specified range of frequencies. Results from recent experimental work are presented to show a difference of some 15 dB between comfortable listening level and loudness discomfort-level, up to 80 dB hearing loss. Results are also shown for the measurement of reaction time to a 2AFC task testing the ability to discriminate initial consonants. Loudness scaling is suggested as a method of eliciting supra-threshold measurements which might be helpful in the fitting of hearing aids.


Requirements on speech processing hearing aids for the profoundly deaf, some preliminary remarks.

Many severely hard of hearing and profoundly deaf persons seem to get very little help from an ordinary hearing aid. This observation has prompted the development of tactile speech processing aids. These aids should however only be used if it can be shown that the subject has no useful residual hearing. In the case of a profound hearing loss the audiogram might in some cases show tactile sensations in the ear. To get a better estimation of the remaining auditory capacity experiments have been made with frequency discrimination measurements, and with speech tests consisting of rhyming word pairs. Results from these different tests are summarized in relation to an audiogram classification system. Two tactile speech processing aids are described. Evaluation studies with these aids show positive results but these results must be compared with the results that can be obtained with ordinary hearing aids and different types of auditory speech processing hearing aids. The possibility to use extreme auditory recording must also be studied. In this case the transmitted signal is not speech-like and it is possible that this limits their usefulness. Experiments with this type of aids might however give valuable information about how the human perceptual systems process signals derived from the original speech signal.


Methods for the evaluation of hearing aids.

Methods of hearing-aid evaluation are sub-divided into four broad categories, prescriptive selective amplification, selection from a set of existing hearing aids, use of a fixed frequency-gain characteristic, and adaptive fitting of a master hearing aid. The advantages, disadvantages, and controversies associated with each approach are reviewed and major research needs are discussed in the light of this review. Among the more important research needs are i) the development of improved speech tests that have low test-retest variability and which can discriminate between hearing aids with much greater precision than existing tests, ii) the development of reliable methods of predicting hearing-aid performance from audiological data, and iii) the determination of the optimum characteristics of the simplest, conventional hearing aid (i.e., a frequency-selective linear amplifier with no distortion other than peak clipping to limit maximum power output). It is also recommended that the simplest conventional hearing aid with gain, frequency-response, and clipping characteristics optimally matched to the subject's residual hearing be used as a reference for assessing more sophisticated hearing aids or alternative auditory prostheses for the hearing impaired. The third part of the paper provides a progress report on the development of a protocol for the prescriptive fitting of a wearable master hearing aid. The results to date show that the parameters of a conventional hearing aid can be adjusted according to well established optimization procedures so as to obtain superior, if not optimum, performance for each subject as a result of such individualized adjustments.


Adaptive testing in audiology.

A general approach to adaptive testing of the up-down type is described. Audiological applications of the procedure are described, including measurement of the auditory threshold, estimating the spread of the subject's response curve, testing for sequential response dependencies, and finding PB-max. Guidelines are provided for the practical implementation of these procedures using relatively simple rules  for controlling stimulus levels and for estimating the parameters of interest. Also described is the Simplical Method, a multivariate adaptive procedure which, like the transformed up-down method, is relatively efficient yet involves simple rules of adjustment and of estimation. The Simplical Method has been used in the adaptive fitting of a wearable master hearing aid and an example of this application is given.


Clinical experiments with hearing aids with amplitude compression.

The model for audiological rehabilitation in Denmark is brief-described. Clinical experiments have not demonstrated a general superiority of the compression hearing aids compared to the conventional hearing aids. In situations with negligible background noise the compression hearing aid tends  to provide the best comprehension of speech. However, in situations with background noise the conventional hearing aid is generally preferred by hearing impaired persons. The attitude with regard to compression hearing aids differs among Danish hearing clinics. One clinic with a very positive attitude delivers compression hearing aids to 27% of the total clientele. In several clinics it has been observed that patients with a perceptive impairment who are using the hearing aid for the first time may benefit from compression during the early stages of hearing aid adjustment. In many cases, however, these patients prefer less compression as they continue to use their hearing aids in daily life. It is pointed out, that in connection with compression hearing aids it is especially important to provide careful instruction about the possibilities and limitations of the hearing aid.


Multichannel compression hearing aids: experiments and consideration on clinical applicability.

The rational for applying multichannel compression in connection with peripheral perceptive hearing loss is briefly discussed. The speech transfer ability of a multichannel compression hearing aid with up to 4 channels is evaluated on high tone hearing impaired subjects. The compression characteristic of each channel is individually fitted to compensate on average the loudness recruitment of the subject's hearing within each individually determined frequency band. Experiments on 5 subjects under several signal-to-noise ratio conditions reveal that 4-channel recruitment compensation is indistinguishable in performance from an "optimally" fitted linear hearing aid which takes advantage of the limited dynamic range of the applied speech signal. Contrarily, 3- and 2-channel configurations investigated show in general degraded performance on speech compared to this condition. The results obtained and their implications for the design objectives of future multichannel compression hearing aids and the clinical fitting of such an aid are discussed.


The influence of attack time and release time on speech intelligibility. A study of the effects of AGC on normal hearing and hearing impaired subjects.

Automatic gain control may be used in hearing aids intended for different types and degrees of hearing loss; to average the levels, to reduce the dynamics of speech or to set the maximum output level of the hearing aid without adding nonlinear distortion. Our study concerns hearing impaired listeners with recruitment. A group of normal hearing subjects was used as a control group. The compression was introduced over a laboratory unit. The compression ratio was set to 30:5 dB. A fixed ratio between the attack time and the release time, tr = 200 x ta, was used. The values used varied from 0.05 to 5 ms for ta and 10 - 1000 ms for tr. The speech material was nonsense syllables of the CVC type presented with S/N = 60 dB and S/N = 5 dB. With S/N = 5 dB a significant deterioration of the discrimination of the nonsense syllables was found at time constant combination ta = 5 ms and tr = 1000 ms. No statistically significant difference in discrimination was found between the different combinations of attack and release times for the S/N = 60 dB condition neither for the normal hearing nor for the hearing impaired group.


Automatic regulation systems with relevance to hearing aids.

Compression amplification in hearing aids has gained much interest in the recent years. Other types of nonlinear signal processing in hearing aids are developing. The principles of nonlinear signal processing through automatic regulation, as opposed to instanteaneously acting nonlinear signal transfer, is described in general. In particular the properties and terminology of amplitude compression and limiting circuits in relation to hearing aids are discussed. Several examples are given of nonlinear regulation systems for hearing aids involving other features than amplitude compression.


The industrial aspect in describing performance characteristics in complex hearing aids.

Performance characteristics of complex hearing aids (e.g. AGC hearing aids) which are measured and presented using existing standards (IEC 118) may mislead the hearing aid fitter. In this paper examples of possible erroneous interpretations of frequency response curve are demonstrated. A test method is proposed to record "instantaneous frequency response" -- the actual response in the AGC mode which corresponds to the perceived tonal balance.


On the problems of fitting an individual hearing aid: a case story.

For a fourteen-year old girl with a pronounced ski-slope bilateral hearing loss of 100-120 dB and with a dynamic range of approx. 10 dB, a tailor-made true binaural hearing aid was made. Establishing the acoustical characteristics of the hearing aid based on details from audiograms proved not to yield satisfactory results. Consequently an adjustment of the acoustical data according to the method of trial and error was initiated. By applying this method, a satisfactory result was obtained. The suitability of an audiogram to describe the optimum characteristics of hearing aids may thus be questioned. Likewise it seems substantiated that it is possible to compensate for even very difficult hearing losses by tailoring the acoustical characteristics of the hearing aid very carefully. Consequences of the individual fitting of hearing aids in general will be discussed.


Comparison of normal and impaired hearing. I. Loudness, localization.

Impaired hearing is characterized by high thresholds and reduced loudness. Loudness, however, may quickly recover as it increases rapidly from an elevated threshold. This rapid growth, known as loudness recruitment, is a sign of cochlear impairment and is generally not seen in conductive or retrocochlear impairment. Loudness recruitment means that the hard-of-hearing person detects small changes in intensity near his elevated threshold but he probably does no better than a normal listener at the same SPLs. Recruitment is often accompanied by reduced loudness summation, which means that the loudness of a band of noise does not increase as much with increasing bandwidth as in normal hearing. This reduced summation of loudness is probably why the cochlearly impaired ear has nearly the same threshold for the acoustic reflex to pure tones as to wide-band noise, whereas the normal ear has a much lower threshold to wide-band noise. Corresponding differences between normal and impaired hearing are not found in auditory localization. Rather, the evidence suggests that persons with residual hearing learn to localize sounds reasonably well. Even the inability of many hearing impaired persons to understand a speaker in a noisy environment may result more from a failure of frequency analysis rather than of localization.


Comparison of normal and impaired hearing. II. Frequency analysis, speech perception.

Frequency analysis covers two separate listening tasks, one involving frequency discrimination, the other frequency selectivity. Discrimination refers to the ability to distinguish one frequency from another. Selectivity refers to the ability to hear one frequency in the presence of other frequencies. Selectivity is critical to the understanding of speech which comprises sounds containing many different frequencies. To understand speech easily, the listener must be able to analyze speech sounds into their component frequencies, especially formants. The hard-of-hearing person is probably less able to make that analysis, but we know surprisingly little about either discrimination or selectivity in hearing impairment. Existing evidence does suggest that both discrimination and selectivity are reduced in cochlear impairment so that such patients need a bigger frequency difference to discriminate between two tones and they have a wider critical band. A widened critical band would be expected to make it very difficult for the severely impaired person to understand speech under all listening conditions; it would make it difficult for the moderately impaired person to understand speech in a noisy background, unless the signal-to-noise ratio is improved as is possible by appropriate amplitude compression in hearing aids.


Peripheral auditory processing in normal and abnormal ears: physiological considerations for attempts to compensate for auditory deficits by acoustic and electrical prostheses.

The processing of complex acoustic signals, such as speech, by the normal peripheral auditory system is described in terms of the properties of individual cochlear nerve fibres and the effects of lateral inhibition in the cochlear nucleus. The implications of the peripheral filtering of signals is indicated in psychophysical terms. In cochlear pathology, these filtering characteristics deteriorate, and the implications arising from the consequent deterioration in auditory frequency selectivity are outlined for the coding of loudness and for the analysis of speech. These considerations suggest theoretically ideal methods of compensation for recruitment by multichannel instantaneous compression, and for deterioration in format analysis by dichotic frequency partition. Finally, the implications for a minimal design for artificial electrical multi-electrode array stimulation of the cochlea in order to impart speech information to the profoundly deaf, are explored. Single wire stimulation, on the other hand, should be able to impart useful prosodic and minimal speech feature information to aid in lip reading and in the acquisition of speech.


A scanning electron microscopic and microradiographic study of pits in fluorosed human enamel.

The aim of the present study has been to correlate the surface appearance of pitted, fluorosed enamel in the scanning electron microscope (SEM) with the microradiographic features of the underlying tissue. Intact enamel surfaces of severely fluorosed teeth appeared similar to that of normal enamel. In the unabraded surfaces discrete pits were sharply demarcated from the surrounding intact enamel surface leaving steep walls of parallel running rods. The microradiographic appearance of sections made through pits indicated that focal loss of surface enamel occurred corresponding to the inner highly hypomineralized part of the fluorotic subsurface lesion. The abrupt wall formation and the finding that the striae of Retzius never changed direction along the margins of the lesions indicated that pits in fluorosed enamel may be secondarily produced defects rather than true hypoplasias. Further evidence of the posteruptive origin of the defects was derived from the observation that enamel lamellae occasionally formed part of the lateral border. The relatively higher degree of radiopacity observed in the tissue surrounding the pit indicates a posteruptively acquired deposition of minerals in the exposed porous enamel.


Determination of fluoride and chlorhexidine from chlorhexidine/fluoride-containing dentifrices.

The intention of the present experiment was to study the effects of a possible interaction between fluoride and chlorhexidine when both agents were incorporated in the same vehicle. The amount of fluoride extractable from dentrifrices containing 0.1% NaF and the fluoride ion activity were not reduced by the addition of 2% chlorhexidine digluconate. Less than 50% of the added chlorhexidine was available when the dentifrices were dissolved in deionized water. This was not affected by the presence of fluoride. The in vitro antibacterial activity of the chlorhexidine-containing dentifrices was not reduced by the addition of fluoride. Approximately 40% of the chlorhexidine was retained in the human oral cavity after brushing for 1 min with both the chlorhexidine- and the chlorhexidine/fluoride-containing dentifrice. Thus the binding of chlorhexidine to vehicle ingredients when dissolved in water is probably too weak to affect the retention in the mouth.


Autoradiography of 90Sr in developing rats.

The distribution patterns of 90Sr in five littermate, 8-day-old Wistar rats were studied by whole body autoradiography. Rats were killed 15 min, 1, 4, 24, and 72 h after a single intraperitoneal injection of the isotope. Immediately after administration, 90Sr was distributed throughout most of the soft tissues of the body. The soft tissue deposits had practically disappeared after 4 h. In the hard tissues of the body 90Sr accumulated up to 24-72 h. Fifteen minutes after injection the uptake of 90Sr in the enamel of the teeth was highest in the occlusal and incisal regions. 90Sr gradually accumulated throughout the enamel and after 72 h its distribution in this layer was fairly uniform. Immediately after injection a narrow zone of radioactivity appeared in the dentin near the pulp. This zone broadened with time towards the dentinoenamel junction and included the entire dentin layer 72 h after injection. Initially, the uptake of 90Sr was higher in the dentin thatn in the enamel, particularly in the cervical areas of the crown. This difference became less apparent with time. There was good correlation between the uptake in teeth and bones, supporting the use of teeth as indicators of the 90Sr body burden.


A comparison between different methods for prediction of caries activity.

The aim of the study was to find out if any of the following factors, alone or in combination, could be used for prediction of caries activity: salivary S. mutans, salivary lactobacilli, buffer capacity, saliva secretion rate, salivary pH, the amount of plaque and the caries frequency. The patient material consisted of 300 children between 9 and 12 years old. The increment of caries after 1 and 2 years was compared with all the above factors registered at baseline. Only the factors caries frequency and S. mutans showed a significant correlation to caries activity. In one of the tests the lactobacillus count approached significance. The combination of high numbers of incipient smooth surface lesions, S. mutans and lactobacilli gave a mean value of caries activity almost three times that of the whole group (8.4 versus 3.1).


Abrasion of class 1 restorative resins.

Studies have been made on the abrasion by food of different restorative resins in standard Class 1 cavities in mandibular first molars of schoolchildren. The abrasion was recorded by measuring the increasing height of the exposed cavity walls. It was found that Sevriton Simplified was abraded more than Adaptic, that no difference in abrasion between Adaptic and Adaptic without filler could be demonstrated, and that Adaptic was abraded more than an experimental composite containing 30% by weight Aerosil, an ultra-fine SiO2 filler with a grain size of the order of 25-50 nm. The results are in accord with the findings in an initial study by one of the present authors, in which the fillings were placed in denture porcelain teeth. It is stressed that the conclusions of the present work are pertinent only to the type of abrasion studied, i.e. chewing of food without the possibility of direct contact between the fillings involved and the corresponding antagonists.


Effect of long-term administration of fluoride on plasma fluoride and calcium in relation to forming enamel and dentin in rats.

Three groups of 1-month-old rats were kept on water containing 0.2, 56.5 and 113 parts/10(6) F-, respectively, for a period of 2 months. These groups were set up in order to study the relationship between long-term administration of high doses of waterborne fluoride, plasma values of fluoride and calcium and changes in forming dentin and enamel of the rat incisor. The microradiographic study revealed that increased levels of fluoride caused a delay in mineralization of the enamel. In the 113 parts/10(6) group the radiolucent areas in the enamel exhibited a distinct periodicity along the tooth, a phenomenon which cannot at present be explained. In the dentin the fluoride interfered with the process of mineralization giving rise to radiolucent and radiopaque banding in the experimental groups. With increasing levels of fluoride in the water supply a significant increase of plasma fluoride was recorded. In contrast the serum calcium values in both experimental groups were reduced to the same extent, which differed significantly from that of the control group. It is concluded that the fluoride doses which are needed to create fluorotic changes in rats may interfere with calcium metabolism and basic mineralization processes.


[Polymorphism of a tumor ce-l population and selection processes. IV. The effect of dibunol and nitrosourea on the variability of Ehrlich-I. Ch. Ph. ascitic strain tumor cells].

A study was made of the effect of dibunol and methyl-N-nitrosourea (MNU) on two tumor cell subpopulations of the Ehrlich-I. Ch. Ph. ascites strain, one of which is characterized with A + B + 2C and A + D + 2C--markers and the other one--with A1 + A2 + 2B + D + C markers. Dibunol that belongs to the class of inhibitors of free-radical processes was shown to bring about changes in cell subpopulations, the mode of changes depending on the dose and regime of treatment. The effect of MNU on the population resulted predominantly in the accumulation of cells with various chromosome aberrations. At early stages of tumor progression, aberrations were more pronounced in cells with marker chromosome "A" than in the cells with 44 chromosomes and markers A1 + A2 + 2B + D + C.


[Splenectomy and polychemotherapy in chronic myelosis].

It is referred to the possibility of the splenectomy in the early phase of the chronic myelosis in the time of the first remission. The intervention cannot be recommended generally and should be used only now and then in centres which have rooms at their disposal which are poor in germs. Own experiences speak for the fact to extend the polychemotherapy of the blast crisis of the chronic myelosis to its prephase, when an increasing therapy resistance occurs, the spleen becomes clearly larger, the number of blasts in the bone-marrow increases and single blasts may be proved in the peripheral blood. Combinations of two or three medicaments, such as Busulfan, Myelobromol, Merkaleukin, Methotrexat, Hydroxyurea, Alexan, Vinkristin or Rubomycin seem to be favourable.


Prognostic signs in chronic myelocytic leukemia.

In a retrospective search for prognostic factors in 127 patients with chronic myelocytic leukemia we found that thrombocytopenic patients had a significantly shortened life-span. Patients with the Philadelphia chromosome lived longer and were significantly younger than those lacking it. Females lived longer than males. Numerous other single features failed to correlate significantly with survival, but several combinations of two did. Patients having a low hematocrit with a low white blood count had an impressively short life-span compared to all others. Those having a high cholesterol and a low uric acid survived significantly longer than all others.


Acute lymphoblastic leukemia--hand mirror variant. An analysis of a large group of patients.

One hundred and thirty-four initial bone marrows and 102 peripheral blood smears were evaluated for hand mirror cells (HMC) on pediatric patients with acute lymphoblastic leukemia (ALL). Twenty (15%) of the 134 patients were found to have greater than 5% HMC in their bone marrows. Only rarely were a few HMC noted in the peripheral blood. Survival did not appear to be related to the number of HMC in the bone marrow. Also, the median survival was not significantly greater in those HMC patients with greater than 40% HMC in their bone marrows when compared to the non-HMC group. From the available data in this study, it would appear that the number of HMC does not affect prognosis in the bone marrows of children with ALL. However, the prognostic significance of the HMC has not been clearly established in adults.


Regional localization of human gene loci on chromosome 9: studies of somatic cell hybrids containing human translocations.

Somatic cell hybrids were derived from the fusion of (1) Chinese hamster cells deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) and human cells carrying an X/9 translocation and (2) Chinese hamster cells deficient in thymidine kinase (TK) and human cells carrying a 17/9 translocation. Several independent primary hybrid clones from these two series of cell hybrids were analyzed cytogenitically for human chromosome content and electrophoretically for the expression of human markers known to be on human chromosome 9. The results allow the assignment of the loci for the enzymes galactose-1-phosphate uridyltransferase (GALT), soluble aconitase (ACONs), and adenylate kinase-3 (AK3) to the short arm of chromosome 9 (p11 to pter) and the locus for the enzyme adenylate kinase-1 (AK1) to the distal end of the long arm of human chromosome 9 (hand q34). Earlier family studies have shown that the locus for AK1 is closely linked to the ABO blood group locus and to the locus of the nail-patella (Np) syndrome. Thus the regional localization of AK1 locus permits the localization of the AK1-Np-ABO linkage group.


Linkage analysis and the inheritance of arches in a Habbanite isolate.

A pedigree and linkage analysis was performed on a corrected version of the Habbanite pedigree 2 of Slatis et al. [1]. The trait "arch on any digit" was examined for major gene inheritance and possible linkage to several blood and serum group markers. The results confirm the proposed dominant major gene inheritance of this trait with almost complete penetrance. In addition, the analysis suggests linkage with the haptoglobin locus with evidence against linkage with Pl and Rhesus. These results are of particular interest in view of recently reported dermatoglyphic associations with haptoglobin.


Leukocyte larceny: a cause of spurious hypoxemia.

Multiple blood specimens with different leukocyte counts from two patients with extreme leukocytosis secondary to leukemia and unexplained hypoxemia were tonometered with a gas of known oxygen concentration and the decay of oxygen tension (PO2) was measured over time. The decay in PO2 in the first 2 minutes for blood with leukocyte counts of between 55.2 X 10(3)/mm3 and 490.0 X 10(3)/mm3 ranged from 13 to 72 torr. The degree of PO2 decay was blunted by placing the blood on ice and was obliterated by adding potassium cyanide. Thus, extreme leukocytosis secondary to leukemia can cause spurious hypoxemia and spurious lowering of the mixed venous PO2 due to oxygen consumption by leukocytes ("leukocyte larceny").


Frequency of tooth extraction in orthodontic treatment.

Orthodontic extraction frequency describes the percentage of an orthodontic patient population experiencing extractions of one or more permanent teeth, excluding third molars. It is a useful statistical measure of the many variables associated with the extraction-nonextraction decision. For a study sample of 537 North American white orthodontic patients, the frequency of tooth extraction was determined as 42.1 percent. This is compared with a range of 6.5 percent to 83.5 percent reported in the world dental literature. Factors responsible for the enormous differences in orthodontic extraction frequency are explored. The study of extraction frequency offers a simple, valuable aid for the clinician to understand better the nature of his patient population, his treatment methods, and his rationalizations.


Preformed arch wires: reliability of fit.

The preformed arch wires currently available are unsuitable for many orthodontic cases, since these arch wires do not take into account all the variations in the size and dimensions of the human arch. In this study, nine theoretical arch wires are derived, based upon two-parameter curves. Tests were conducted which demonstrated that arch wires performed to these nine sets of dimensions would correspond with a reasonable degree of accuracy to a large section of the orthodontic population.


Patient radiation dose in conventional and xerographic cephalography.

A comparison of the radiation doses for xeroradiographic and conventional film screen cephalography was made. Alderson tissue-equivalent phantoms were used for patient stimulation. An optimum technique in terms of patient dose and imaqe quality was established for the xeroradhe data indicated that the dose for the Xerox process ranged from five to eleven times greater than that for the conventional process for entrance and exit exposures, respectively. The most commonly reported dose, the entrance dose, was found to be 206 mrad, which is five Imes that for the conventional cephalogram. This dose, however, falls within an acceptable range for other dental and medical radiation doses. It is recommended that conventional cephalography be used for routine purposes and that xeroradiography be reserved for situations requiring the increased image quality that the process affords.


The functional compensation: an interceptive procedure in the mixed dentition.

Functional compensation, like the morphologic one which accompanies it, is to be encountered naturally in each system or organ. It facilitates the performance of the required activities in spite of the defectiveness of one or more of their biocomponents. If the pathologic forms of compensation or simply their perturbing effects are noticed early enough, deleterious and irreversible effects on growth and development of the orofacial zone may be avoided. Instead of simply supervising dental evolution or limiting intervention to early procedures (extractions) until the time for corrective treatment, we propose functional interception. Inducing a therapeutic compensation by removable appliances, this therapy aims to modify the equilibrium of displaying forces and to favor a more physiologic alveolar and sutural growth. The proposed appliances are able to intervene in the postural and dynamic position of the mobile organs in the oral cavity. They compensate their action; that is, they correct, complete, or replace it. The bimaxillary appliance is called bioactivator "biprax". Based simultaneously on the principles of the activator and bionator, it exploits a normal display of the two praxisms-speech and deglutition. Being worn day and night, it is able to guide efficiently the evolution of dental, alveolar, and even basal relationships. Its interceptive use does not interfere in any way with the onset of a corrective treatment in the adult dentition.