Repair of ultraviolet light-damaged deoxyribonucleic acid in sbc-A strains of Escherichia coli K-12. An Escherichia coli strain carrying both rec+ and sbcA has been constructed. Repair of ultraviolet light-induced deoxyribonucleic acid damage was examined by measuring survival and thymine-dimer excision in the rec+ sbcA strain as well as rec+ sbcA+ and recB recC sbcA strains. The sbcA mutation restores normal survival in both recB recC uvrB and recB recC uvr+ strains. Excision of thymine-containing dimers does not occur in uvrB mutants, regardless of the rec or sbcA genotype. Survival, after ultraviolet-light damage, of a rec+ sbcA strain is quantitatively similar to rec+ sbcA+ and recB recC sbcA strains.


Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli. The lipid phase transition of Escherichia coli phospholipids containing cyclopropane fatty acids was compared with the otherwise homologous phospholipids lacking cyclopropane fatty acids. The phase transitions (determined by scanning calorimetry) of the two preparations were essentially identical. Infection of E. coli with phage T3 inhibited cyclopropane fatty acid formation over 98%, whereas infection with mutants which lack the phage coded S-adenosylmethionine cleavage enzyme had no effect on cyclopropane fatty acid synthesis. These data indicate that S-adenosylmethionine is the methylene in cyclopropane fatty acid synthesis.


Alteration of the fatty acid composition of Escherichia coli by growth in the presence of normal alcohols. The addition of normal alcohols in the series n-butanol to n-octanol to cultures of Escherichia coli ML308 grown on defined or lipid-free medium (at 17, 27, and 37 degrees C) caused an alteration in the fatty acid composition of this organism: the ratio of saturated to unsaturated fatty acids increased. Changes in the relative quantities of individual fatty acid species elicited by increasing concentrations of these alcohols were as follows: (i) myristic acid remained constant: (ii) palmitic acid increased; and (iii) the combined amount of palmitoleic plus cis-methylene hexadecanoic acids changed in a way which was reflected inversely by changes in the amount of cis-vaccenic acid. Comparable changes were not observed when cells were grown in the presence of n-nonanol and n-decanol in the concentration range tested. The changes observed upon addition of normal alcohols (n-butanol to n-octanol) paralleled, in part, the alterations in fatty acid composition observed when growth temperature was increased.


Mating pheromones of Saccharomyces kluyveri: pheromone interactions between Saccharomyces kluyveri and Saccharomyces cerevisiae. Saccharomyces kluyveri is a heterothallic yeast with two allelic mating types denoted as a-k and alpha-k by analogy with Saccharomyces cerevisiae and from the work described here. S. kluyveri produces mating pheromones analogous to those of S. cerevisiae, but which appear to have different specificity. S. kluyveri thus differs from S. cerevisiae, Hansenula wingei, and Schizosaccharomyces pombe in that it exhibits both strong constitutive agglutination and mating pheromones. alpha-k cells produce a pheromone ("alpha-k-factor") which causes a-k cells to arrest in the G1 phase of the cell cycle and to undergo a morphological change. After a period of time dependent on the concentration of alpha-k-factor, cells exposed to the factor resume cell division. alpha-k-factor has no effect on a-k/alpha-k diploids or on alpha-k cells, but at high concentration does induce G1 arrest of S. cerevisiaea cells (a-c). a-k cells produce a pheromone ("a-k-factor") which causes alpha-k cells to exhibit a morphological change. In addition, a-k cells exhibit the Bar phenotype with respect to alpha-k-factor. Partially purified preparations of S. cerevisiae alpha-factor are more active in inducing G1 arrest of a-k cells than of a-c cells. A more purified preparation of alpha-c-factor is less active against a-k cells than a-c cells, suggesting that an additional factor (KRE, kluyveri response enhancer) may be lost during purification. Attempts to mate S. kluyveri and S. cerevisiae cells by prototroph selection and by cell-to-cell mating have been unsuccessful with all combinations of mating types. Thus, S. cerevisiae and S. kluyveri are incompatible for mating even though their pheromones exhibit some physiological cross-reaction.


Regulation of nonspecific acid phosphatase in Salmonella: phoN and phoP genes. Mutations in Salmonella typhimurium strains lacking nonspecific acid phosphatase mapped in two unlinked loci. One of these, phoP, was cotransducible by phage P22 with purB, whereas the second, phoN, was cotransducible by phage P1 with purA. Mutants with temperature-sensitive nonspecific acid phosphatase activity (measured in whole cells) were also isolated. A phoN mutant with thermolabile whole-cell activity was isolated directly from wild-type LT-2. Several other mutants with temperature-sensitive enzyme activity were also isolated as revertants of phoN mutants. These data suggest that phoN might be a structural locus for nonspecific acid phosphatase. The observation that a mutation resulting in high level of nonspecific acid phosphatase mapped in phoP suggests a possible regulatory role for this locus.


Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae. Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited.


Length growth of two Escherichia coli B/r substrains. Length growth of synchronized Escherichia coli B/r substrain A (ATCC 12407) and B/r substrain F26 (Thy his) was followed with an electron microscope. Cells were grown with doubling times (tau) of 60 min (B/rA) and of 82 and 165 min (B/rF26). Different length growth patterns were found for the two substrains. In B/rF, the length growth rate increased about midway in the cell cycle. For tau = 165 min, the rate increase was preceded by a short period of slow growth. For B/r A (r = 60 min), this period seemed to occur at the beginning of the cell cycle. The possibility is raised that the different length growth patterns are related to different deoxyribonucleic acid replication patterns of the respective strains.


Meiotic mapping of yeast ribosomal deoxyribonucleic acid on chromosome XII. We have used meiotic mapping techniques to locate the position of the repeating ribosomal DNA (rDNA) genes of the yeast Saccharomyces cerevisiae. We found that the rDNA genes are located on the right arm of chromosome XII, approximately 45 map units centromere distal to the gene gal2. Together with mapping data from previous studies, this result suggests that the tandem array of rDNA genes contains at least two junctions with the non-rDNA of the yeast chromosome. In addition, we observed segregation patterns of the rDNA genes consistent with meiotic recombination within the rDNA gene tandem array in 3 of the 59 tetrads examined.


Characterization of arithmetic deoxyribonucleic acid synthesis at restrictive temperature in a dnaE mutant of Escherichia coli K-12. The dna-293  mutation is shown to be a dnaE allele. The linear deoxyribonucleic acid synthesis previously observed in this mutant has been further characterized. The production of small deoxyribonucleic acid intermediates and their subsequent joining were identical in the mutant and its dnaE+ parent at 42.5 degrees C. Though the mutant cells continued to divide at the nonpermissive temperature, the rate of division was reduced. The data are consistent with a lack of production of replicationally active deoxyribonucleic acid polymerase III at the restrictive temperature.


Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli. Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.


Relationship between deoxyribonucleoside triphosphate pools and deoxyribonucleic acid synthesis in an nrdA mutant of Escherichia coli. A shift to 42 degrees C in an nrdA mutant causes a decrease in deoxyribonucleic acid synthesis without a concomitant decrease in deoxynucleotide triphosphate pools.


Cross-linking analysis of the two forms of protein I, a major outer membrane protein of Escherichia coli. The two forms of protein I were cross-linked to molecules of the same species, even when both were present simultaneously. This suggests that they form separate multimers in the outer membrane.


Rapid methods for generalized transduction of Salmonella typhimurium mutants. A procedure has been developed that allows the propagation of generalized transducing phage directly on cells growing on solid media. After the donor cells are killed with chloroform, the phage can be transferred directly to recipient cells and transductants can be selected.


Mapping hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase, in Escherichia coli. The structural gene for histidyl-tRNA synthetase was localized to 53.8 min on the Escherichia coli genome. The gene order in this region was determined to be dapE-purC-upp-purG-(guaA, guaB)-hisS-glyA.


Resolution of glpA and glpT loci into separate operons in Escherichia coli K-12 strains. The independent insertion of bacteriophage Mu into the gene coding for anaerobic sn-glycerol 3-phosphate dehydrogenase (glpA) or into the genes coding for sn-glycerol 3-phosphate transport (glpT) suggested that these two closely linked loci are in separate operons.


Increased amplification of pBR322 plasmid deoxyribonucleic acid in Escherichia coli K-12 strains RR1 and chi1776 grown in the presence of high concentrations of nucleoside. When pBR322 plasmid-harboring Escherichia coli strains RR1 or chi1776 were grown in the presence of 1 mg of uridine or cytidine per ml and later treated with chloramphenicol, as much as three times more plasmid deoxyribonucleic acid was recovered than would normally be obtained by routine plasmid amplification procedures.


Distribution of cell lengths in cultures of a lexA mutant of Escherichia coli K-12. Distributions of cell lengths in lexA+ and lexA mutant cultures during normal growth and under thymidine starvation conditions are presented. During normal growth lexA mutant cells were slightly shorter, on the average, than were lexA+ cells. lexA mutant cells were also shorter in comparison with lexA+ cells after a period of thymidine starvation. These results are consistent with the hypothesis that the lexA gene is involved in the coordination of cell division with DNA repair.


Gene copy number effects in the mer operon of plasmid NR1. The level of resistance to Hg2+ determined by the inducible mer operon of plasmid NR1 was essentially the same for three gene copy number variants in Escherichia coli, less in Proteus mirabilis, and intermediate in P. mirabilis "transitioned" to a high r-determinant gene copy number. Cell-free volatilization rates of radioactive mercury indicated increasing levels of intracellular mercuric reductase enzyme from low- to high-gene copy number forms in P. mirabilis and from low- to high-copy number forms in E. coli, but the additional enzyme in E. coli was effectively cryptic.


Receptor for bacteriophage lambda of Escherichia coli forms larger pores in black lipid membranes than the matrix protein (porin). The receptor for phage lambda in Escherichia coli was isolated by cholate extraction and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands corresponding to the monomer and the dimer were eluted from the gel and tested for their activity to inactivate phage lambda and to form pores in black lipid membranes. It was found that only the dimer inactivated phage lambda, whereas both the monomer and the dimer were active in forming pores. The pore characteristics were similar to those exhibited by the matrix protein (porin) (R. Benz, K. Janko, W. Boos, and P. Läuger, Biochim. Biophys. Acta 511:305--319, 1978). In comparison, the lambda receptor showed a somewhat higher degree of cation specificity, and its pore size was larger. Assuming that the thickness of the outer membrane is 7.5 nm and that the pore is an ideal hydrophilic channel, the pore diameter in vivo was estimated to be 1.6 nm for the lambda receptor and 1.2 nm for the matrix protein.


Polypeptides expressed in Escherichia coli K-12 minicells by transposition elements Tn1 and Tn3. Escherichia coli K-12 minicells were employed to examine polypeptides encoded by plasmids carrying wild-type and mutant Tn1 or Tn3 transposition elements. Tn1- and Tn3-containing minicells express high levels of four transposon-specified polypeptides. Three, of molecular weights 30,000, 28,000, and 25,000, are related immunologically to beta-lactamase, the enzyme responsible for ampicillin hydrolysis. A fourth polypeptide of molecular weight 19,000 is encoded by the Tn1 or Tn3 region which spans the BamHI cleavage site. Mutant transposons which no longer produce this polypeptide transpose at higher than wild-type frequencies to give aberrant transposition products (Gill et al., J. Bacteriol. 136: 742--756, 1978; Heffron et al., Proc. Natl. Acad. Sci U.S.A. 72:3632--3627, 1975). No expression could be detected from a region of the transposons extending from the inverted repeat sequence distal to the beta-lactamase gene to more than half the distance into the Tn1 or Tn3 sequence.


Two-dimensional protein patterns during growth and sporulation in Saccharomyces cerevisiae. Proteins synthesized by Saccharomyces cerevisiae in presporulation and sporulation media were compared by using sporulating (a/alpha) and nonsporulating (a/a and alpha/alpha) yeast strains. Total cellular proteins were labeled with [35S]methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms and/or fluorograms showed some 700 spots per gel. Nine proteins were synthesized by a/alpha cells which were specific to vegetative, log-phase conditions. During incubation in sporulation medium, sporulating (a/alpha) cells synthesized 11 proteins not present in vegetatively growing cell. These same 11 proteins, however, were synthesized by nonsporulating (a/a and alpha/alpha) cells on sporulation medium as well. Nonsporulating diploids (a/a and alpha/alpha) were also examined with the electron microscope at various times during their incubation in sporulation medium. Certain cellular responses found to be unique to meiotic yeast cells in previous studies were exhibited by the nonsporulating controls. The degree to which all cell types (a/alpha, a/a, and alpha/alpha) were committed to sporulation was also determined by shifting cells from sporulation medium to vegetative medium. Some commitment to the meiotic pathway was observed in both the a/alpha and the a/a, alpha/alpha cells.


Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19. Plasmid R1drd-19 is present in a small number of copies per cell of Escherichia coli. The plasmid was reduced in size by in vivo as well as in vitro (cloning) techniques, resulting in a series of plasmid derivatives of different molecular weight. All plasmids isolated contain a small region (about 2 x 10(6) daltons of deoxyribonucleic acid) of the resistance transfer factor part of the plasmid located close to one of the IS1 sequences that separates the resistance transfer factor part from the resistance determinant. All these derivatives were present at the same copy number, retained the incompatibility properties of plasmid R1drd-19, and were stably maintained during cell division. Genes mutated to yield copy mutations also were found to be located in the same region.


Dependency of size of Saccharomyces cerevisiae cells on growth rate. The mean size and percentage of budded cells of a wild-type haploid strain of Saccharomyces cerevisiae grown in batch culture over a wide range of doubling times (tau) have been measured using microscopic measurements and a particle size analyzer. Mean size increased over a 2.5-fold range with increasing growth rate (from tau = 450 min to tau = 75 min). Mean size is principally a function of growth rate and not of a particular carbon source. The duration of the budded phase increased at slow growth rates according to the empirical equation, budded phase = 0.5 tau + 27 (all in minutes). Using a recent model of the cell cycle in which division is thought to be asymmetric, equations have been derived for mean cell age and mean cell volume. The data are consistent with the notion that initiation of the cell cycle occurs at "start" after attainment of a critical cell size, and this size is dependent on growth rate, being, at slow growth rates, 63% of the volume of fast growth rates. Previous reports are reanalyzed in the light of the unequal division model and associated population equations.


Genetic mapping of the chromosomal replication origin of Salmonella typhimurium. Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.


Genetic studies of an Escherichia coli K-12 temperature-sensitive mutant defective in membrane protein synthesis. The mutant divE42(Ts) of Escherichia coli K-12, defective in the synthesis of membrane proteins and in the transcription of the lac operon at high temperature, has been further characterized. It was found that a mutation (divE42) located at about min 22 on the E. coli chromosome map is responsible for the Lac- phenotype and temperature-sensitive growth. The mutation could be contransduced with serC, pyrD, or pyrC by phage P1 at a frequency of 4, 16, or 0.5%, respectively, the gene order being serC-pyrD-ompA-sulA-divE-pyrC. Examination of temperature-independent revertants and Pyr+ transductants revealed that all the mutant phenotypes examined (deficiencies in the increase of activities of some membrane enzymes, expression of the lac operon, and synthesis of several other proteins) are due to a single mutation (divE42) which is recessive to the wild-type (divE+) allele. Protein synthesis in the mutant was also analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis. Synthesis of a number of proteins, including membrane proteins, was found to decrease significantly, whereas that of an elongation factor, EF-Tu, increased upon transfer of a log-phase culture to high temperature (42 degrees C). These effects of temperature shift-up on protein synthesis were evident within 5 min under the conditions used.


Rifampin disrupts conjugal and chromosomal deoxyribonucleic acid metabolism in Escherichia coli K-12 carrying some IncIalpha plasmids. The effects of rifampin and chloramphenicol on the transfer of ColIdrd-1 have been examined to determined whether transfer requires the synthesis of an untranslated species of ribonucleic acid (RNA), as proposed in models for the transfer of another IncIalpha plasmid, R64drd-11. When RNA synthesis was inhibited throughout mating by rifampin, ColI transfer between dna+ bacteria occurred at the normal rate for about 10 min and then stopped abruptly. Conjugational deoxyribonucleic acid (DNA) synthesis in dnaB mutants indicates that plasmid DNA was made in the rifampin-treated donors to replace the transferred material but the DNA tended to be unstable. In the presence of chloramphenicol, transfer of ColI gradually diminished over a longer period. Rifampin, but not chloramphenicol, was found to have unpredicted effects on chromosomal DNA metabolism in unmated dna+ and dnaB bacteria when they harbor any of three IncIalpha plasmids (ColIdrd-1, R144drd-3, and R64drd-11). Replication of the bacterial chromosome in such cells stopped abruptly about 15 min after the addition of rifampin, and at 41 degrees C, but not 37 degrees C, this was followed by extensive DNA breakdown. These findings suggest that curtailment of IncIalpha plasmid transfer by the drug results from a general disruption of DNA metabolism rather than from inhibition of a species of RNA essential for transfer.


Multiple loci affecting photoreactivation in Escherichia coli. Sutherland et al. mapped a phr gene in Escherichia coli at 17 min and found that induction of an E. coli strain lysogenic for a lambda phage carrying this gene increased photoreactivating enzyme levels 2,000-fold. Recently, Smith and Youngs and Sancar and Rupert located a phr gene at 15.9 min. We have therefore investigated the properties of photoreactivating enzyme and cellular photoreactivation in cells containing deletions of the gene at 17 min. Cells with this deletion photoreactivated ultraviolet-induced killing at a rate 20% of normal; they also contained approximately 20% of the normal photoreactivating enzyme level. The residual enzyme in these cells was characterized to determine whether the reduced cellular photoreactivation rate and photoreactivating enzyme levels resulted from reduced numbers of normal enzymes or from an altered enzyme. Photoreactivating enzymes from strains carrying a deletion of the region at 17 min had an apparent Km about two- to threefold higher than normal enzyme and showed markedly increased heat lability. The gene at 17 min thus contains information determining the function of the E. coli photoreactivating enzyme rather than the quantity of the enzyme. It is proposed that the gene at 17 min be termed phrA and that located at 15.9 min be termed phrB.


Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B. A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.


Individual proteins are synthesized continuously throughout the Escherichia coli cell cycle. The pattern of synthesis of about 750 individual polypeptides was followed throughout the cell cycle of Escherichia coli B/r. Samples taken at different times in the cell cycle exhibited the same pattern of protein synthesis. No protein could be identified that was synthesized at different rates during different parts of the cell cycle.


Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid. Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.


Phospholipid synthesis during the cell division cycle of Escherichia coli. Stepwise changes in the rate of phosphatidylethanolamine and phospholipid synthesis during the cell division cycle of Escherichia coli B/r were observed. The cell ages at the increases were found to be a function of the growth rate. At each growth rate, the increase occurred around the time new rounds of chromosome replication were inaugurated in the cycle.


Ionizing radiation damage to the folded chromosome of Escherichia coli K-12: sedimentation properties of irradiated nucleoids and chromosomal deoxyribonucleic acid. The structures of the membrane-free nucleoid of Escherichia coli K-12 and of unfolded chromosomal deoxyribonucleic acid (DNA) were investigated by low-speed sedimentation on neutral sucrose gradients after irradiation with 60Co gamma rays. Irradiation both in vivo and in vitro was used as a molecular probe of the constraints on DNA packaging in the bacterial chromosome. The number of domains of supercoiling was estimated to be approximately 180 per genome equivalent of DNA, based on measurements of relaxation caused by single-strand break formation in folded chromosomes gamma irradiated in vivo and in vitro. Similar estimates based on the target size of ribonucleic acid molecules responsible for maintaining the compact packaging of the nucleoid predicted negligible unfolding due to the formation of ribonucleic acid single-strand breaks at doses of up to 10 krad; this was born out by experimental measurements. Unfolding of the nucleoid in vitro by limit digestion with ribonuclease or by heating at 70 degrees C resulted in DNA complexes with sedimentation coefficients of 1,030 +/- 59S and 625 +/- 15S, respectively. The difference in these rates was apparently due to more complete deproteinization and thus less mass in the heated material. These structures are believed to represent intact, replicating genomes in the form of complex-theta structures containing two to three genome equivalents of DNA. The rate of formation of double-strand breaks was determined from molecular weight measurements of thermally unfolded chromosomal DNA gamma irradiated in vitro. Break formation was linear with doses up to 10 krad and occurred at a rate of 0.27 double-strand break per krad per genome equivalent of DNA (1,080 eV/double-strand break). The influence of possible nonlinear DNA conformations on these values is discussed.


Ionizing radiation damage to the folded chromosome of Escherichia coli K-12: repair of double-strand breaks in deoxyribonucleic acid. The extremely gentle lysis and unfolding procedures that have been developed for the isolation of nucleoid deoxyribonucleic acid (DNA; K. M. Ulmer et al., J. Bacteriol. 138:475-485, 1979) yield undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically significant doses of ionizing radiation. Repair of ionizing radiation damage to folded chromosomes of Escherichia coli K-12 strain AB2497 was observed within 2 to 3 h of post-irradiation incubation in growth medium. Such behavior was not observed after post-irradiation incubation in growth medium of a recA13 strain (strain AB2487). A model based on recombinational repair is proposed to explain the formation of 2,200 to 2,300S material during early stages of incubation and to explain subsequent changes in the gradient profiles. Association of unrepaired DNA with the plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (greater than 3,100S) during the later stages of repair. Direct evidence of repair of double-strand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from ribonuclease-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2 to 3 h of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells that survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that one to two double-strand breaks per genome are repairable in E. coli K-12 strain AB2497.


Protein and ribonucleic acid syntheses in heat-damaged and heat-killed Escherichia coli. Protein and ribonucleic acid (RNA) syntheses were measured in both lethally injured and thermally damaged viable cells after heating at lethal temperatures. Immediately after heating, cells were incubated in growth media containing either [14C]leucine or [3H]uracil. The labeled cells were subsequently treated with penicillin. Viable cells were shown to lyse, and the intact nonviable cells were collected by centrifugation. The results showed that after heating, protein and RNA synthesis were reinitiated in the penicillin-sensitive cell population, but there was no detectable protein or RNA synthesis in the heat-killed cells which were resistant to penicillin. The lack of protein or RNA synthesis in lethally damaged cells during the entire recovery period may be interpreted to reflect the lethal thermal damage.


Inhibition by itaconate of growth of methylotrophic bacteria. The effects of the isocitrate lyase-directed growth inhibitor itaconate on the growth of certain methylotrophic organisms was investigated. It was found that growth of those organisms possessing the Icl(+)-serine pathway of one-carbon metabolism was inhibited during growth on methylamine and on acetate, but not on glucose. Organisms possessing the Icl(-)-serine pathway pathway were unaffected. Organism PAR, an Icl(-)-serine pathway type, was not specifically inhibited during growth on acetate. This finding further substantiates previous reports of the lack of isocitrate lyase in this organism, indicating a totally new pathway for acetate assimilation.


Light effects in yeast: evidence for participation of cytochromes in photoinhibition of growth and transport in Saccharomyces cerevisiae cultured at low temperatures. Visible light of moderate intensity inhibits growth, respiration, protein synthesis, and membrane transport in bakers' yeast and has a deleterious effect on membrane integrity. The results of this study indicate that these effects require the presence of cytochromes b and a/a(3). The light sensitivities of growth rate and [(14)C]histidine uptake in wild-type rho(+) Y185 and D225-5A strains of Saccharomyces cerevisiae were compared with those in a variety of mutants lacking cytochrome b or a/a(3) or both; a close correlation was found between the presence of these respiratory pigments and photosensitivity. Thus, strain TL5-3C, a nuclear petite lacking cytochromes b, a, and a(3), was resistant to light; strain GL5-6A, another nuclear petite having reduced amounts of cytochromes a and a(3), was partially resistant; strains MB127-20C and MB1-6C, nuclear petites lacking only cytochrome b, were also only partially resistant to light; whereas mutants containing all three cytochromes but having their respiratory chain either nonfunctional (strain ZK3-6B) or uncoupled (strain 18-27/t12) were fully sensitive to light. Finally, an equal-energy, broad-band action spectrum for the light inhibition of growth and transport indicated that blue light (408 nm) was most effective; these wavelengths correspond to the Soret region of the cytochrome absorption spectrum. The results suggest, therefore, that the yeast cytochromes b, a, and a(3) are the primary photoreceptors for the inhibitory effects of light and, perhaps, for other processes, such as the entrainment of biological rhythms in this species.


Escherichia coli mutant containing a large deletion from relA to argA. A mutant of Escherichia coli has been isolated that contains a large deletion (about 3 X 10(7) daltons of deoxyribonucleic acid) encompassing argA, fuc, and relA. This mutant strain (AA-787) is also cold sensitive for growth at 18 degrees C. Strain AA-787 was obtained fortuitously as a cold-sensitive pseudorevertant of a strain having a heat-sensitive peptidyl-transfer ribonucleic acid hydrolase. Genetic analysis, using transduction and interrupted mating, showed the cold sensitivity mutation to be located adjacent to relA. Further analysis demonstrated loss of relA, fuc, and argA gene functions but retention of eno and recB, closely linked genes adjacent to relA and argA, respectively. Unusually high cotransduction of flanking markers (cysC and thyA) indicated loss of approximately 1 min of the E. coli genetic map in strain AA-787. Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) was synthetized in mutant strain AA-787 at basal levels, and ppGpp synthesis was stimulated by carbon-source downshift. No ppGpp synthesis could be obtained using ribosomes isolated from strain AA-787. These findings, taken together, show that deletion of relA in E. coli does not completely abolish ppGpp synthesis and suggests that another enzyme system must also be responsible for ppGpp synthesis.


Structure of polyadenylic acid in the ribonucleic acid of Saccharomyces cerevisiae. Investigations of the structure of polyadenylic acid [poly(A)] in yeast have shown that there are two classes of poly(A) distinguished by size and kinetics of synthesis. Each class is found directly on the 3' end of messenger RNA. One class contains poly(A) molecules ranging from 60 to less than 20 nucleotides long. The longest molecules in this poly(A) class are the first to become labeled when cells are exposed to [3H]adenine. Label then appears in progressively smaller molecules. The second class of poly(A) is about 20 nucleotides long. The length homogeneity of this class and the presence in nuclear DNA of many copies of a polythymidylate sequence which is the same length suggests that this poly(A) is synthesized by transcription from DNA.


Comparative ultrastructure of Mycobacterium leprae and Mycobacterium lepraemurium cell envelopes. The structural properties of the cell envelopes of Mycobacterium leprae and Mycobacterium lepraemurium were investigated by freeze-fracture, freeze-etching, and negative-staining techniques. Freeze-fracture split the cell wall and exposed the internal features of the peptidoglycolipid mycosidic filamentous network. The cell membrane was also split into two asymmetric faces. The external fracture face was characterized by linear arrays of intramembranous particles, whereas the protoplasmic fracture face showed randomly distributed clusters of particulate entities. Comparative analysis of the ultrastructural features observed in M. leprae and M. lepraemurium indicated that the organization of the cell envelope in these two species differed particularly with respect to the amount and complexity of the superficial peptidoglycolipid and mycosidic integument, which is poorly developed in the mycobacterium responsible for human disease.


Mutants of the mini-F plasmid pML31 thermosensitive in replication. Hydroxylamine mutagenesis was used for the induction of thermosensitive replication mutants of the mini-F plasmid pML31. Replication mutants were characterized by studying the segregation kinetics and the incorporation of [3H]-thymidine into plasmid deoxyribonucleic acid at the nonpermissive temperature. Based on these experiments two types of mutants could be distinguished. Mutants of type I are fast segregating with the kinetics expected if plasmid replication was blocked immediately. Double-label experiments showed a rapid shut-off of replication in these mutants at 42 degrees C. Mutants of type II segregate slower, showing only a partial inhibition of plasmid deoxyribonucleic acid synthesis at the nonpermissive temperature. The label incorporated at 42 degrees C was predominantly found in open circular plasmid molecules.


Biochemical and topographical studies on Escherichia coli cell surface. IV. Giant spheroplast formation from a filamentous cell. Long, nonseptate filamentous cells consisting of 5 to 40 single-cell unit lengths were formed from Escherichia coli surface mutant ONT-3 by treatment with a sublethal concentration of sodium dodecyl sylfate. As distinct from several other elongated cells (e.g., thymine-starved filaments), it was found here that stable giant spheroplasts, 5 to 10 micrometers in diameter, were produced by the action of lysozyme in the presence of bovine serum albumin via the gradual fusion of distinct spheroplasting bulbs.


Molecular nature of two Haemophilus influenzae R factors containing resistances and the multiple integration of drug resistance transposons. The 36-megadalton Haemophilus influenzae R plasmid pHK539 was found to specify resistance to tetracycline (Tc) and ampicillin (Ap). It was shown by molecular hybridization studies and by electron microscopy that the plasmid pHK539 contained the tetracycline translocation deoxyribonucleic acid (DNA)segment (TnTc) as well as the ampicillin translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both tetracycline and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a chloramphenicol resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from different countries. The tetracycline-chloramphenicol resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing tetracycline. The presence of the two copies of the transposon was correlated with higher minimum inhibitory concentrations against tetracycline as well as against chloramphenicol. After its growth in medium containing tetracycline, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained one, two, three, or even four copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against tetracycline.


Morphological stabilization of capsules of group B streptococci, types Ia, Ib, II, and III, with specific antibody. Antibody prepared to the type-specific capsular polysaccharides of group B streptococci was used to demonstrate a stabilizing effect on the capsular glycocalyx. This permitted visualization by electron microscopy of the size of the capsule relative to the rest of the bacterial cell, and clear differences in the dimensions of untreated and antibody-treated capsular material were noted. Antibodies produced against group B streptococci types Ia, Ib, II, and III were used to demonstrate morphologically that the organization and extent of the capsular glycocalyx more closely resembles its natural state on stabilization by reaction with specific antibody.


Suppressor of deoxythmidine monophosphate uptake in Saccharomyces cerevisiae. Although yeast cannot normally incorporate exogenous deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid, mutants able to do so have been isolated. We have characterized a recessive suppressor of dTMP uptake (sot1) that prevents strains carrying either tup1, tup2, or tup4 from growing on selective medium. The sot1 mutation maps between rad1 and the centromere of chromosome XVI, and is unlinked to any of the tup mutations. The sot1 mutation does not suppress the other pleiotropic effects of the tup1 mutant, notably the lack of mating of tup1 MATalpha strains. The sot1 mutation specifically blocks the uptake of dTMP into tup strains. After growing a sot1 strain in medium containing [3H]dTMP, we showed that the medium still contained more than 90% of the original [3H]dTMP and that this medium could support the incorporation of [3H]dTMP by a tup2 strain. Therefore, sot1 strains do not degrade dTMP in the medium. The sot1 mutation had no effect on the uptake of other nutrients essential for growth, including several amino acids, adenine, and uracil.


Clozapine versus chlorpromazine for the treatment of schizophrenia: preliminary results from a double-blind study. Fifteen schizophrenic inpatients were treated in a double-blind comparison study of clozapine versus chlorpromazine. Clozapine appeared to be at least as effective as chlorpromazine without producing any extrapyramidal reactions. Although bone marrow toxicity associated with clozapine has led to the drug's withdrawal from clinical use, data from studies of clozapine demonstrate that a drug can be effective antipsychotic without producing neurologic reactions.


Effect of deglycosylation of yeast invertase on its uptake and digestion in rat yolk sacs. The uptake by rat yolk sacs of native invertase and invertase which was deglycosylated by treatment with endo-beta-N-acetylglucosaminidase was compared. The initial rate of uptake of the deglycosylated enzyme was severalfold greater and its accumulation leveled off much earlier than that of the native enzyme. Uptake rates of the deglycosylated and native forms of the enzyme were proportional to their concentration in the medium in the range employed and were inhibited about 85% by 10(-6) M glucagon in both cases. After preloading of yolk sacs with native invertase, the tissue level of activity remained relatively constant over a subsequent 6-h time period, while with the deglycosylated form, activity declined substantially. Since this difference appears not to be attributable to differences in thermal stability, it is suggested that the deglycosylated form of the protein is more susceptible to intracellular proteolytic digestion. In vitro studies on the digestion of these two forms of invertase by trypsin are consistent with this suggestion.


Nucleoside transport in cells and membrane vesicles from Escherichia coli K12. Osmotic shock treatment of cells of Escherichia coli K12 caused a reduction in the transport of nucleosides into the cells. The strains used carried mutations in the nucleoside catabolizing enzymes. This indicated that the decrease in transport capacity was not due to loss of these enzymes during the shock treatment. Membrane vesicles, prepared from the same strains, showed a limited transport of cytidine, deoxycytidine, and uridine. Transport of purine nucleosides and of thymidine was very low in vesicles lacking the appropriate nucleoside phosphorylases and no significant stimulation was observed if the nucleoside phosphorylases were present in the membrane vesicles. These results all indicate that components outside the cytoplasmic membrane are important for nucleoside transport. Selection for resistance to fluorodeoxycytidine yielded mutants which were unable to transport any nucleoside, even when the nucleoside phosphorylases were present in high amounts. This finding is consistent with a requirement for a specific transport process prior to the initial enzymatic attack on the incoming nucleoside.


The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid. A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by differential centrifugation of lysed spheroplasts. The preparation, a modified 100,000 x g supernatant fraction, contains ribosomes and monosomes, ribosomal subunits, translation factors, and aminoacyl-tRNA synthetases, but no polysomes. After removal of small amounts of remaining mRNA with micrococcal nuclease, protein synthesis is stringently dependent on the addition of mRNA, as well as amino acids and an energy-generating system. The 5'-cap analogue, 7-methylguanosine 5'-phosphate, inhibits translation of several natural mRNAs, but has no effect on chain elongation. Incubation of the polysome-free extract with natural mRNA leads to the formation of protein-synthesizing polysomes and eventually, to the release of protein; the molecular weight of the protein synthesized in the presence of BMV (brome mosaic virus) RNA is consistent with that of BMV coat protein.


Oxygen-sensitive and -insensitive nitroreduction by Escherichia coli and rat hepatic microsomes. Nitrofurazone is shown to undergo an initial 1-electron (oxygen-sensitive) or 2- or more electron (oxygen-insensitive) reduction by partially purified nitroreductases from Escherichia coli. Nitrofurazone (50 micronM) is reduced by the oxygen-sensitive reductase to a nitro anion free radical as indicated by ESR and visible spectroscopy. The visible spectrum of the nitro anion free radical is characterized by an increase in absorption at 406 nm. In the presence of the oxygen-sensitive reductase, nitrofurazone stimulates superoxide formation and oxygen consumption. This enzyme gives a steady state radical concentration which is proportional to the square root of the enzyme concentration, suggesting that the nitrofurazone anion radical is an obligate intermediate in the reduction and that the radical decays by a nonenzymatic second order process. The oxygen-insensitive reductase does not form the nitro anion free radical nor in the presence of nitrofurazone does it stimulate oxygen consumption. Visible spectroscopy shows that nitrofurazone is reduced by the oxygen-sensitive reductase to a species with an absorption maximum at 335 nm, which has been previously identified as the amine. The oxygen-insensitive reductase reduces nitrofurazone to a previously identified cyano derivative with an absorption maximum at 280 nm. Rat hepatic microsomes appear to metabolize nitrofurazone in a manner similar to the oxygen-sensitive E. coli reductase.


The purification of orotidine-5'-phosphate decarboxylase from yeast by affinity chromatography. We have prepared an affinity column for the purification of orotidine-5'-phosphate decarboxylase from yeast. The column effects a 3200-fold purification from yeast homogenate in one pass; simple additional steps produce enzyme that has been purified 6700-fold and is not contaminated by any other protein that can be detected by sodium dodecyl sulfate-acrylamide gel electrophoresis. Overall, 35% of the activity present in the yeast is recovered as pure enzyme. The resin for the column is synthesized by attaching the ethylenediamine amide of 5-(2-carboxyethyl)-6-azauridine 5'-phosphate to carboxymethyl-agarose.


Purification of the o-dianisidine peroxidase from Escherichia coli B. Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities. Extracts of aerobically grown Escherichia coli B exhibit both catalase and dianisidine peroxidase activities. Polyacrylamide gel electrophoresis demonstrates two distinct catalases which have been designated hydroperoxidases I and II (HP-I and HP-II) in order of increasing anodic mobility. HP-I has been purified to essential homogeneity and found to be composed of four subunits of equal size. Its molecular weight is 337,000, and it contains two molecules of protoheme IX per tetramer. Its amino acid composition is unusual, for so large a protein, in lacking half-cystine. HP-I is a very efficient catalase with an activity optimum at pH 7.5, a Km for H2O2 of 3.9 mM, and a turnover number of 9.8 x 10(5) per min. It is also a broad specificity peroxidase capable of acting upon dianisidine, guaiacol, p-phenylenediamine, and pyrogallol. Dianisidine acted as a powerful reversible inhibitor of the catalatic activity of HP-I and as a suicide substrate when HP-I functioned in its peroxidatic mode.


Purification and characterization of protease III from Escherichia coli. An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).


Annular constricting bands.

Annular constricting bands. We reviewed forty-five patients with congenital constriction bands with an average follow-up of 15.8 years. The family history and associated anomalies were recorded and particular attention was paid to temperature gradients and neurocirculatory deficits with respect to band location. No family history of band constrictions was elicited. Most of our patients were products of first pregnancies and of young mothers. Deformity of the nails was a constant finding in patients with distally located bands. All patients demonstrated at least one other anomaly, most commonly a malformation of the hand. Neural deficits were noted in 23 per cent of the patients, while circulatory findings appeared in 16 per cent. A significant temperature gradient averaging 2.38 degrees Celsius was noted in twenty patients in whom the bands were located proximally. All patients with neural deficits demonstrated a significant temperature gradient. Staged z-plasty was the procedure of choice in surgical release of constriction bands.


The presence and significance of the Thomsen-Friedenreich antigen in breast cancer. I. Serological studies. Serum levels of antibodies against the Thomsen-Friedenreich (TF) antigen were determined for a group of patients with malignancies of the breast, as well as a control group of healthy persons and patients with no breast disease. Despite recent claims of anti-TF levels being severely depressed in patients with breast cancer, we were unable to show any significant differences between the two groups. Standardised anti-TF containing sera or solutions of the TF specific agglutinin from peanuts (PNA) were absorbed with membrane preparations from normal and cancerous breast tissue, but again no differences in absorption were noted. Histological examination, by contrast, showed both free and sialic acid-covered receptors to be present on both normal and malignant breast epithelium. Based on these findings it was concluded that determination of serum anti-TF is an unsatisfactory method of diagnosing breast cancer and that the TF antigen cannot be considered to be carcinoma associated.


Human islet cell tumor storing pancreatic polypeptide: a light and electron microscopic study. A 45-year-old man was operated for surgical treatment of a long-standing peptic ulcer disease and upon inspection of the pancreas for suspected Zollinger-Ellison syndrome, tumor nodules were found in this organ. The tumor tissue examined by immunofluorescence showed specific staining only after incubation with anti-pancreatic polypeptide. Negative results were obtained with antisera directed against insulin, pancreatic glucagon, somatostatin, GLI, VIP, secretin, and gastrin. Examination of the tissue by electron microscopy revealed a homogeneous population of small granule-containing cells. This case, therefore, illustrates a tumor composed of one single hormone-producing cell type and allows definition of the ultrastructural features of human pancreatic polypeptide-containing cells.


Requirement of extracellular complement and immunoglobulin for intracellular killing of micro-organisms by human monocytes. The role of serum factors in the intracellular killing of bacteria by monocytes was studied on the basis of an assay independent of phagocytosis. After 3 min of phagocytosis of preopsonized bacteria and removal of noningested bacteria, the monocytes containing bacteria are reincubated for various periods and the number of unkilled bacteria is determined by a microbiological method after lysis of the cells. Evidence that this assay measures the killing of ingested bacteria was provided by scanning electron microscopy, lysostaphin treatment, and the effect on the rate of intracellular killing of inactivated serum lacking specific opsonic activity. Intracellular killing of Staphylococcus aureaus, S. epidermidis, and Escherichia coli by human monocytes does not occur or is low in the absence of serum, and maximal killing is only reached when fresh serum is present; intermediate values are obtained in the presence of heat-inactivated serum. These findings indicate that complement stimulates intracellular killing. Isolated heterogeneous immunoglobulin (Ig)G, pFc fragments of heterogeneous IgG, and both IgG1 and IgG3 stimulate intracellular killing of S. aureaus by monocytes to the same degree as heat-inactivated serum. Sphingomyelinase, which decreases the number of Fc receptors, and neuraminidase, which increases these receptors, respectively, decreased and increased the intracellular killing, whereas anti-monocyte serum completely abolished the stimulation of intracellular killing by inactivated serum. These results prove that interaction of the Fc receptor with the Fc part of IgG is required for the intracellular killing. Inhibition of the activation of complement components via the alternative pathway gave a considerable reduction in the intracellular killing of S. aureaus; impairment of the activation via the classical pathway had no effect. The addition of complement components to heat-inactivated serum showed that intracellular killing is maximal only when C3b is generated. Reduction of the number of C3b receptors in the membrane by trypsin or pronase decreased intracellular killing in the presence of fresh serum; anti-monocyte serum completely abolished the stimulation of intracellular killing by fresh serum. These results lead to the conclusion that intracellular killing is also dependent on the interaction between C3b and its receptor in the membrane.


A rapid method for electron microscopic examination of blood cells. A method is described whereby the time required for the processing of white blood cells for electron microscopy can be shortened from 36 to 3 1/2 hours. Because cells of the peripheral blood are not attached to each other, fixation, dehydration, and infiltration of the embedding medium is more rapid. This makes it possible for clinicians to use fine structural data for diagnosis as illustrated in three cases.


A comparison of thiomersal and 50% alcohol as preservatives in urinary cytology. The efficacy of 50% ethyl alcohol and of thiomersal as preservatives in urinary cytology were compared. In both methods over 80% of the cells were sufficiently well preserved after three days to allow cytomorphological evaluation, and over 50% on the seventh day. In the specimens without preservative, only 54% and 28% were intact after the same time intervals. In contrast with 50% ethyl alchohol, thiomersal is a more effective bactericide, it does not increase the volume of the sample, it is cheaper, and it does not affect the cytomorphology.


Evaluation of acetaminophen, propoxyphene, and their combination in office practice. Thirty-two patients with chronic painful disease treated in office practice willingly participated in a double-blind, crossover analgesic study. They were informed about (1) the medications--placebo, 650 mg acetaminophen, 100 mg propoxyphene napsylate, and the combination of the two active drugs; (2) the regimen--four to six single-tablet doses per day for two days, crossover; (3) the reporting system--estimates of pain intensity and scores for other symptoms to be reported daily by telephone; and (4) return of the unused medication on the next office visit. In comparison to placebo, propoxyphene provided statistically significant analgesia, but acetaminophen did not. Significant adverse effects were not present with any test drug when compared to test placebo.


Treatment of severe hypertension with minoxidil: advantages and limitations. Minoxidil, a new peripheral vasodilator, was used in the therapy of 26 hypertensives who were previously uncontrolled on conventional medications or who had dose-limiting side effects. Minoxidil provided (1) therapeutic advantages in all patients, regardless of the etiology of their hypertension, (2) no symptoms of orthostatic hypotension or sympathetic nervous system depression, (3) a simplification of medical regimens and perhaps costs, and (4) regression of impotence in four out of seven patients. The major limiting factors encountered were (1) fluid retention with the development of congestive heart failure in three patients, (2) pericardial effusion in three patients, and (3) hypertrichosis, which reduced its acceptability in female patients.


Medieval arabic views on speech disorders: al-Razi (c. 865--925). This paper discusses al-Razi's views on speech defects of lingual origin. It begins with a background sketch of the man, his times, and his major work, with its various translations. There follows translated extracts and analysis from his section on the causes and treatment of speech defects, seen in the light of the humoral theory of disease. Information is derived from the recently published Arabic version of Rzi's al-Hawi, which reveals that diagnosis was more advanced than is indicated in the Latin translations, which have been the principal source of reference in the West to date.


Preprocessing in computed tomography picture analysis: a "bone-deleting" algorithm. A procedure for automatic deletion of extracranial parts and bone in computed tomography (CT) pictures is presented. Such a procedure is a prerequisite for many types of quantitative image analysis. The algorithm can delineate complex bone contours and is completely independent of information outside the skull. Special options allow, in addition, the deletion of calcified parts of the brain and of some artifacts. The program is implemented in a general program system (PICPRO) developed for CT image analysis and picture manipulation.


Factors affecting tissue aluminum concentration. Elevated concentration of aluminum has been reported in serum, whole blood and tissue samples of patients with renal insufficiency. Evidence incriminating aluminum as a neurotoxin among the dialysis population is strong. The source of this aluminum has not been clearly defined, although both gastrointestinal and parenteral routes may be involved. Data from this laboratory suggest that an effect of parathyroid hormone on tissue aluminum burdens may, in part, explain why only certain patients exposed to high dialysate aluminum develop the dialysis encephalopathy syndrome and the occasional occurrence of this syndrome in the absence of dialysis or increased dialysate aluminum.


Effect of two antibacterial mouth sprays and dentifrices on dental plaque and gingivitis in beagle dogs. Plaque inhibition by dilute mouth sprays and dentrifrices containing benzethonium chloride and chlorhexidine gluconate was compared in beagle dogs. Agents with chlorhexidine gluconate produced less plaque than their benzethonium chloride or placebo counterparts, but the differences were not significant when compared to mean control scores registered during interexperimental recovery periods.


Effect of autogenous marrow and calcitonin on reactions to a ceramic. The effect of autogenous marrow and calcitonin on reactions to a tricalcium phosphate ceramic was studied by subcutaneous implantation in rats. No bone formation was evident in association with uncoated and calcitonin-coated ceramic. At four weeks and thereafter bone formation was evident in association with all marrow-coated implants.


Dental resins with reduced shrinkage during hardening. Unsaturated spiro-orthocarbonates are monomers that expand on polymerization and will co-polymerize with conventional BIS-GMA based resins. This combination has good physical properties and bonding to enamel with some polymerization expansion evident at 50 C. Dispersion of the carbonate in microcrystalline form in liquid monomer appears to be a method to allow its reaction in the initial polymerization.


Clinical placement and performance of composite resin restorations. The most frequent complaints registered against composite restorations are stained margins and a rough surface. The stained margins have many causes such as (1) fracturing or crazing of enamel during preparation of the cavity, (2) using a composite having an excessive curing shrinkage, (3) manipulating too slowly, which permits a loss of plasticity during placement, (4) maintaining inadequate pressure during setting or curing of the composite, and (5) using finishing instruments which cause defects in the margin area of the tooth and/or composite. All of these the operator can control, and if not, the defect will be built into the restoration. The roughness of composites can be reduced by polishing with the finest grit available in disks and by coating with a glaze. Today's composite restorations placed with care will provide long lasting esthetic restorations providing they are not placed in areas subjected to load bearing abrasion.


The supply of dental manpower in the United States. This description of the supply of dental services in the United States addresses the number, kind, distribution, and training of dentists and dental auxiliaries, and the organizational factors that affect the production of dental services. Beginning with a brief historical review, the paper gives a general overview of the different types of dental personnel including the dentist, dental hygienist, dental assistant, and laboratory technician. The discussion of these categories of providers includes consideration of manpower planning as it has evolved over the past two decades, National manpower legislation is mentioned first as a reaction to the projected dentist shortage and then in response to the issues of geographic maldistribution and the effects of specialization. The second section of the paper discusses the dynamics of the dental care market. The distribution of the supply of services is identified and related to patterns of utilization and productivity. These factors are considered to be part of the set of dynamic relationships that help explain the current manpower problems of geographic and specialty maldistribution. A concluding section superficially discusses policy implications regarding the potential for increasing supply by: (1) increasing the number of dentists, (2) increasing the numbers and functions of auxiliaries, (3) increasing practice efficiency through group practice, and (4) reducing the restrictions that result from current state dental practice acts.


Immediate repair of wounds following operations by Mohs' fresh-tissue technique. With the introduction of the Mohs fresh-tissue technique, immediate repair of surgical wounds has become feasible in selected cases. In this article several case reports are presented to illustrate the use of primary closures, skin grafts, and flaps in an office facility.


Acute gastro-intestinal bleeding in the critically ill patient. Endoscopic studies have shown that all critically ill patients are liable to a degree of stress ulceration. Diffuse erosions appear first in the fundus and then spread to the corpus and antrum within 48 h. Duodenal disease is particularly common in burns patients. Discrete ulceration occurs in most severely injured patients. Mucosal damage is probably initiated by ischaemia but only develops in the presence of acid. Although related to the severity of underlying illness, haemorrhage is unpredictable. Mortality is high and largely unaffected by treatment. Emphasis should therefore be made on prophylaxis. Improved intensive reducing intra-luminal acid by antacid or H2 receptor antagonists appears to be the most effective measure, but controlled studies are required.


Double-blind and triple-blind assessments of medication and placebo responses in hyperactive children. Hyperactive boys taking psychostimulant medication were studied using a randomized, placebo-controlled, crossover design. Behavior ratings and medication guesstimates were obtained for the boys when they were given methylphenidate (Ritalin) and when they were given a placebo. The ratings showed positive medication-related changes, and the guesses, done by independent judges, were significantly better than chance. The pattern of ratings for double-blind and triple-blind raters was identical. These results imply that positive psychostimulant effects are not attributable to rater sensitization or expectancy. The medication-placebo differences were highly reliable for the group comparisons and were in the predicted direction for 21 of 22 individuals, but the magnitude of the change for many individuals was not dramatic. Implications for child psychopharmacology research and differences between clinical and experimental significance are discussed.


A comparative study of two pit and fissure sealants: two-year results in Augusta, Ga. Two pit and fissure sealants, an autopolymerized unfilled resin, and an ultraviolet-light-curing unfilled resin, applied to the permanent first molars of children are compared in terms of retention and efficacy.


United States Air Force survey of dental needs, 1977: methodology and summary of findings. This study represents the first, comprehensive baseline survey of dental needs conducted in the US Air Force. The air force population was stratified by command, base, and grade, and 5,805 active duty air force members, aged 17 to 57 years (mean, 27.83), were examined at random to determine their overall oral health status. Data were collected from 42 air force bases, representing 11 worldwide commands, which comprised 90% of all active duty members. This article presents the methodology for the study, a summary of the findings, and a comparison of dental needs among recruits and all other grades of active duty air force personnel. Results indicate a significant level of unmet dental needs in the US Air Force. For example, the average active duty individual presently serving in the air force would require eight hours and 48 minutes of chairside care to provide comprehensive treatment. Each entry level recruit would need an average of ten hours and 23 minutes of dental treatment.


Is atherosclerosis reversible?

Is atherosclerosis reversible? In this review, the author recapitulates the mechanisms involved in the development of atherosclerotic plaques. The relationship of high plasma cholesterol and the per cent of caloric intake from saturated fat is noted. Can intervention on risk factors prevent or reverse atherosclerosis? In animal studies, regression of atherosclerotic lesions has been achieved when dietary factors were manipulated. Recent studies in man have also pointed to the effectiveness of reducing cholesterol and cholesteryl esters in atherosclerotic plaques.


Comparative tolerance of elderly from differing ethnic backgrounds to lactose-containing and lactose-free dairy drinks: a double-blind study. Milk intolerance was investigated in 87 healthy elderly individuals with a mean age of 77 years who were given 240 ml of a chocolate dairy drink twice in one week with a light lunch. No significant differences in symptomatic responses distinguished the subjects consuming a lactose-free (LF) drink from those consuming a drink containing 4.5% lactose (LC) under double-blind study conditions. Breath hydrogen analysis during lactose tolerance testing identified 23 malabsorbers, none of whom responded exclusively to the LC drink, although five were symptomatic on both days, and two had symptoms only on the day the LF drink was served. A similar percentage of absorbers (72%) and malabsorbers (70%) were asymptomatic on both days. Factors other than lactose malabsorption appeared to be responsible for the symptoms of intolerance reported, and most may have been psychosomatic in origin.


An automated microscope for quantitative cytology combining television image analysis and stage scanning microphotometry. This paper describes an automated microscope developed for operation in conjunction with the Leyden Television Analysis System. It features automated control of magnification, illumination, movement of scanning stages, and fine focus. These functions are controlled by means of a microcomputer. This enables a flexible design and relieves the supervising computer of simple but time consuming tasks. The combination of an automated microscope and Leyden Television Analysis System provides a powerful tool in quantitative cytological research. The flexible design permits other microscopic functions to be added with relatively little effort.


A double scanning microphotometer for image analysis: hardware, software and biomedical applications. A new image processing system designed for densitometry and pattern analysis of microscopic specimens is described with special regard to the hardware, the software and the biologic applications. The data acquisition procedure involves the combination between the scanning of the preparation by means of a motorized stage and the scanning of successive fields by a mechanical device. The signal provided by the photomultiplier is converted into digital values which are directed to an on-line computer. The data processing is based on a one-pass computation involving automata theory and therefore it avoids the storage of the image in the computer memory. In so doing, an entire and continuous image of the whole preparation can be processed at the highest magnification of the microscope whatever the size of the analyzed specimen may be. A biologic application of the system is reported and concerns the automatic identification and counting of cells in the various phases of the mitotic cycle.


Ultrafast laser scanner microscope.

Ultrafast laser scanner microscope. Advances in monolayer deposition of cervical cells have removed one of the last serious obstacles to the design of high-resolution automated diagnostic assessment systems. In this article, we describe the design considerations for a system that is capable of acquiring, within 60 sec, a 0.5 micron digitized image of a 4 cm2 area on a standard glass slide. The most feasible approach is found to be a system using a rotating polygon to sweep the focused spot from a laser across a 2-mm scan line while the slide is uniformly translated perpendicular to the scan direction the use of laser sources (a helium-neon laser at 632 nm and a krypton ion laser at 568 and/or 476 nm) as compared to the incoherent light sources used in conventional microscope systems alleviates many of the optical design problems and provides the proper wavelengths needed for recognition of Papanicolaou stained cells. We also find that focus control of the scanning spot should be achievable using a technique involving a holographic grating. Other relevant considerations such as sample heating problems, multiphoton absorption by the sample, detector signal-to-noise ratios, laser amplitude noise control, and the digitization and buffering of the data stream are also discussed.


A practical application of computer pattern recognition research: the Abbott ADC-500 differential classifier. The ADC-500 is a new blood cell differential classifier manufactured by Abbott Laboratories. It performs 500-cell leukocyte differentials on both normal and abnormal cells, evaluates red cell morphology and estimates platelet sufficiency at a rate of 40 to 50 samples per hour in stand-alone operation. The ADC-500 system consists of a spinner which prepares a uniform blood monolayer on a slide, a stainer which reproducibly stains the slide with Wright's stain, an encoder which attaches an instrument and human readable identification to the slide and an analyzer which accepts a stack of up to 50 slides, evaluates these slides and prints the results and the slide identification on report forms. The system's analysis rate, which represents a 5- to 10-fold increase over other commercially available differential counters, requires a number of specialized techniques for its realization. One key to this performance is the development of a high speed X-Y slide positioning stage which can move to a new cell and settle in 50 msec. Another is the high degree of parallelism used in the system structure and the pipelining of the data processing. A third is the development of uniform and repeatable sample preparation modules. Within the analyzer module, the autofocus, white cell acquisition and high resolution cell analysis systems are independent and operate in parallel. At the same time within the high resolution cell analysis system, one cell is acquired; the digitized image of a second processed; and a third is classified using pattern recognition techniques. All of these tasks, except focus, are under the control of a minicomputer system. Tests of the system reveal good accuracy and an improvement in precision due to the increase in the number of counted cells.


Rapid analysis of hematology image data: the ADC-500 preprocessor. A sequential, pipeline processor (that we have named the ADC-500 preprocessor) has been developed which scene segments the three color image data from the ADC-500 optics one image element at a time, groups together image elements from each object in the scene and extracts features from each object. The processing occurs at television frame rates, requiring 16.7 msec to process the entire image. This speed was instrumental in allowing the ADC-500 automated differential analyzer to perform routine 500-cell differentials. The preprocessor also contains hardware which simplifies compilation of the three color histograms. The segmentation algorithms implemented in the preprocessor are multicolor extensions of the classical monochrome density histogram threshold method. For most cell image analysis tasks, a sequential pipeline processor of this type should be more economical and as fast or faster than a parallel processor.


A thresholding method for automatic cell image segmentation. An algorithm for automatic segmentation of PAP-stained cell images and its digital implementation is described. First, the image is filtered in order to eliminate the granularily and small objects in the image which may upset the segmentation procedure. In a second step, information on gradient and compactness is extracted from the filtered image and stored in three histograms as functions of the extinction. From these histograms, two extinction thresholds are computed. These thresholds are suitable to separate the nucleus from the cytoplasm, and the cytoplasm from the background in the filtered image. Masks are determined in this way, and finally used to analyse the nucleus and the cytoplasm in the original image.


Correlation between nuclear morphology and rate of deoxyribonucleic acid synthesis in a normal cell line. Chinese hamster fibroblasts were investigated for the existence of correlations between proliferative activity and nuclear morphology. As a proliferative parameter, the rate of DNA synthesis of individual cells was determined by quantitative 14C-autoradiography. In a second step the images of the Feulgen-stained nuclei were digitized for extraction of features of morphology and texture. These features were correlated with the corresponding DNA synthesis rate values. The following relationships were found: Round nuclei have higher rates of DNA synthesis than flat ones. The more chromatin is packed at the nuclear rim, possibly representing heterochromatin, the lower the rate of DNA synthesis. The DNA synthesis rate also correlates with the graininess of chromatin. Larger areas of condensed chromatin are associated with lower rate values. A fine and irregular network of chromatin, as is typical of immature cell types, is associated with a high rate of DNA synthesis. Although these results are presently confined to the cell line investigated, parallels seem to exist to other cell types, such as erythropoietic cells, which await further investigation.


Texture analysis of cervical cell nuclei by segmentation of chromatin patterns. Texture parameters of the nuclear chromatin pattern can contribute to the automated classification of specimens on the basis of single cell analysis in cervical cytology. Current texture parameters are abstract and therefore hamper understanding. In this paper texture parameters are described that can be derived from the chromatin pattern after segmentation of the nuclear image. These texture parameters are more directly related to the visual properties of the chromatin pattern. The image segmentation procedure is based on a region grow algorithm which specifically isolates high chromatin density. The texture analysis method has been tested on a data set of images of 112 cervical nuclei on photographic negatives digitized with a step size of 0.125 micron. The preliminary results of a classification trial indicate that these visually interpretable parameters have promising discriminatory power for the distinction between negative and positive specimens.


Computer aided analysis of chromatin network and basophil color for differentiation of mononuclear peripheral blood cells. Computer aided differentiation of plasmoblasts, Pfeiffer cells, immunoblasts, lymphocytes and centrocytes is achieved with the parameters of chromatin network arrangement and structure, and multispectral cytoplasm color. The digital methods involve: (a) segmenting the nuclear image into topographic sections and analyzing the optical density distribution from the chromatin in these sections; (b) determining the nuclear structure with a 7 x 7 median filter, gradient filter and contour following algorithms; and (c) clustering two-dimensional chromatic data from panoptically stained cellular components. The parameters reported here are a subset of those needed for the automated diagnosis of many hematologic diseases especially the leukemias.


Effectiveness of feature groups for automated pairwise leukocyte class discrimination. Automated blood cell differentials using statistical classification techniques have been implemented in several commercial machines. The machine-derived features used to classify leukocytes resemble the descriptors used by humans performing visual classification, e.g. size, content, shape, color, and texture. However, because of our crude modeling of vision there is no universally accepted measure of characteristics such as shape, color, or texture. One expects, therefore, that features which are powerful discriminators for humans may perform poorly when quantified by machine, while other parameters may be more precisely measured automatically and so prove more useful in cell classification. This paper reports the results of a feature evaluation study for automated discrimination between all pairs of a large set of leukocyte classes consisting of both normal and abnormal types. To provide a framework for comparing automated feature ordering with the ranking attached by medical technologists, the machine-derived features were divided into six groups: size and content, mean and mode, cytoplasm/nucleus comparison, contrast and texture, color, and nuclear shape. A sequential procedure was used to select the best five-feature set from each group and the globally best five-feature set for each pairwise classification. The results illustrate the strengths and weaknesses of machine-derived descriptors for each type of decision.


Automated quantitative analysis of single and double label autoradiographs. A method for the analysis of silver grain content in both single and double label autoradiographs is presented. The total grain area is calculated by counting the number of pixels at which the recorded light intensity in transmission dark field illumination exceeds a selected threshold. The calibration tests included autoradiographs with low (3H-thymidin) and high (3H-desoxyuridin) silver grain density. The results are proportional to the customary visual grain count. For the range of visibly countable grain densities in single labeled specimens, the correlation coefficient between the computed values and the visual grain counts is better than 0.96. In the first emulsion of the two emulsion layer autoradiographs of double labeled specimens (3H-14C-thymidin) the correlation coefficient is 0.919 and 0.906. The method provides a statistical correction for the background grains not due to the isotope. The possibility to record 14C tracks by shifting the focus through the second emulsion of the double labeled specimens is also demonstrated. The reported technique is essentially independent of size, shape and density of the grains.


The automated multiparameter analyzer for cells (AMAC) IIA, a true bridge circuit Coulter-type electronic cell volume transducer. A true bridge Coulter effect (electronic cell volume) transducer has been developed. All resistances of this bridge are now the result of current flow through saline channels. Contamination by electrode products including gas bubbles has been completely eliminated since both power electrodes are now remote from the flow chamber. Since the orifice is in series with an approximately 10 K ohm resistance generated by a gel-filled capillary and a displacement rheostat, it floats electrically, at virtual ground. The other side of the bridge consists of a fluid side-wire. Removing the power electrode from the orifice outlet makes possible downward flow and the use of a single outer sheath, and eliminates noise generated by gas bubbles which could possibly be trapped. It should now be possible to combine this design with that of the AMAC III square orifice, to produce an electro-optical sorter where all parameters are measured simultaneously. This true bridge circuit possesses the further advantage that noise due both to the power supply and to overvoltage at the power electrodes is common-mode rejected, and any drift due to changes in electrode polarization is eliminated. Preliminary experiments confirm results with the AMAC II that hemoglobinopathies can be recognized by the increased coefficient of variation (CV) of the erythrocyte spectra.


Flow-system measurement of cell impedance properties. A flow-system technique has been developed that detects the high-frequency resistance and capacitance changes in a sensing orifice due to the passage of cells through the orifice. The resistance and capacitance changes are related to cell properties such as size, plasma membrane capacitance, and electrical resistivity of the cell interior. The relationship between measured impedances and cellular properties is discussed, and the prototype instrument for making such measurements is described. The instrument can simultaneously detect the dc Coulter volume and two ac parameters related to the complex ac impedance change. Some initial tests of the instrument using plastic microspheres and Chinese hamster ovary cells are described.


A comparison of mercury arc lamp and laser illumination for flow cytometers. Optical differences between a mercury arc lamp and a laser-illuminated flow cytometer are compared. The distributions of spectral intensities of the two light sources are shown in relation to the excitation characteristics of the fluorescent dyes acriflavine, chromomycin A3, mithramycin, ethidium bromide, Hoechst 33258, and 4,6-diamidino-2-phenylindole (DAPI). Fluorescence intensities of microspheres and Hoechst 33258-stained mouse sperm are compared in the two cytometers. The optical efficiencies are similar and depend on the match of the excitation characteristics of the stain with the emission spectra of the light source.


Fluorescence intensity resolution in flow systems. The factor which can limit fluorescence intensity resolution in a flow cytometer of the type in which cells pass perpendicularly through a focussed laser beam depends on signal intensity. For the brightest sources (e.g. fluorescent DNA stains), the coefficient of variation (CV) is limited in our system to around 3% by stream hydrodynamics, unstable illumination intensity, nonstoichiometric staining, etc. The weakest detectable sources (e.g. fluorescent cell-surface labels) are limited in coefficient of variation by shot noise in the photomultiplier due to constant background light levels. Finally, over a fairly wide brightness range between these extremes, resolution is determined primarily by photoelectron statistical variation on the signal itself (i.e. "photon statistics"). Thus photon collection and detection efficiency (solid angle, barrier filter passband, detector quantum efficiency) become of primary importance.


Light scattering with stream-in-air flow systems. Both forward angle and 90 degrees light-scattering measurements have been used for cell sizing with stream-in-air flow systems with very little theoretical base for the measurements. Mie theory calculations are compared with measurements on plastic microspheres. Detector response for homogeneous spheres is shown to be sensitive to refractive index.


A statistical approach to the classification of biological cells from their diffraction patterns. An effective method of graphically representing complex biological data is presented, and a statistical classification technique is outlined including an example involving exfoliated cervical cells.


Dual-laser flow cytometry of single mammalian cells. An improved dual-laser flow cytometric system for quantitative analysis and sorting of mammalian cells has been developed using a low-power argon and high-power krypton laser as illumination sources, thus permitting the excitation of fluorescent dyes having absorption regions ranging from the ultraviolet to infrared. Cells stained in liquid suspension with fluorescent dyes enter a flow chamber where they intersect two spatially separated laser beams. Separate pairs of quartz beam-shaping optics focus each beam onto the cell stream. Electro-optical sensors measure fluorescence and light scatter signals from cells that are processed electronically and displayed as frequency distribution histograms. Cells also can be electronically separated and microscopically identified. The ease and versatility of operation designed into this system represent a marked technological improvement for dual-laser excited flow systems. Details of this instrument are described along with illustrative examples of cells stained with mithramycin and rhodamine and analyzed for DNA content, total protein, and nuclear and cytoplasmic diameter.


A real-time delay monitor for flow-system cell sorters. For optimum performance in cell sorting, it is critical to assure proper timing in the charging of droplets to be deflected. A method for determining the transiet delay time in cell sorters has been devised and applied to daily operation in the Los Alamos sorter systems. This delay monitor relies on detection of either scattered or absorbed light from cells in the fluid stream near the point of droplet breakoff.


Investigations in high-precision sorting. We have investigated the accuracy with which droplets containing cells can be sorted individually onto known and thus relocatable positions. The presence and random arrival of cells and particles in the sorter jet disturbs the orderly production and deflection of droplets, causing a dispersion of sorted droplet trajectories. The magnitude of this dispersion is a function of the phase relationship between the arrival of a cell at the end of the jet and the droplet formation. Using a modified Becton Dickinson Fluorescence-Activated Cell Sorter, we selected for sorting only those droplets that formed with a cell near the center of the droplet. We used this technique to sort Lewis lung tumor cells. The dispersion of droplet positions was reduced from over 3% to about 1% of an average deflection of typically 15 mm for a nozzle with a 50-micron diameter orifice. Sorting onto a surface such as magnetic tape or a microscope slide introduces another uncertainty in position because the cell may be located anywhere within the wetted radius of the droplet on the slide. Sorting onto less-wettable surfaces reduces the wetted radius and thus the variation in cell position.


Individual cell sorting.

Individual cell sorting. Current cell sorting machines do not preserve the individual identity of processed cells; after analysis, the cells are assigned to a subpopulation where they are pooled with other similar cells. This paper reports progress on a system that sorts cells individually to precise locations on a microscope slide and preserves them for further observation with a light microscope while recording flow measurement data for each cell. Various electronic and mechanical modifications to an existing sorting machine are described that increase drop placement accuracy and permit individual cell sorting.


Nonparametric flow cytometry analysis.

Nonparametric flow cytometry analysis. A nonparametric statistical test for the analysis of flow cytometry derived histograms is presented. The method involves smoothing and translocation of data, area normalization, channel by channel determination of the mean and S.D., and use of Bayes' theorem for unknown histogram classification. With this statistical method, different sets of histograms from numerous biological systems can be compared.


Selection of optimal model for the DNA histogram by analysis of error of estimated parameters. The ability of four different mathematical models of the DNA histogram to give accurate estimates for the fractions of cells in G1, S, and G2 + M has been investigated. The models studied differ in the form and number of parameters of the function used to represent cells in S-phase. Results obtained from simulated DNA histograms suggest that the standard deviations of the model parameters increase exponentially with the width of the G1 and G2 + M peaks of the histogram. Error analysis is presented as a method to select a model of optimal complexity in relation to the resolution provided by the data in a given set of DNA histograms. Introduction of additional parameters improves the agreement between model and data but may result in a less well-posed model. A model with an optimal number of parameters can therefore be found that will yield parameter estimates with the smallest possible standard deviations.


The development of a new technology for controlled cell fixation. A methodological pilot study. Fixation in a traditional sense means the immersion of biological material into a chemical fluid. For permanent preservation (1) the fixative is always supplied in excess of the cell sample, and (2) the process of fixation is influenced by chemical impurities of the fixative fluid. Both factors influence the subsequent staining of cells. In order to avoid these uncontrolled influences, a new technology for controlled cell fixation has to be developed, whereby freshly prepared formaldehyde gas in an "inert" gas-flow of helium was applied to thin membranes by use of a capillary flow-in technique. The amount of fixative gas supplied, adsorbed, absorbed, diffused, and desorbed after saturation of the membranes could be reliably measured with an on-line operating "inert" mass spectrometer of the Omegatron type.


A template method for decomposing flow cytometry histograms of human chromosomes. A new method for decomposing flow cytometry histograms of isolated human metaphase chromosomes is described and tested. The method is based on fitting a template, composed of the means of all chromosomes of a normal karyotype to the flow histogram. The utility of the method is demonstrated by application to flow measurements of chromosomes from a normal person and comparing the results with those obtained by conventional cytophotometry. The power of the method for detecting gross chromosomal abnormalities, such as trisomy 21, as well as more subtle variations such as a single translocation, is determined for simulated data.


Signal analysis of slit-scan contour data. As a method for the preselection of alarms in gynecological cell samples, the Battelle Cytophotometry Research Group uses the slit-scan technique to obtain various cell parameters, such as the N/C ratio and the relative DNA content, from fluorescently stained cells, which are aligned one-dimensionally in the tape system designed at Battelle. The system developed at Battelle Institute analyzes all signals that exceed the background noise. As the first step in processing the slit-scan data, several threshold levels permit the separation of various artifacts. In subsequent steps, the nuclear peak is recognized, the nuclear boundaries are calculated, and seven cell parameters are determined. For the alarm detection at present only one parameter, DNA fluorescence, is used for these determinations. Visual assignment of these data to definite objects on the tape makes it possible to obtain frequency distributions of: (a) all recorded objects within the sample on the tape; (b) all signals that are classified as cells; and (c) all types of objects that preferentially cause alarms.


Laser flow cytometric light scatter and fluorescence pulse width and pulse rise-time sizing of mammalian cells. In laser flow cytometry, an increasingly popular technique of analytical cytology, quantitative measurements of interest include cell and nuclear diameters. Electronic circuitry for a new cell sizing technique has been developed which measured the time that signal pulses from either fluorescence or light scatter sensors exceed a preset constant fraction of the peak signal amplitude (pulse width) or the time that it takes a signal to rise between constant fractions of the peak signal amplitude on the rising side of the pulse (pulse rise-time). These pulse width or pulse rise-time measurements were related to cell or nuclear diameters and were used in combination to determine nuclear size to cell size ratios. This method of sizing was found to be independent of fluorescent or light-absorbing stain intensity, linearly related to cell or nuclear diameter, and capable of resolving small diameter differences.