Uroporphyrinogen III cosynthase-deficient mutant of Salmonella typhimurium LT2. A new type of heme-deficient mutant of Salmonella typhimurium LT2 was isolated using neomycin. The mutant, designated as strain SASY74, accumulated uroporphyrin I and coproporphyrin I. Extracts of the mutant converted 5-aminolevulinic acid to uroporphyrin I. Extracts of the mutant SASY74 and of the uroporphyrinogen synthase-deficient mutant SASY32 complemented each other and converted, when incubated together, 5-aminolevulinic acid to protoporphyrin. This finding excludes the possibility that uroporphyrinogen I synthase in strain SASY74 is deficient in its cosynthase-binding ability. Hence, the most probable explanation for the accumulation of uroporphyrin I and coproporphyrin I by the mutant is the lack of the uroporphyrinogen III cosynthase activity. This mutant is the first isolated in bacteria with such deficiency, and the mutation is analogous, as far as porphyrin synthesis is concerned, to human congenital porphyria. Mapping of the corresponding gene (hemD) by conjugation and P22-mediated transduction suggests the following gene order on the chromosome: ilv....hemC, hemD, cya....metE. The hemC and hemD genes are probably adjacent; this is the first case in which two hem genes of Enterobacteriaceae are contiguous on the chromosomal map.


Homoazasterol-mediated inhibition of yeast sterol biosynthesis. A naturally occurring azasterol has been shown to inhibit sterol transmethylation in both in vitro and in vivo in the yeast Saccharomyces cerevisiae. The inhibition was competitive, with a calculated dissociation constant of 43 muM. The compound prevented the accumulation of ergosterol in aerobically adapting cells. Cultures forced to gain energy by respiration were found to be much more sensitive to growth inhibition by the azasterol than those cells fermenting glucose. The growth inhibition is reversible at low concentrations of the azasterol.


Protein synthesis and degradation in a leucine auxotroph of Escherichia coli. The synthesis and degradation of the soluble and sodium dodecyl sulfate-(SDS)-solubilized protein fractions of Escherichia coli were studied in both growing and nongrowing cultures. When separated according to molecular weight on SDS-polyacrylamide gels, the proteins of both fractions of growing cells undergo no measureable differential synthesis or degradation during logarithmic growth. However, when a leucine auxotroph is suspended in medium containing 5.3 muM leucine (a level that will not sustain growth), the SDS-solubilized protein of such a nongrowing culture shows a rapid synthesis of two protein components (32,000 and 12,000 daltons) found only in the out membrane.


Use of bacteriophage Mu to isolate deletions in the his-nif region of Klebsiella pneumoniae. Klebsiella pneumoniae M5a1 is naturally resistant to infection by bacteriophage Mu. Mutants of K. pneumoniae sensitive to Mu infection were isolated and found to support both lytic and lysogenic development of Mu. K. pneumoniae lysogens containing a heat-inducible Mu prophage integrated in his were isolated. Strains carrying deletions extending from his into nif were obtained after heat treatment of these lysogens. Such deletions should be useful for determining the map order and cistronic organization of the nif genes.


Salmonella typhimurium mutants generally defective in chemotaxis. The mutations of eight chemotaxis-deficient strains of Salmonella typhimurium, including five new mutants in strain LT2, were mapped by P22 transduction in relation to various fla mot deletions in S. abortus-equi. Seven recessive che mutations mapped between motB and flaC: three, all nontumbling, the che region I, adjacent to motB, and four, including one ever-tumbling, in che region II, adjacent to flaC. Mutant che-107, never-tumbling and dominant to wild type, mapped at flaAII, other mutations of which cause either absence of flagella or lack of locomotor function. We surmise that gene flaAII specifies a protein that polymerizes to form an essential component of the basal apparatus (so that absence of gene product prevents formation of flagela); that a component built up from certain mutationally altered proteins cannot transmit (or generate) active rotation of the hook and flagellum, and so causes the Mot (paralysis) phenyotype; and that a component built up from protein with the che-107 alteration permits only counterclockwise rotation, so that the tumble, normally produced by transient clockwise rotation, cannot be effected.


Role and location of "protease I" from Escherichia coli. Pacaud and Uriel described an enzyme from Escherichia coli ("protease I") that hydrolyzes acetyl phenylalanine naphthyl ester (APNE). We examined the possible involvement of this enzyme in intracellular protein degradation, its subcellular distribution, and its proteolytic activity. Although the APNE-hydrolyzing activity is localized primarily in the periplasm, proteolytic activity against casein was found in the periplasm, membrane, and cytoplasm with similar specific activities. The APNE-hydrolyzing enzyme did not appear to contribute to the proteolytic activity of the periplasm. A mutant deficient in APNE-hydrolyzing activity lacked all activity in the periplasm but showed a slight percentage of residual activity in the cytoplasm. Extracts of such cells were normal in their ability to hydrolyze casein. The mutant was indistinguishable from wild-type cells in its rate of protein degradation during growth or glucose starvation and in the ability to rapidly degrade puromycin-containing polypeptides. Nitrogen starvation, which increased protein breakdown severalfold, affected neither the total amount nor the distribution of APNE-hydrolyzing activity. The mutant showed no defect in its ability to cleave small phenylalanine-containing peptides released during protein degradation. The mutant and wild-type cells are equally able to hydrolyze exogenously supplied phenylalanyl peptides. These experiments suggest that the APNE-hydrolyzing enzyme is not required for protein degradation and that "protease I" is probably not a protease.


3-Deoxy-3-fluoro-D-glucose-resistant Salmonella typhimurium mutants defective in the phosphoenolpyruvate:glycose phosphotransferase system. Three classes of phosphotransferase system mutants in Salmonella typhimurium were selected through their resistance to 3-deoxy-3-fluoro-D-glucose (DFG). Strains with mutations in the ptsH (HPr) and/or pts I (enzyme I) genes were selected on medium containing lactate plus DFG. Strains with mutations in ptsH but not pstI were selected on medium containing fructose plus DFG. Clones isolated from fructose plus DFG semisolid plates and selected for ability to swarm were mutant in either ptsH or ptsG. Mutants of the latter class were defective in enzyme IIB', a membrane component of the glucose transport system. Some pleiotropic properties of one representative ptsG mutant are described.


Deoxyribonucleic acid synthesis in a temperature-sensitive Escherichia coli dnaH mutant, strain HF4704S. The dnaH mutant strain HF4704S, isolated by Sakai et al. (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis. It was found to carry two mutations affecting DNA synthesis. One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C. The mutant marker cotransduced with ilvD at a frequency of about 9%. It seems likely that this mutation is in the dnaA gene. The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml. Both effects could be overcone by adding excess exogenous thymine. We were not able to unambiguously determine the map position of this mutant locus. Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.


Mapping of nrdA and nrdB in Escherichia coli K-12. The structural genes coding for the B1 and B2 subunits of the enzyme ribonucleoside diphosphate reductase, nrdA (formerly designated dnaF) and nrdB, respectively, have been mapped in Escherichia coli. They are located at approximately 48 min. The gene order in this region of the E. coli chromosome was found to be purF glpT nrdB nrdA nalA cdd dcd his.


Diploidy for a structural gene specifying a major protein of the outer cell envelope membrane from Escherichia coli K-12. Homogenotes, heterogenotes, and intergeneric hybrids have been studied that are diploid for the structural gene of a major outer cell envelope membrane protein (protein II) from Escherichia coli. This protein can act as a phage receptor. In wild-type homogenotes, diploidy for the gene did not cause a gene dosage effect. It could be shown with two heterogenotes that both the chromosomal mutant and the episomal wild-type genes are expressed, and in each case more of the mutant than the wild-type protein species was found in the cell envelope. In on case of 21 phage-resistant mutants missing protein II was a trans effect observed of the mutant gene on the expression of the episomal wild type gene. Transfer of E. coli episomes carrying the protein II structural gene into Salmonella typhimurium and Proteus mirabilis resulted in intergeneric hybrids that became sensitive to the relevant phage and harbored the E. coli protein II in their cell envelopes. The results may be taken as suggestive evidence for a simple feedback mechanism for the regulation of synthesis of protein II, and they show that there are no highly specific requirements on protein primary structure for incorporation into an outer cell envelope membrane.


Complexity of the ultraviolet mutation frequency response curve in Escherichia coli B/r: SOS induction, one-lesion and two-lesion mutagenesis. Three distinct sections of the ultraviolet mutation frequency response (MFR) curve toward tryptophan prototrophy have been demonstrated in Excherichia coli B/r WP2 trp thy and its uvrA derivative in log-phase growth in minimal medium. The initial section, which appears fluence-squared, may reflect the necessity, if mutation is to result, for induction of two lesions, one located within the potentially mutated genetic locus and the other damaging deoxyribonucleic acid replication and resulting in inducation of the error-prone SOS repair function. A second linear section is ascribed to the continued induction, after exposure above that sufficient for complete SOS expression, of isolated lesions which lead to mutation in potentially mutated loci. The third section demonstrates an increased rate of mutagenesis and suggests the induction of two lesions in proximity which result in additional mutations. Split-exposure studies support the inducible nature of the SOS function and suggest that mutation frequency decline (MFD) is due to exicion resulting from or related to the prevention of SOS induction by inhibition of protein synthesis. Preirradiation tryptophan starvation of the uvr+ strain for 30 min decrease MFR in the first and second sections of the curve. Reduction of MFR in the third section requires more prestarvation time and is blocked by nalidixic acid. The decreased MFR of the first and second sections ascribed to promotion of postirradiation MFD based on excision and that of third section to completion of the chromosome during the prestarvation period.


Detection, with the dye phloxine B, of yeast mutants unable to utilize nitrogenous substances as the sole nitrogen source. Yeast mutants unable to degrade certain nitrogen compounds produce characteristic small red colonies on an agar medium containing the red dye phloxine B, galactose, the test nitrogen compound, and a small amount of ammonium chloride.


Adenine methylation of Okazaki fragments in Escherichia coli. In Escherichia coli polA lig-4 bacteria, the moles percent 6-methyladenine content of 10S deoxyribonucleic acid (Okazaki fragments) is 0.96 compared with 1.4 for bulk desoxyribonucleic acid.


Regulation of thiamine transport in Saccharomyces cerevisiae. Yeast cells were found to be repressed for the uptake of both thiamine and pyrithiamine by growth with exogenous thiamine, and they appeared to regulate the activity of the binding protein for these compounds.


Interconvertible forms of Escherichia coli dihydrofolate reductase with different affinities for analogs of dihydrofolate. Dihydrofolate reductase from Escherichia coli exists as two species, which show large differences in their affinities for trimethoprim and for pyrimethamine. The two species are present in approximately equal proportions. Each possesses one binding site per mol with dissociation constants (KD) of 14 and 1400 nM, respectively, for the binding of trimethoprim in the binary complex, and of 5 and 47 nM for the pyrimethamine binary complex. In the formation of the ternary complex with NADPH, trimethoprim bound to dihydrofolate reductase as if all the enzyme existed as a single species with a KD for trimethoprim of 1.9 nM. Formation of the trimethoprim NADPH ternary complex thus involves strong cooperative effects, and interconversion of the two species. The binding of pyrimethamine in the ternary complex was indistinguishable from its binding in the binary complex, showing neither the cooperative effects, nor the interconversion of the two species observed with trimethoprim. The species with a low KD for trimethoprim and pyrimethamine could be isolated by selective proteolysis. It was quite stable, but could be converted to a mixture of the original species via formation of the ternary complex with trimethoprim, as predicted from the binding data. The results are interpreted in terms of a model in which the two species can be interconverted only via the formation of a common ternary complex, which can be formed after the binding of trimethoprim or dihydrofolate, but not pyrimethamine. The model is shown to be consistent with all data from the measurements of both binding and inhibition by both ligands.


Interaction of tetraiodofluorescein with yeast hexokinase. The dye tetraiodofluorescein (TIF) was found to be an effective inhibitor of yeast hexokinase. It is a competitive inhibitor relative to MgATP2- and a noncompetitive inhibitor of glucose binding, a kinetic pattern consistent with the previously proposed random kinetic mechanism. TIF interacts directly with the native dimeric protein to give a difference spectrum with a maximum at 543 nm. Monomeric protein (produced by addition of 0.6 M NaCl) interacts with TIF to give a slightly altered difference spectrum, with the gammamax at 545 nm. The difference spectrum of the dimeric form is not perturbed by the addition of substrates but the absorbance with the monomer is lowered by MgATP2-. The Kd for MgATP2- was estimated to be 7 nM for monomeric hexokinase. These results suggest that results of previous binding studies with hexokinase at high concentrations which have been interpreted as being at variance with kinetic studies are due likely to different conformations of the protein under different experimental conditions.


DNA "melting" proteins. II. Effects of bacteriophage T4 gene 32-protein binding on the conformation and stability of nucleic acid structures. Bacteriophage T4-coded gene 32-protein is an essential component of the T4 replication and recombination systems. Alberts and co-workers (Alberts, B.M., Amodio, F.J., Jenkins, M., Gutmann, E.D., and Ferris, F.L. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, 289-305) have shown that the major physiological activity of the protein involves preferential and cooperative binding to single-stranded DNA. In this paper, the physiochemical parameters characterizing this "melting" protein system are quantitatively determined. Boundary sedimentation velocity experiments are used to measure the interaction of gene 32-protein with native DNA. The binding is shown to be non-cooperative and involves an overlapping site size (nh) of approximately 10 nucleotide residues (or approximately 5 nucleotide pairs). In analogy with the ribonuclease results (Jensen, D.E., and von Hippel, P.H. (1976) J. Biol. Chem. 251, 7198-7214), the logarithm of the association constant (Kh) is found to be linerarly related to log [Na+]. The binding of gene 32-protein to denatured (single-stranded) DNA involves appreciable distortion of the polynucleotide backbone from the unliganded conformation; binding totally unstacks the bases of both ribose- and deoxyribose-containing polynucleotides at 10 degrees, and results in a hyperchromic change exceeding that which can be induced by heating. This hyperchromism induced in poly(dA) on binding gene 32-protein under low salt (tight binding) conditions is used to determine a value of nc (the single-stranded DNA site size) of approximately 6.7 nucldotide residues per protein. In addition, gene 32-protein binding to single-stranded polynucleotide induces an unusual circular dichroic spectrum characterized principally by a marked decrease in the magnitude of the positive CD band centered at approximately 265 nm. This spectral change is attributed to significant uncoupling of the transition moments of the vicinal bases of the single-stranded polynucleotide on gene 32-protein binding, in accord with the ultraviolet hyperchromism observed. Binding of gene 32-protein to double helical DNA has virtually no effect on the spectral properties of this conformation...


DNA "melting" proteins. III. Fluorescence "mapping" of the nucleic acid binding site of bacteriophage T4 gene 32-protein. The intrinsic tryptophan fluorescence of bacteriophage T4-coded gene 32-protein is found to be partially quenched on binding a variety of mono-, oligo-, and polynucleotides. This phenomenon is exploited to partially "map" the nucleic acid binding site of the protein. The intrinsic fluorescence spectrum of the protein peaks at about 347 nm, compared to 359 nm for the fully solvated model fluorophore, N-acetyl-L-tryptophanamide. Nucleotide binding, or collisional quenching by iodide ion, reduces the intensity of the fluorescence, with little or no peak shift. Small ligands, ranging in size from ribose- and deoxyribose-phosphate to tetranucleotides, quench the fluorescence by 2 to 6%; larger ligands quench from 20 to 35% of the intrinsic protein fluorescence. Iodide quenching experiments subjected to Stern-Vollmer analysis suggest that the binding of short nucleotide-containing ligands brings about a conformational change in the protein, fully exposing a tryptophan side chain to the solvent environment. The fluorescence of this tryptophan is fully quenched by the binding of d(Ap)2, but is largely unaffected by the binding of d(ApA) or d(pA)2, indicating both that this (tryptophan) "reporter" residue is located in the nucleic acid binding site and that binding is polar, i.e. polynucleotide chains of only one orientation are complexed. Long oligonucleotides fully quench the fluorescence of this binding site tryptophan. At high salt concentration (2 M NaCl), gene 32-protein forms self-limited dimers (Carroll, R.B., Neet, K.E., and Goldthwait, D.A. (1972) Proc. Natl, Acad. Sci. U.S.A. 69, 2741-2744; (1975) J. Mol. Biol. 91, 275-291). These dimers, in either high salt or in low salt after cross-linking, fail to bind nucleotides, suggesting that dimer formation partially occludes the nucleic acid binding site and thus that these dimers are probably not involved as intermediates in cooperative protein binding to the DNA. On the other hand, dimerization apparently results in a conformational change which fully exposes the "reporter" tryptophan to iodide quenching. These results are used to formulate a model of some of the nucleic acid-protein and protein-protein interactions involved in the cooperative binding of gene 32-protein to single-stranded DNA.


Regulation of gene 32 expression during bacteriophage T4 infection of Escherichia coli. The gene 32 protein of the bacteriophage T4 plays an important role in genetic recombination, DNA repair, and DNA replication; the protein functions in these processes by virtue of a strong binding capacity for single-stranded DNA. During infections of Escherichia coli by bacteriophage carrying amber of temperature-sensitive mutations in gene 32, the altered gene 32 protein (that is, the amber fragment of the missense polypeptide) is synthesized at greatly elevated rates. During infections by phages that are mutant in other genes (and wild type in gene 32), gene 32 expression is coupled to the quantity of single-stranded DNA produced during the infection. The data are consistent with a model in which the gene 32 protein binds preferentially to all available single-stranded DNA. When all available single-stranded DNA is complexed with gene 32 protein, free gene 32 protein represses its own synthesis. The high level expression of altered gene 32 proteins (amber fragments or missense polypeptides) is a direct consequence of the proposed autoregulation.


Translational, autogenous regulation of gene 32 expression during bacteriophage T4 infection. Functional half-life measurements of the bacteriophage T4 gene 32 messenger RNA indicate that this mRNA is extremely stable. Regulation of gene 32 expression at the transcriptional level cannot account for the rapidity with which P32 synthesis can be repressed. Furthermore, derepression of P32 synthesis occurs in the presence of rifampicin, a drug which inhibits transcriptional initiation. In addition, T4-infected cultures in which P32 expression is repressed possess almost as much gene 32 mRNA as derepressed cultures. We conclude that expression of the T4 gene 32 protein is regulated at the level of translation.


Selective inhibition of protein synthesis initiation in Saccharomyces cerevisiae by low concentrations of cycloheximide. We have previously determined the amounts of time required to complete various macromolecular synthetic processes needed for induction of allophanate hydrolase in Saccharomyces cerevisiae. This information provided a means of testing, in vivo, an early hypothesis suggesting that cycloheximide inhibited the initiation as well as elongation steps of protein synthesis. Our data suggest that initiation of protein synthesis in yeast may be inhibited by low concentrations of cycloheximide which do not significantly affect polypeptide chain elongation.


Gastrointestinal abnormalities in renal homotransplant patients. Of 93 patients who received a total of 110 consecutive renal transplants, 83 had gastrointestinal contrast studies. These were of value during treatment, management and follow-up of the renal homotransplant patients. Barium studies are most helpful in the diagnosis of unexpected gastrointestinal disease in asymptomatic patients, but of little value in cases of massive gastrointestinal hemorrhage. Common abnormalities include coarsening of the mucosal folds in the stomach and duodenum. Duodenal ulceration is the most commonly diagnosed abnormality in gastrointestinal hemorrhage. Most abnormalities are demonstrated during the first 100 days after transplantation at which time the patient is most susceptible to gastrointestinal disease.


Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions. The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anti-cytochrome b5/anti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome b5. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product.


Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody. The distribution of cytochrome b5 in rat liver microsomes, and in two microsomal subfractions isolated by density equilibration in a linear sucrose gradient, was studied under the electron microscope by means of a ferritin-labeled hybrid anti-cytochrome b5/anti-ferritin antibody. Results of this study show that cytochrome b5 is present in essentially all microsomal vesicles derived from endoplasmic reticulum (ER), whether rough or smooth. Thus, the dissociation of ER constituents into two groups (b and c), achieved by subfractionating microsomes by isopycnic centrifugation (Beaufay, H., A. Amar-Costesec, D. Thines-Sempoux, M. Wibo, M. Robbi, and J. Berthet. 1974. J. Cell Biol. 61:213-231), does not reflect the association of each group with distinct microsomal particles but reflects rather an enzymatic heterogeneity of the ER: the ratio of group c to group b enzymes increasing with the density and ribosome load of the particles.


Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets. Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium-dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.


Mycotic aneurysm of the innominate artery with supravalvular aortic stenosis. A case of mycotic innominate artery aneurysm in association with supravalvular aortic stenosis has been presented with successful resection as the first such case documented. Blood flow was restored with low cervical autogenous internal jugular vein, as an end-to-side vein graft from the left common carotid and end-to-end to the right subclavian and right common carotid arteries. The future repair of the supravalvular aortic stenosis could then be more easily accomplished.


Microtubules and pancreatic amylase release by mouse pancreas in vitro. The effects of vinblastine and colchicine on pancreatic acinar cells were studied by use of in vitro mouse pancreatic fragments. Vinblastine inhibited the release of amylase stimulated by bethanechol, caerulein, or ionophore A23187. Inhibition required preincubation with vinblastine,and maximum inhibition was observed after 90 min. Inhibition was relatively irreversible and could not be overcome by a high concentration of stimulant. Inhibition could also be produced by colchicine although longer preincubation was required and inhibition was only partial. Uptake of [3H]vinblastine and [3H]colchicine by pancreatic fragments was measured and found not to be responsible for the slow onset of inhibition by these drugs. In incubated pancreas, microtubules were present primarily in the apical pole of the cell and in association with the Golgi region. Vinblastine, under time and dose conditions that inhibited the release of stimulated amylase, also reduced the number of microtubules. The only other consistent structural effects of vinblastine were the presence of vinblastine-induced crystals and an increased incidence of autophagy. The remainder of cell structure was not affected nor were overall tissue ATP and electrolyte contents or the stimulant-induced increase in 45Ca++ efflux. It is concluded that the antisecretory effects of vinblastine and colchicine are consistent with a microtubular action, but that acinar cell microtubules are more resistant to the drugs than many other cell types.


Clinical evaluation of the minitek differential system for identification of Enterobacteriaceae. Forty-one stock organisms and 581 fresh clinical isolates were used in comparing the Minitek system to conventional tubed media to determine if this system is feasible and accurate for a high-volume clinical microbiology laboratory. In addition to comparison with tubed media, the following parameters were tested: (i) reproducibility of disks, (ii) the effect of variation in inoculum size, (iii) the effect of the age of culture, and (iv) the effect of predispensing disks. A total of 5,947 disks were compared with tube reactions; 95.9% agreed on the initial reading. After repeating questionable tube and disk reactions, this agreement rose to 98.2%. Using the disk reactions and the identification schema currently in use with tubed media, 94.9% of the Minitek final identifications agreed with those of tubed media; 1.5% gave differing identifications and 3.6% were inconclusive. This system is accurate, inexpensive, flexible, and convenient.


Evaluation of the fluorescent gonococcal test-heated. In a 1-year evaluation of the indirect fluorescent antibody test for gonorrhea, 16,127 specimens from low-and high-risk groups were examined, and the results were compared with culture, history of exposure to a known patient, and clinical signs and symptoms. The results confirmed the usefulness of the indirect fluorescent antibody test for gonorrhea as a screening procedure, especially for women in the low-risk group. The test is more economic and probably more sensitive than culture for mass screening of asymptomatic women.


Evaluation of four methods for detection of group B streptococcal colonization. Four methods (streak plate, pour plate, selective broth, and direct fluorescent-antibody staining) were evaluated for their ability to detect group B streptococcal colonization in parturient women and their offspring. When colonization was defined as a positive culture by any method from any site, selective broth was the most sensitive method, detecting 100% of colonized mothers and infants at birth and 48 h of age. This method failed to detect only one colonized individual (infant at 24 h of age). The other three methods detected from 20 to 56% of colonized individuals.


Hemolysis-in-gel test for demonstration of Chlamydia antibodies. Sheep erythrocytes were coated with antigens of a strain of Chlamydia trachomatis serotype D. Chlamydial antibodies in sera from patients with salpingitis or pneumonia were demonstrated by passive hemolysis in agarose gel.


Basophils in tuberculin and "Jones-Mote" delayed reactions of humans. Jones-Mote reactions are delayed, erythematous, and mildly indurated cutaneous reactions originally described in humans sensitized by skin injection of heterologous proteins. Similar reactions in guinea pigs contain many basophils and are called cutaneous basophil hypersensitivity. In contrast, guinea pigs immunized with mycobacterial adjuvants have classical tuberculin-type delayed hypersensitivity reactions, which contain few basophils. This has led to a new classification of delayed responses, based largely on the presence or absence of basophils. We induced sensitization for Jones-Mote reactions in 20 normal humans by intradermal injections of keyhole limpet hemocyanin. Skin tests with KLH 1 wk later showed erythematous and indurated delyaed reactions in all subjects. Rebuck skin windows showed specific accumulations of basophils with a delayed time-course in 18 of 20 subjects. In 12 normals sensitized with oxazolone-keyhole limpet hemocyanin conjugates, skin reactions and in vitro lymphocyte stimulation showed carrier and not hapten specificity, suggesting that cutaneous responses were probably mediated by T cells. A comparative study of strongly positive PPD skin tests in patients with tuberculosis showed significant basophil accumulations in five of nine subjects. Thus, basophils occurred in human tuberculin and Jones-Mote reactions and were not a distinguishing feature of Jones-Mote reactions. We suggest that the occurrence of basophils at delayed reactions is under complex regulation and that basophil accumulations are an aspect of delayed hypersensitivity, rather than an indication of a distinctive and separate response.


Cutaneous antigen-stimulating lymphokine production by lymphocytes of patients with progressive systemic sclerosis (scleroderma). Cell-mediated immunity to skin extracts was studied by the macrophage migration inhibition test, lymphocyte transformation, and direct cytotoxicity to skin fibroblasts, in normal individuals and patients with progressive systemic sclerosis. The latter included 18 individuals with diffuse scleroderma and 12 with the CREST syndrome, a variant form of systemic sclerosis in which there is more limited involvement of the skin. Controls consisted of 13 patients with other connective tissue diseases and 16 normal individuals. Phosphate-buffered saline and 3 M KCl extracts of both normal and sclerodermatous skin were used as antigens. No evidence of lymphocyte reactivity was found by the lymphocyte transformation and direct cytotoxicity test procedures. However, the lymphocytes of patients with diffuse scleroderma did respond to extracts of both normal and sclerodermatous skin in the migration inhibition assay. 10 of 16 patients (62.5%) had migration indices below 2 SD of the normal range, 1 of 10 CREST patients and 1 of 13 patients with other connective tissue diseases showed similar reactivity. Antisera specific for immunoglobulin-bearing lymphocytes (B lymphocytes) and T lymphocytes were used to characterize the lymphocytes found in skin biopsies of patients with diffuse scleroderma. T lymphocytes made up the majority of lymphocytes in the skin infiltrates. These findings suggest that lymphocytes sensitized to skin extracts are present in patients with diffuse scleroderma. The cell-mediated immune reaction to skin antigens may be a factor in the pathogenesis of diffuse scleroderma.


Testing therapeutic measures for controlling chronic gingivitis in man: the results of two studies. A previous paper described a suggested protocol which could be used for testing therapeutic measures. In this paper the protocol has been used as a basis for testing experimental toothpastes over short and medium periods of time. The effects on plaque and gingivitis of a chlorhexidine paste used for 4 weeks, and of two other experimental products used over 8 weeks, have been investigated. Statistically significant improvements in gingivitis were observed during the use of chlorhexidine and one of the experimental pastes compared with their respective placebo pastes. The benefit observed for the experimental paste became evident when the data were analyzed considering only participants with 'frank' gingivitis. The findings have also been presented in various ways in attempts to express the benefits of the therapeutic measures in clinical terms by using arbitrarily determined success criteria.


Subepidermal bullous disease. A correlated clinico-pathologic study of 51 cases. The histologic features of 51 cases of subepidermal bullous disease were evaluated and correlated with the clinical features including the course of the disease and the response to therapy. There was a good clinico-pathologic correlation, 46 out of 51 cases showing concordant clinical and histologic diagnoses. We conclude that although immunofluorescence is a confirmatory procedure histologic diagnosis is still of great value, particularly in the early stages of the disease when the clinical features may not yet have evolved sufficiently to allow for a confident diagnosis.


Combined routes of administration to assay oral analgesia in postoperative pain. To increase the sensitivity of the method for evaluating oral analgesics in postoperative patients, we designed a combined oral/parenteral bioassay. Drugs studied were parenteral morphine, parenteral propiram, and oral codeine at two dose levels each and oral propiram at four dose levels. Results from data on 308 patients suggest that future studies designed to establish the relative potencies of oral analgesics should use parenteral morphine as the standard in a combined oral/parenteral study because this approach provides a very sensitive measure of analgesia. Further, with one drug as the reference compound, results from many sources would be more readily compared.


Effect of food on the bioavailability of prednisone. Two commercial prednisone tablets were studied which had previously been shown by Sullivan et al.5,6 to have the slowest and fastest in vitro rates of dissolution, and the slowest and fastest rise to peak plasma prednisolone concentrations in human beings. The effect of food on the adsorption of these two tablets was studied in a crossover study, which also repeated the fasting conditions used by Sullivan et al.6 Marked differences in mean prednisolone plasma concentrations during the 0- to 2-hour absorption phase were observed between the two tablets again, but food did not affect either tablet with respect to mean plasma prednisolone concentrations.


Action of milk-clotting enzymes on beta-caseins from buffalo's and cow's milk. beta-Caseins isolated from buffalo's and cow's milk were hydrolysed either with rennet or with microbial proteases from Mucor miehei, M. pusillus Lindt or Endothia parasitica. The degradation products were separated by polyacrylamide-gel electrophoresis and the residual beta-casein was determined quantitatively after various times. The electrophoretic patterns of the degradation products of buffalo and bovine beta-casein produced by the different enzymes were not identical. beta-Casein of buffalo's milk was hydrolysed by rennet and M. miehei protease at a slower rate than that of cow's milk. The reverse was found with E. parasitica and M. pusillus Lindt proteases. Carbamylation of buffalo beta-casein was found to retard its proteolysis by all the enzymes but particularly by rennet and M. miehei protease.


Parkinsonism and levodopa: a five-year experience. Seventy parkinsonian patients were treated continuously with levodopa for five years. During the first year, sixty-three patients (90 per cent) improved. After five years, however, only thirty-seven patients remained improved while thirty-three patients (48 per cent) experienced progressive disease. Complications of treatment, albeit nonfatal, increased in frequency during the five-year interval. The reason for early improvement and subsequent deterioration of parkinsonian symptoms and signs in spite of levodopa therapy remains unexplained. It suggests that Parkinson's disease may not be simply a striatal dopamine deficiency syndrome and that treatment with levodopa is more than replacement therapy.


The use of epidermis over a keloid as an autograft after resection of the keloid. A technique is described using the epithelium over a keloid as a graft after excision of the keloid. Advantages of this procedure are availability of skin and avoidance of the danger of keloid formation in a distant donor site. Results in a series of five patients followed for five years were satisfactory in three.


The mode of action of aspirin and similar compounds. Aspirin-like drugs inhibit the biosynthesis of prostaglandins in all tissues and species so far studied. The concentrations necessary for this action are achieved after therapeutic dosage. The evidence is reviewed which establishes this biochemical action as the basic mode of therapeutic action of aspirin-like drugs as anti-inflammatory, analgesic, and antipyretic compounds. It may also account for the shared side effects, including the hypersensitivity to aspirin-like drugs shown by some allergic patients.


Identification and study of species specificity of antiphage lipopolysaccharides found in the preparations of bacterial DNA. A method of isolation of the antiphage agent found in the preparations of bacterial DNA was developed. Chemical analysis of the preparations has shown that according to their qualitative and quantitative composition they are identical to the lipopolysaccharide of the bacterial membrane. On the basis of data on the antiphage activity of the D-LPS from E. coli B and E. coli K12 and on the basis of presumed analogy between the inactivation of the phage by the D-LPS preparations and the phage--cell interaction it is believed that different parts of the LPS serve as receptors for the phages T7 and T4: O-specific polysaccharide for T4 and core LPS for T7. On the basis of data on the activity of D-LPS of two species of the genus Aerobacter against the phage T7 it is presumed that Aerobacer and Escherichia are related according to the structure of their core LPS.


The agglutinating and immunofluorescent activities of antileptospiral antibodies of human sera and of immunoglobulins M and G. Microagglutinating and immunofluorescent activities of sera of nine patients with clinically diagnosed leptospirosis were investigated. L. grippotyphosa was found in seven patients, L. sejroe in one and L. icterohaemorrhagiae in one patient. The agglutinating activities of IgM approximated the activities of the whole sera. In five patients the agglutinating activities were found in IgG; in four of them two to three months after the onset of the disease, in one patient a month after its onset. In one patient only the activity of IgG reached the activity of IgM after six months. Immunofluorescent activity was found in IgG in eight patients two to five weeks after the onset of the disease. In seven of these patients the activity of IgG reached or exceeded the activity of IgM three to ten weeks after the onset of the disease. IFAT was found to be poorly specific because of high heterologous titres. In 5 out of 7 patients with L. grippotyphosa, heterologous IFAT titres were reduced or eliminated by carrying out the reaction with isolated IgM and IgG. However, in patients with L. icterohaemorrhagiae and with L. sejroe heterologous IFAT activity persisted in both IgM and IgG. The study points out the possibility of differentiating an early stage of the infection from convalescence.


Antibody response to lymphoma antigens in syngeneic and H-2 compatible mice. The antibody response to immunization with A/J lymphoma L1117 cells by syngeneic A/J mice and by H-2 compatible B10.A mice has been show- to be highly specific for tumor antigens of the lymphoma cells. In both strains the response exhibits a very slow rise in cytotoxic antibody levels reaching a maximum only after 3 months of biweekly injections. It is also unusual in that cytotoxic activity is primarily due to IgM antibodies even after 20 weeks of immunization. The similarity between these characteristics and those reported for "T-independent" antigens suggests that tumor antigens on L1117 cells may be unable to stimulate helper T cells.


Binding of IgE myeloma proteins to human cultured lymphoblastoid cells. The binding of human IgE myeloma proteins to 16 human cultured lymphoblastoid cell lines was studied by measuring specific uptake of radiolabeled deaggregated IgE myeloma proteins and/or E-IgE rosette formation. Eight lines, RPMI-8866, Wil-2WT, RPMI-6410, RPMI-1788, RPMI-4265, Clowers, COLO-59 and Victor, bound IgE as shown by at least one of these methods. The lines, RPMI-4098, SCRF-5004, NC-37, Daudi, Raji, P3JHR-1, RPMI-1301 and Molt-4 did not bind IgE. Of the positive cell lines, 58 to 98% of the cells formed E-IgE rosetts. The binding of IgE was Fc fragment specific. It could only be inhibited by human IgE and its Fc fragment but not by IgE Fab fragments and Ig of other classes. The binding of IgE also appeared to be species specific, since a rat IgE myeloma protein did neither bind to the cells nor inhibit the binding of human IgE. The binding of IgE was relatively temperature independent and was abolished by trypsin and pronase pretreatment of the cells. Most of the cell lines binding IgE did not bind IgG but had surface immunoglobulin and did not form spontaneous E rosettes. These data suggest that certain lymphoblastoid cells may have receptors for IgE.


Specific lysis of human colon tumor cells by antibodies to CEA and isoantigen A: dependence on rabbit serum or neuraminidase. The 51Cr cytotoxicity test was used to measure specific antibody reactions against carcinoembryonic antigen (CEA) and isoantigen A on the surface of human colon tumor cells. When human serum or guinea pig serum was used as a source of complement, no anti-CEA or anti-isoantigen A cytotoxicity was demonstrable despite binding of specific antibodies and activation of complement at least through the C3 component on the cell surface. In contrast, specific anti-CEA and anti-isoantigen A cytotoxicity was demonstrated when rabbit serum was used as a source of complement. Specific antibody-mediated cell lysis was also achieved with guinea pig complement if the cells were treated with neuraminidase before testing. These results support the concept that certain tumor cells have surface properties that render them resistant to immune lysis.


Anti-lymphokine antibody. II. Specificity of biological activity. The present study was designed to explore the range of biologic activity of an anti-lymphokine antiserum (ALKS). This preparation, which was produced by a two-stage immunization procedure, was used to prepare Sepharose bead immunoadsorbent columns. Columns made with ALKS, but not with anti-control antiserum, could specifically remove MIF activity, but not LT, MF, or NCF activity from activated lymphocyte culture supernatants. MIF but not LT, MF, or NCF could be recovered from the beads by acid extraction. These findings, taken in conjunction with the previously described capacity of ALKS to remove MCF and SRF activity from supernatants, demonstrated that ALKS although prepared against a lymphokine preparation with multiple activities, has restricted specificity. Thus far, its traget appear to be those mediators that affect macrophages either in vitro or in vivo. The capacity of ALKS to suppress delayed hypersensitivity reactions in the guinea pig is consistent with these observations, since the macrophage is the predominant infiltrating cell in those reactions.


Triggering of human peripheral blood B cells: polyclonal induction and modulation of an in vitro PFC response. In the present studies we utilized polyclonal activators to analyze the signals required to induce antibody synthesis by subpopulations of human peripheral blood lymphocytes. The in vitro synthesis of antibody directed against sheep erythrocytes was measured by a modification of the Jerne plaque assay. The plaques observed contained central lymphoid cells, could be inhibited by anti-human immunoglobulin, and were strictly dependent on both the addition of complement and viable B lymphocytes actively synthesizing protein. Kinetic analysis showed that the PFC response developed over time during cell culture with peak activity occurring between days 6 and 7. It was found that human peripheral blood B lymphocytes, isolated by Sephadex G-200 anti-Fab column chromatography, were nonspecifically induced by either PWM or soluble products of antigen-activated human T cells to differentiate into PFC. In contrast, unfractionated peripheral blood lymphocytes. Ig- cells isolated from immunoabsorbant columns, and B cells enriched by nylon wool adherence were not triggered to antibody synthesis even in the presence of polyclonal activators. B cells isolated by nylon wool adherence and further fractionated by binding and elution from anti-Fab columns were then readily triggered by PWM to differentiate into PFC. These results suggest that the interaction of human B cells with anti-Fab columns, presumably at the Ig receptor, serves as a signal important in the differentiation events leading to PFC activity. The anti-Fab interaction alone, although critical, was not sufficient to induce the differentiation of precursor cells into PFC since subsequent triggering by antigen-activated T cell supernatants or PWM was required for the development of an optimal PFC response. Additional experimental data demonstrated that the PFC response could be regulated by autologous peripheral Ig- lymphoid cells in vitro. At low concentrations of PWM, Ig- cells consistently augmented the B cell PFC response whereas at high concentrations of PWM, suppression was observed. Depletion of E rosetting cells from the Ig- population eliminated the subset responsible for augmentation but did not eliminate the suppressor cell activity.


Lack of antibody activity directed against the most common human bacteria in human myeloma protein. The fact that sera of germ-free animals contain very little Ig suggests that most Ig probably correspond to antibacterial antibodies. We have detected an antibacterial activity against 40 bacterial strains, pathogenic and nonpathogenic, in 85 M-component of human origin. This work has been conducted by indirect immunofluorescence withipure labeled antibodies. The reasons for which indirect immunofluorescence seems to be the best technique for this kind of research are discussed. No activity whatsoever could be detected. The reasons of this lack of activity remain to be explained.


Genetic control of B cell activation by bacterial lipopolysaccharide is mediated by multiple distinct genes or alleles. Eleven closely related C3H mouse strains were examined for differences in their DNA synthetic response to a highly discriminatory endotoxin, LPS Escherichia coli K-235. Strains of the C3H line were found to be either high, intermediate, or low responders to endotoxin. Three stains, C3H/St, C3H/Bi, and C3H/CR were high responders. Six strains, C3H/He, C3HfB/He, C3H/Hen, C3Heb/Fej, C3H/DiSn, and C3H/Avy were intermediate responders, suggesting that a mutation producing decreased LPS responsiveness occurred in the C3H/He(C3H/An) strain between 1931 and 1945. Two strains, C3H/HeJ and C3H/Bts were unresponsive due to a mutation that occurred between 1960 and 1968. Breeding experiments among high, intermediate, and low responding strains documented probable codominant genetic control by the genes or alleles in the C3H/HeN and C3H/HeJ strains. In the C3H/HeN x C3H/St cross, dominance of the C3H/St gene over the C3H/He gene was documented, whereas the C3H/HeJ x C3H/St cross indicated codominance of these two genes. These findings may represent either three alleles or three distinct genes in C3H mice. A second X-linked gene locus was documented in the CBA/N strain which also causes impaired B cell responsiveness to LPS E. coli K235 in serum-free conditions. The abnormal gene products in the CBA/N and C3H/HeJ strains exhibit complementarity since F1 female animals from the cross between these two unresponsive strains are responsive to LPS. We conclude that there is at least one distinct X-linked gene locus and either three additional autosomal genes or three possible alleles at one or more autosomal loci which determine the LPS sensitivity of murine B cells.


Antibody-induced modulation of Leishmania donovani surface membrane antigens. The effects of specific antibodies on the surface membrane antigens of Leishmania donovani (strain 1S, clone 2D) amastigote and promastigote developmental stages was detected in vitro by using several direct and indirect immunofluorescence methods. Stage specific anti-L. donovani sera and sera from L. donovani-infected hamsters induced parasite surface membrane antigens to aggregate, move along the longitudinal cell axis, form polar cell caps, and subsequently disappear. No membrane fluorescence was observed in cells treated identically with normal sera. In amastigotes in a single anterior cell pole cap was formed after antibody treatment. However, antibody-treated promastigotes showed tripartite membrane antigen capping consisting of a major anterior cell pole cap with minor caps at the posterior cell pole and flagellar tip regions. Loss of surface fluorescence from the two latter capped cell regions was frequently concurrent with the formation of extracellular fluorescent plasmanemes. The cell-capping process was antibody concentration, temperature, time, and energy dependent. No cell caps were formed at low temperature or in the presence of several metabolic inhibitors. Cell cap formation was also selectively inhibited by certain types of antibody-induced intercellular agglutination. Parasite membrane antigens removed by capping were regenerated and detectable at the cell surface only after a 3.5 to 4-hr period. Antibody-induced membrane antigen movement in L. donovani is apparently similar to pheonmena observed with mammalian cells. Results of direct cross-staining and cross-absorption cell-capping experiments showed that the two parasite developmental forms shared some common or at least cross-reacting membrane antigens and that each form also possessed some unique stage-specific surface antigens. Cell-capping results with sera from L. donovani infected hamsters showed that this host produces some IgG which is directed specifically against certain parasite surface membrane antigens. The results suggest that host antibody-induced parasite membrane antigen modulation might have a role in the infectious process of this human pathogen.


Activation of human B lymphocytes. II. Cellular interactions in the PFC response of human tonsillar and peripheral blood B lymphocytes to polyclonal activation by pokeweed mitogen. A recently described system for the induction and assay of plaque-forming cells (PFC) following polyclonal stimulation of human B lymphocytes was employed to delineate the cellular requirements for pokeweed mitogen (PWM)-induced activation of human tonsillar and peripheral blood B lymphocytes. It was found that the process was not dependent on the presence of monocytes, but was dependent on the presence of T cells. T cell depleted suspensions of tonsillar as well as peripheral blood lymphocytes had markedly diminished if not absent PFC responses following PWM stimulation. The PFC responses of T cell depleted cultures of tonsil and blood could be reconstituted by adding back T cells. T cell supernatants could also reconstitute the PFC response of T cell depleted tonsillar lymphocytes but not of T cell depleted blood lymphocytes, thus suggesting a more stringent T cell requirement of the PWM-induced PFC response of blood lymphocytes. Enrichment for T cells on the other hand enhanced PFC responses in both organs, particularly in those cultures which had low responses in unfractionated suspensions demonstrating the requirement of optimal T cell help for maximal PFC responses. This study demonstrates the feasibility and effectiveness of this PFC system in elucidating some of the complex regulatory processes associated with the activation of human B lymphocytes.


The regulation of the immune response to T-independent antigens by prostaglandins and B cells. The effect of inhibitors of prostaglandin synthesis on the in vitro primary immune response to two T-independent antigens has been investigated. The results, with PVP or DNP-Ficoll, suggest that the prostaglandins play an important role in the regulation of the immune response to these antigens. Furthermore, it appears that B cells may be involved in their own regulation by synthesizing and responding to prostaglandins. Although it has been previously established that T cells exert a regulatory influence on the response to PVP and DNP-Ficoll, this is the first demonstration of an apparent B cell regulation of B cell function via the production of a specific hormone.


Subpopulations of human T cells identified by receptors for immunoglobulins and mitogen responsiveness. Human T lymphocytes in peripheral blood have been shown to have receptors for IgG (Tgamma) or IgM (Tmu). Cultured Tgamma cells do not express receptors for IgM and purified Tmu cells do not have receptors for IgG, thus they appear to be distinct T cell populations. Although the two subpopulations show similar response patterns to concanavalin A, Tmu and Tgamma cells exhibit different dose-response curves to phytohemagglutinin. The normal response pattern to phytohemagglutinin requires a mixture of T cell subpopulations suggesting that synergistic interactions may occur.


Autoimmunity and aging: the age-related response of mice of a long-lived strain to trinitrophenylated syngeneic mouse red blood cells. Mice of 1.5, 9, 22, and 31 to 32 months of age were injected with the thymus-dependent antigen, TNP-SRC, or the thymus-independent antigen, TNP-SRC, TNP-MRC. The anti-SRC and TNP immune responses to TNP-SRC were markedly reduced in older mice, whereas the anti-TNP response to the TNP-MRC showed no substantial decline. Young mice produced higher anti-TNP plaque-forming cell responses after injection of TNP-SRC than after TNP-MRC, whereas in older mice the reverse obtained. Old mice but not young mice displayed a high anti-SRC cross-reactive response after injection of TNP-MRC. The avidity of anti-TNP antibody of young mice immunized with TNP-SRC was higher than that following immunization with TNP-MRC, whereas the avidities of anti-TNP antibodies from old mice injected with these two reagents were the same. Those individual mice which showed a poorly regulated immune response also displayed an autologous anti-MRC plaque-forming cell response after injection of either TNP-SRC or TNP-MRC. It is suggested that mechanisms mediated by suppressor T cells may be responsible for regulating the autoimmune response to modified self antigens, and that these are severely impaired in age individuals.


Hemolytic plaque inhibition: the physical chemical limits on its use as an affinity assay. An analysis of the fundamental physical chemical limits of hemolytic plaque inhibition as a method for obtaining thermodynamic and kinetic information is presented. It is shown that inhibition curve characteristics will be sensitive functions of reaction affinities only when the antibody inhibitor and antibody red blood cell reaction mechanisms are related in certain ways. It is further shown that conditions which are most sensititive to IgG affinity changes will generally not be the best for detecting changes in IgM affinity. The apparently conflicting reports on IgM maturation are completely explicable in terms of experimental requirements imposed by physical chemical characteristics of the reaction. Experiments in which maturation is observed are found to conform to the most sensitive conditions of the assay, whereas those in which it is not observed are found to conform to relatively insensitive conditions, and would therefore be capable of registering changes only when affinity shifts are large.


Experimental autoimmune thyroiditis in the guinea pig: characterization of infiltrating lymphocyte populations. Experimental autoimmune thyroiditis was induced in out-bred guinea pigs by isoimmunization with thyroid extract in complete Freund's adjuvant. A digestion procedure using collagenase and deoxyribonuclease was used to make viable single-cell suspensions of pooled thyroid glands from groups of animals with advanced degrees of thyroiditis. Thymus-derived or T lymphocytes, identified by their capacity to form E rosettes with rabbit erythrocytes, were found to be the predominant (75%) infiltrating lymphocyte; bone marrow-derived or B cells consitituted most of the remainder. The infiltrates of inbred animals with thyroiditis were demonstrated to contain cells capable of mediating antibody-dependent lymphoid cell-mediated cytotoxicity.


Preparation and use in immunohistology of antibodies specific for type I and type III collagen and procollagen. Antibodies to bovine type I and type III collagen and their precursor form procollagen were produced in rabbits and rendered specific for the immunizing antigen by immunoadsorption. These purified antibodies showed distinct immunofluorescence staining on frozen sections of both bovine and human connective tissue at concentrations as low as 1-10 mug/ml. Antibodies to type III collagen and procollagen reacted with reticulin in liver and spleen, with fascicles around tendons and with the upper portion of the dermis. Antibodies to type I collagen and procollagen reacted with skin and fiber bundles in tendon but did not stain reticulin. No reaction was observed with cartilage collagen or with kidney glomerular basement membrane.


A simple screening assay for substances affecting lymphocyte reactivity. A simple method is described for assaying substances modifying lymphocyte reactivity in vitro to mitogens and antigens. This procedure employes whole blood, microtiter plates and an automated cell harvester. The simplicity and expediency afforded by this assay, which requires very little blood, enables the large-scale screening of different test materials which may be of experimental or clinical merit.


A rapid and sensitive technique for the detection of Fc receptors on macrophages. A rapid sensitive technique for the production of macrophage EA rosettes employing centrifugal sedimentation of opsonized erythrocytes is described. Using this method, a sensitive rosette formation-inhibition assay for the determination of anti-macrophage antibody titers has been developed.


Iodinated versus fluorescent labelling in the radioallergosorbent test (RAST) for the determination of serum IgE levels. Experiments were performed to determine the feasibility of replacing the radioactive 125I tag in the radioallergosorbent test (RAST) with a new fluorescent compound, 2-methoxy-2, 4-diphenyl-3 (2H)-furanone (MDPF). It was possible to discriminate a grass-allergic from a grass-non-allergic patient using the traditional RAST procedure with 125I-ant-IgE. However, when a fluorescent label, MDPF was attached to IgE using the same RAST procedure, it was not possible to discriminate between allergic and non-allergic responses. Additional experiments, using BSA and anti-BSA as a model, showed that it was possible to detect 0.025 ppm antigen using iodinated antiserum while the fluorescent tag had a lower limit of antigen detection of 0.20 ppm. It was possible to distinguish between allergic and non-allergic responses using 125I-N-succinimidyl-3(4-hydroxyphenyl) propionate (SHPP) which binds to protein in a manner similar to MDPF. These results suggested that the unsatisfactory fluorescence results were not due to an alteration of protein structure as a result of MDPF labelling. Although the fluorescence work was performed using a fluorimeter with optics and electronics optimized for detection of MDPF, sensitivity of the method was limited by high fluorescent background.


Reproducibility, efficacy and methodology of mitogen-induced lymphocyte transformations by the whole blood assay. For the past few years, the whole blood culture method has been scrutinized for its usefulness and reproducibility in evaluating the immunologic status of patients. This method, used in our laboratory mainly screening specimens for evidence of immunodeficiencies, has been systematically evaluated for reproducibility, and factors affecting normal response through testing specimens from over 70 normal persons. Three mitogens (phytohemagglutinin, Concanavalin A, and pokeweed) were studied; peak time responses occurred later than in the separated cell culture method, and they were different for each mitogen. The dose response curves also depended on the mitogen tested, with pokeweed giving a sharp curve and the others a broad plateau. The harvesting procedure was studied by varying reagents and was optimized for speed and efficiency. The mononuclear cell count of the specimen had little effect on the results as long as it remained less than 3.5 X 10(6)/ml. The results for all mitogens followed a similar distribution curve irrespective of whether the results were expressed as cpm or cpm divided by the mononuclear cell count. The use of the stimulation ratio method to express results was less satisfactory. The mean coefficient of variation for all triplicate samples remained 20% or less and for all conditions tested. In evaluating the immunodeficient patient, the whole blood culture method was found to be equally as informative and easier to perform than the separated cell method.


A simple method for isolation of human properdin by affinity chromatography. Human properdin was prepared from the zymosan eluate or euglobulin fraction of serum by immunoadsorbent column chromatography. An immunoadsorbent column containing antibody to human serum was employed to remove serum proteins other than properdin from zymosan eluates. Immunoadsorbent with antibody to properdin was used for direct isolation of properdin from the euglobulin fraction. Properdin isolated by these methods showed satisfactory purity with high activity. No immunochemical differences between the two types of preparation were found.


Measurement of anti-enzyme antibodies using an active-site directed radiolabel. A new technique is described for measuring antibodies to an enzyme which is not available in pure form. The secretions of the rat parasitic nematode, Nippostrongylus brasiliensis, were treated with tritiated diisopropylfluorophosphate. Only one component, an acetylcholinesterase, was radiolabelled. Antibodies to this enzyme in rat antisera were estimated by the Farr technique using the labelled enzyme as antigen. The acetylcholinesterase secreted by Necator americanus, the human hookworm, was similarly specifically labelled.


Simultaneous quantitation of two antigens in mixture in individual cells by microimmunofluorimetry. Double immunofluorescence has so far only been used qualitatively. In present work the possibility of quantitative double immunofluorescence was evaluated in model systems with the ultimate goal to enable simultaneous measurements of different antigens in mixture in biological objects. In one test system a conjugate of fluorescein isothiocyanate (FITC) was studied in combination with conjugates of dimethyl-amino-naphthalene-sulphonyl-chloride (DANSC) or with tetramethyl-rhodamine-isothiocyanate (TRITC) in solutions. In another test system FITC + DANSC antibody conjugates were analyzed after reaction with antigen mixtures in insolubilized form. Fluorescence intensities were measured in a microspectrofluorimeter using narrow band excitation and emission filters of appropriate types for different fluorochromes. Fading was no problem in this system. A formula was developed to calculate the antibody proportions from the fluorescence contribution of either fluorochrome in the region of optimal fluorescence of other fluorochrome. Experimental values obtained from analyses of FITC + DANSC and FITC + TRITC combinations in solutions agreed excellently with theoretically expected values. In the second model system polyacrylic beads were coated with different proportions of two antigens, one labelled with 125I and the other with 131I. After radioactivity measurements the beads were stained with appropriate antisera conjugated with FITC and DANSC, respectively. "Double fluorescence" intensities of individual beads were measured and a very strong correlation was found between the radioactivity and the fluorescence values. The results indicate that quantiation of double fluorescence is possible and meaningful for measurements of two different antigens in mixture on individual particles.


Insolubilization of protein antigens on polyacrylic plastic beads using poly-L-lysine. A new model system for quantitation of immunofluorescence on single cells is described using poly-L-lysine (PLL) coated polyacrylic plastic beads of approximately cell sizes as carriers for protein antigen. By increasing PLL concentration on the beads increased amounts of 125I labeled antigen were attached to the particles. Excess binding sites of PLL could be completely blocked by unrelated proteins. After staining with FITC-conjugated antibodies and quantitative fluorescence measurements of individual beads using a microspectrofluorimeter, strong correlations were found between antibody and antigen concentration on the beads. Neither repeated washings with PBS nor storage of the beads for two months caused detectable shedding of antigen-antibody complexes. There was a strong linear correlation between fluorescence intensity and the volume of beads, but the correlation between surface area and fluorescence was nonlinear. The described procedure was shown to be a simple method for quantitative and stable coating of particles with proteins. It can be applied as a useful model system for quantitative immunofluorescence studies on intracellular antigens.


Amphotericin B and amphotericin B methyl ester ascorbate. I. Chemotherapeutic activity against Candida albicans, Cryptococcus neoformans, and Blastomyces dermatitidis in mice. Amphotericin B methyl ester (AME) has been reported to possess in vitro antifungal activity similar to that of amphotericin B and to have less intrinsic toxicity in mice and dogs. For these reasons AME has been porposed as an alternative to amphotericin B in the therapy of deep mycoses. For comparison of the therapeutic efficacy of the two polyenes in laboratory animals before initiation of studies in humans, groups of mice were infected with Candida albicans, Cryptococcus neoformans, and Blastomyces dermatitidis. Treatment consisted of two or more doses of each drug given by the intravenous route. Concurrently, studies of subacute toxicity were conducted in the same species to permit calculation of therapeutic indices. These studies have shown that AME, as the ascorbate salt, is substantially less efficacious than amphotericin B (in colloidal dispersion with sodium deoxycholate) for treatment of the fungal infections and that amphotericin B had a higher therapeutic ratio for all infections studied than did AME.


Rapid detection of measles virus in skin rashes by immunofluroescence. Specific immunofluorescence was used to detect measles virus antigen in skin rashes. Cryostat sections of punch-biopsy specimens of the skin were stained with use of hyperimmune rabbit and horse antisera to measles virus, also conjugated with fluorescein isothiocyanate, served as controls. Measles virus was specifically demonstrated in 20 of 21 biopsy specimens taken within four days after the onset of exanthema. Measles virus antigen was also found in three of five biopsy specimens from nonexanthematous skin during the first four days of the exanthematous phase. The viral antigen was found in single cells or in clusters of cells in the surface epithelium, skin appendages, and corium. No viral antigen was detected in biopsy specimens taken five to six days after the onset of rash.


Alterations in the fecal flora of an individual frequently lacking coliforms. An individual who usually completely lacked or had very low numbers of coliforms in his stool over a period of 19 months was identified. The subject's levels of clostridia and aerobic lactobacilli were elevated as compared with those of our control group. However, these elevated populations of microorganisms were not related to the number of coliforms present in stool samples. The number of coliforms was inversely related to the number of anaerobic lactobacilli. In addition, the absence of coliforms was always accompanied by the absence of fungi. Populations of other prominent groups of intestinal flora were not significantly affected by the number of coliforms present in the intestnal tract. Since ankylosing spondylitis was the only physical abnormality of the subject, the intestinal flora of other individuals positive for histocompatibility antigen B27 who had this disease were examined but were found to be normal. The absence of coliforms from the normal flora of the intestinal tract of the subject had no apparent effect on his general health.


Amikacin Therapy of Urinary tract infections. The efficacy and safety of amikacin in treatment of patients with urinary tract infections were evaluated. Underlying urologic disease was prevalent in the population studied. Amikacin was administered to 50 patients in a dosage of 7.5 mg/kg twice daily. In a subsequent study, amikacin was compared with gentamicin in the treatment of 63 patients; amikacin was administered in a dose of 150 or 200 mg every hr, and gentamicin was administered in doses of 60-80 mg every 8 hr. In the initial study, amikacin produced a bacteriologic cure rate of 86%. In the comparative study, cure rates for gentamicin and amikacin were 85% and 81% respectively. No significant renal toxicity or audiometric changes were attributed to amikacin.


Comparative effectiveness of combinations of amikacin with penicillin G and amikacin with carbenicillin in gram-negative septicemia: double-blind clinical trial. Preliminary results are presented for an ongoing, double-blind, clinical trial, in which the efficacy of amikacin plus penicillin G (Amik-Pen) and amikacin plus carbenicillin (Amik-Carb) is compared in treatment of severe gram-negative infections superimposed on serious underlying disease. All clinical isolates were sensitive to amikacin in vitro (minimal inhibitory concentration, less than 12 mug/ml). Results in 50 patients with cancer and documented gram-negative infection, 29 of which involved septicemia, were analyzed. In the Amik-Pen group, 40% of 15 cases of septicemia responded favorable to therapy, as compared with 86% of 14 cases of septicemia in the Amik-Carb group; this difference is statistically significant (P less than 0.02). When all patients were considered together, the outcome appeared more favorable (1) in infections caused by pathogens sensitive to both antibiotics used then in those caused by organisms sensitive to amikacin only (83% vs. 43%); (2) when the combined antibiotics demonstrated synergy in virto against the offending pathogen than when the combination was nonsynergistic (83% vs. 38%); and (3) when the peak serum antimicrobial dilution titer was larger than or equal to 1:8 than when titers were lower. The results of this study suggest that routine use of an antibiotic combination that has demonstrable in vitro synergy against the offending pathogen should be considered for the treatment of proven or suspected severe infections due to gram-negative bacilli.


A review of results of clinical trials with amikacin. Case records of 1,098 patients treated with amikacin at 79 research centers in 10 countries in a program of worldwide clinical trials were reviewed. Of the 697 patients eligible for use in evaluation of efficacy of the drug, 81% were cured, as evidenced by clinical remission and eradication of the infecting pathogen. The usual dosage was 7.5 mg/kg administered intramuscularly at 12-hr intervals. This dosage was modified in patients with renal impairment. Amikacin was effective in 90% of 322 patients with genitourinary infections, 85% of 97 patients with septicemia, 70% of 73 patients with infections of skin, soft tissue, or bone (excluding burns), and 69% of 68 patients with infections of the lower respiratory tract. Amikacin was effective in treatment of 88% of 85 infections due to gentamicin-resistant pathogens. The drug was generally well tolerated, and no side effects were reported in 80.6% of the 1,098 patients evaluated. Amikacin shares with other aminoglycosides the risk of ototoxicity and nephtotoxicity; previous exposure to gentamicin was a major factor in the development of such adverse effects. Other adverse reactions were relatively infrequent and in most cases were characterized as mild and transient.


[Studies on the contract of the casting. (I). Influence of casting condition (author's transl)]. A study was made of the contraction of the Silver-Indium alloy due to the difference of the casting condition, by mean of the quartz pipe, whose thermal-expansion coefficient was very small. The results were as follows. 1) The higher the mold temperature, the smaller the casting shrinkage. 2) When the casting pressure was extremely low the casting shrinkage was great. But if the casting pressure was over 0.16 kg/cm2, the casting shrinkage didn't change. 3) The bigger the sprue size, the smaller the casting shrinkage. 4) The casting temperature of the alloy, the cooling system, the amount of the metal and the form of the wax pattern showed that the casting shrinkage remain the same.


[Studies on the pour type resin for denture. (I) Mechanical properties (author's transl)]. The influence of crosslinking agents in syrup type resins on the several mechanical properties was studied. These data were compared with those commercially available heat-curing resin and powder-liquid pour type resins. Addition of 2 to 3 mol% of crosslinking agents to the syrup gave best results, which were between those of the heat-curing resin and the powder-liquid pour type resin.


[Studies on the pour type resin for denture. (II) Curing shrinkage (author's transl)]. This study was to investigate the influence of concentration of crossliniking agents in the monomer and pressure applied on the curing shrinkage in pour type resins. A pressure could improve the dimentional accuracy in both case of the syrup and the power-liquid type resins, on the other hand crosslinking agents gave adversed effect on the dimentional accuracy of the pour type resins, especially on that of the group.


Metabolic consequences of low-temperature kidney preservation. The metabolic consequences of low-temperature kidney preservation were investigated. A comparison was made between kidneys which were immediately preserved and kidneys which had been ischemic for 1 hour. Two types of preservation techniques were used: (1) continuous perfusion with oxygenated plasma as described by Belzer and (2) a single flush with potassium-containing perfusate as suggested by Collins. Slices of renal cortex were removed at varying times during preservation and analyzed for a variety of metabolic intermediates. ATP levels were markedly reduced from normal. The Belzer technique was associated with higher ATP levels and ischemia lowered the ATP level. Kidneys perfused by the Belzer technique had lower ADP levels than those by the Collins method. Preservation caused marked elevation of tissue lactate, irrespective of ischemia or the technique used. We conclude that low temperature kidney preservation has profound effects on cellular metabolism. Therefore, the measurement of metabolic intermediates may provide a rational approach to the prediction of organ survival.


Establishment of the origin of a porcine cell line by chromosome banding techniques and scanning. Use of cultured cells in diagnostic virology implies the necessity to establish the genetic origin of the cells. Chromosome analysis, performed on a cell line of pig embryo kidney cells (PEK), made the porcine origin of the cells plausible. Definite proof, however, was furnished by chromosome banding and scanning. A comparison was made between PEK-cells and normal pig chromosomes. One of the chromosomes No. 1 had an interstitial deletion, which provided an useful marker for identification purposes.


Mycotic flora in tonsils and adenoids. A microbiological and histological evaluation. The tonsils of 147 patients with chronic tonsillitis and the adenoids of 134 children with recurrent respiratory infections were cultured for fungal organisms and studied microscopically. A control group consisted of 68 healthy persons. Positive cultures were obtained in 64% of patients with chronic tonsillitis, in 21% of children treated with adenoidectomy, and in 69% of the control group. Candida albicans, Saccharomyces sp. and Aspergillus were the most common organisms. The histological investigations revealed no evidence of pathogenecity in these organisms. They were found in the tonsillar crypts, and no granulomatous inflammation was seen surrounding them.


Genetics of the idotype of BALB/c myeloma S117: multiple chromosomal loci for Vh genes encoding specificity for group A streptococcal carbohydrate. A small proportion of the antibodies to Group A streptococcal carbohydrate (A-CHO) elicited in BALB/c mice by immunization with Group A streptococci, has idiotypic determinants in common with the BALB/c myeloma protein S117 which has specificity for N-acetyl-glucosamine, the major antigenic determinant of A-CHO. The expression of these idiotypic determinants is under the control of a gene which is linked to the Ig-1a+ allotype locus in strain BALB/c and in other strains carrying the same Ig-1 haplotype. This gene (S117+) segregates in breeding experiments as if it were an allele to the gene A5A+ which controls the expression of the A5A idiotype in association with antibodies to A-CHO in strain A/J and which is linked to the Ig-1e allotype locus. Another possible allele, linked to the Ig-1c allotype locus, controls the expression of both S117 and A5A cross-reactive determinants (S117cr, A5Acr). The distribution of these idiotypic determinants in various lines that carry recombinant Ig-1 haplotypes suggests that the A5A and S117 loci are nonallelic and map at different positions in the Ig-1 region. The data suggest complex pseudollelic relationships between different Ig-1 haplotypes that allow the expression of the same genes in allelic and in nonallelic fashion.


Connective tissue origin of the amyloid-related protein SAA. Protein SAA is a serum protein related to the major constituent of secondary amyloid fibrils, protein AA, and has been suggested to be a precursor of the amyloid protein AA. In the present study, the origin of SAA was investigated by studying human fetal tissues and cultured human fetal fibroblasts with the indirect immunofluorescence technique. Anti-SAA antibodies reacted strongly with cultured fibroblasts producing a fine fibrillary cytoplasmic staining and with extracellular fibrils in loose connective tissues and vessel walls. The reactions were specifically inhibited by absorption with degraded amyloid material, isolated protein AA, isolated protein SAA, and sera from patients with elevated levels of SAA. In contrast, no inhibition was seen with amyloid fibril material devoid of AA protein or by human sera in which SAA was not detectable by double-diffusion tests. These observations showed that SAA-like material is produced by fibroblasts and indicate that it is a normal constituent of developing extracellular connective tissue fibers.


Continuing medical education in America. Some of what may be perceived as today's failures in continuing medical education may have been caused by lack of sound educational principles in the medical education process. Others may be due to changing times and expanding knowledge. New methods need to be established which include education based on physician audit and self-assessment. Learning outcomes should be evaluated in order to assess physicians' abilities to render better patient care. The formal graduate educational program is seen as the base for the new method of delivery of continuing medical education. The residency has the ability to evaluate advances in medicine and distill them for the practicing clinician. It may also assist him with office systems which will enable him to monitor his practice and needs. Linkages with residency programs will benefit the practitioner and resident alike. In the future, other community facilities may be needed to handle problem-centered continuing medical education.


Diagnostic procedures of the skin. Part one: Wood's light, KOH slide, Gram's stain, and cultures. The diagnosis of skin lesions involves the same priniciples and methodology required in other medical problems. Visual recognition alone and "shotgun" therapy is not a satisfactory clinical approach. A disciplined and careful examination of lesions, establishment of a differential diagnosis, and selection of appropriate procedures are frequently necessary for cutaneous diseases. The indications, limitations, interpretation, and techniques of diagnostic procedures must be well understoood to obtain reliable information. Not all tinea capitis will reveal fluorescence with Wood's light examination, but the Wood's light may be particularly helpful in the diagnosis of tinea versicolor, erythrasma, porphyria, and tuberous sclerosis. Bacterial growth on cultures taken from the skin does not necessarily mean infection. Because the eczematous skin teems with bacteria, there must be a careful interpretation of the cultures results within the context of the clinical situation. This paper is the first in a two-part series dealing with selected cutaneous procedures which are useful to the family physician in everyday practice.


Amino-acid pool composition of Saccharomyces cerevisiae as a function of growth rate and amino-acid nitrogen source. The composition of the amino-acid pool of Saccharomyces cerevisiae is markedly influenced by the amino-acid nitrogen source. The yeast tends to accumulate the amino acid supplied and those closely related to it metabolically. A relatively high concentration of glutamic acid is maintained in the pools of all cultures irrespective of the nitrogen source, reflecting the central role of glutamic acid in nitrogen metabolism. The total amino-acid pool concentration increases as a function of growth rate, although differences exist in the behaviour of individual amino acids.


Haemagglutinating and adhesive properties associated with the K99 antigen of bovine strains of Escherichia coli. The K99 antigen common to some bovine strains of Escherichia coli caused mannose-resistant haemagglutination of sheep erythrocytes and was shown to be responsible for the attachment of K99-positive bacteria to calf brush-border preparations because (i) strains grown at 18 degrees C did not produce K99 antigen, cause haemagglutination, or attach to brush borders; (ii) a K12 (K99+) recombinant strain showed both haemagglutinating activity and attachment to brush borders whereas, before it received the K99 plasmid, the recipient strain was negative in both respects; and (iii) cell-free extracts of K99 antigen showed haemagglutinating activity and inhibited the attachment of K99-positive organisms to brush borders. K99 antigen appears to be a virulence determinant in the pathogenesis of neonatal calf diarrhoea. It is readily demonstrated by haemagglutination and brush-border attachment tests.


Induction of the mycelial form of Candida albicans by hydrolysates of peptides from seminal plasma. Previous work led to the separation from seminal plasma of a peptide fraction which promoted a high rate of germination of blastospores of Candida albicans. It has now been shown that an acid hydrolysate of this material is also highly active. A minimal amino-acid mixture consisting of aspartic acid, lysine, histidine, threonine, proline and beta-alanine gave 90% germination in 4 h with 17 out of 28 strains examined. Glucose and inorganic phosphate were also required. Phosphate was not required for the activity of the original peptide fraction.


Uptake of fructose by the sorbitol phosphotransferase of Escherichia coli K12. Strains of Escherichia coli that are unable to grow on fructose because they lack the phosphoenolpyruvate: fructose phosphotransferases specified by ptsF and ptsX mutate to grow on media containing fructose as sole carbon source, but do not regain the function of either of the missing phosphotransferases. Instead, fructose is taken up and phosphorylated to fructose 6-phosphate by a phosphoenolpyruvate: sorbitol phosphotransferase which, in wild-type cells, is induced by sorbitol but not by fructose, but which is constitutively expressed in these mutants. The regulatory gene srlC controlling enzymes of sorbitol uptake and catabolism has been located on the E. coli genome as part of the linkage group cysI srlC attI86 pheA.


Effects of inhibitors of protein, RNA and DNA synthesis on heat-injured Salmonella typhimurium LT2. The role of protein, RNA and DNA synthesis in the repair of thermal injury in Salmonella typhrimurium was investigated. Thermal injury was assessed by the 'minimal medium recovery' system: after heat treatment, higher viable counts are obtained on minimal-medium agar than on complex-medium agar, and the ability of heated bacteria to form colonies on complex-medium agar is recovered when they are incubated in liquid minimal medium. This recovery is inhibited by rifampin and chloramphenicol, but not by nalidixic acid. In addition, rifampin causes a loss in viability. Alkaline sedimentation analyses of radioactively labelled DNA showed that hydroxyurea and rifampin, unlike chloramphenicol and nalidixic acid, cause DNA breaks in heated bacteria. The results indicate that rifampin is lethal to heated bacteria and that chloramphenicol, though not lethal, prevents repair of thermal damage.


Location of the Aspartase Gene (aspA) on the linkage map of Escherichia coli K12. The aspartase (L-asparatate ammonia-lyase, EC. 4.3.I.I) structural gene, aspA, was mapped by two-factor and three-factor transductional crosses using phage PI. Cotransduction frequencies between aspA and other markers were: ampA (69%); frdA (48 TO 67%) mel (35%); purA (17%); fdp (I-6%). The sequence of markers within the corresponding segment (91 to 95 min) of the Escherichia coli linkage map was shown to be mel-aspA-ampA-frdA-purA-fdp.


Polypeptides specified by bacteriophage T1. The proteins synthesized during the replication of phage TI in u.v.-irradiated Escherichia coli strain B have been examined by polyacrylamide gel electrophoresis of polypeptides pulse-labelled with 14C-amino acids. Up to 50 discrete bands were identified of which about 30 were sufficiently distinct to be classified in terms of time of synthesis. Three polypeptides were synthesized only during the first 6 to 8 min post infection (Class I, Early); 16 or 17 were synthesized predominantly during the later stages of replication starting from 6 to 8 min after infection (Class III, Late); three classes of proteins were made continuously, two at constant rate (Class II, Continuous), five at decreasing rate (Class IV, Early-continuous) and five at increasing rate (Class V, Late-continuous). Of the 14 polypeptides identified as structural components of the virion, three (P7, PI0 and PI) account for about 85% of the particle weight with P7 comprising 50% of the particle. P7 and PI0 appear to result from the cleavage of larger polypeptides. Preliminary studies with amber mutants suggest that normal levels of TI DNA synthesis are not required for the manufacture of late proteins and that a phage-controlled function may control the switch-off of proteins made early and the switch-on of proteins made late.


Antibody-induced redistribution of virus antigens on the surface of influenza virus-infected cells. The behaviour of virus-specific antigen-antibody complexes at the plasma membrane of HeLa cells infected with influenza virus (A0PR8) was studied by immunofluorescence- and immunoelectron-microscopy. As early as 7 h after infection the virus-induced plasma membrane antigens are evenly distributed over the surface of the infected cells. The addition of antiviral antibodies interferes with this distribution. The appearance of a subsequent patchlike and 'capping' distribution, followed by the disappearance of virus antigen-antibody complexes from the cell surface, is described. These findings demonstrate the potential mobility of virus-induced antigens in the plane of the cell surface.


Double-blind clinical trial of 5-hydroxytryptophan in a case of Lesch-Nyhan syndrome. 5-Hydroxytryptophan (5-HTP) treatment of a single case of Lesch-Nyhan syndrome showing compulsive self-mutilation, athetoid movements, and characteristic clinical biochemical picture was studied on a double-blind basis. 5-HTP or placebo was administered for seven fortnightly treatment blocks. 5-HTP produced a significant reduction of athetoid movement and a sedative effect but did not improve the patient's mood or reduce self-mutilation.


Sensitivity of the Limulus test and inhibitory factors in the radiopharmaceuticals. The basic sensitivity of the Limulus test and the inhibitory factors were examined in 21 radiopharmaceuticals commonly used in Japan. The sensitivity of the Limulus test using pre-gel was found to be 1 mug/ml for Escherichia coli endotoxin. This sensitivity is about ten times that of the rabbit test adopted by USP and JP. The Limulus test was applicable, with full sensitivity and without inhibitory reaction, for the evaluation of 99mTcO4-, 99mTc-albumin, 99mTc-MAA, 99,Tc-Sn-colloid, 131I-Hippuran, Na131I, Na251CrO49, 67Ga citrate, and 57Co-bleomycin as commercially supplied. On the other hand, with 111In-DTPA, 99mTc-phytate, 99mTc-pyrophosphate, 99mTc-DTPA, 131I-polyvinylpyrrolidone (PVP), 59FeCl3, Na232PO4, 198Au colloid, and selenomethionine (Se-75), the pH required adjustment to avoid inhibition of the gelation reaction. Benzyl alcohol showed an inhibitory effect on the gelation reaction at concentrations of more than 1%. Iodine-131-Bromsulphalein (BSP) and 131I-rose bengal showed intense inhibition of the gelation reaction.


Concentrations of erythromycin in serum and tonsil: comparison of the estolate and ethyl succinate suspensions. Concentrations of erythromycin were measured in serum and tonsil from children who had received either the estolate or ethyl succinate suspension before surgery. The in vitro assay measured total erythromycin activity against a group A beta hemolytic streptococcus. Levels of erythromycin in serum and tonsil after single and multiple doses of the estolate were significantly higher than those after administration of the ethyl succinate. The therapeutic implications of these findings are unknown.


Erythromycin: a review of its uses in pediatric practice. Erythromycin continues to be a valuable and useful antimicrobial agent in children. Its low index of toxicity, freedom from sensitization, and reliable absorption and when administered orally contribute to make it an attractive agent in the treatment of a variety of minor respiratory and skin infections, especially in those situations where real or potential allergy to penicillin exists. Additional major uses are in the eradication of the carrier state in whooping cough and in diphtheria, especially in those instances when oral therapy can be tolerated. Dispite use over more than two decades, resistance developing in formerly susceptible organisms has not been a problem and thus seems unlikely to become so in the future.


Toxigenic bacterial diarrhea: nursery outbreak involving multiple bacterial strains. An outbreak of watery diarrhea occurred in 14 of 15 infants in a special care unit over a four-day period. Using the CHO cell assay for enterotoxin, we found that 11 of these patients had toxigenic bacteria in their stools. These bacteria comprised nine different serotypes of three species of organisms: Escherichia coli, Klebsiella, and Citrobacter. None of the three serotypes of E. coli were classic enteropathogenic serotypes. Rectal swab specimens from all 15 infants were examined for the presence of viruses by electron microscopic and cell culture techniques as well as by studies in suckling mice. None had parvovirus- or reovirus-like agents and two had adenoviruses. No other viral agents were detected. Of 38 bacterial strains isolated from ten control infants without diarrhea, three Klebsiella strains from two individuals were found to be toxigenic. Analysis of a total of 136 enteric isolates showed that toxigenicity as measured by the CHO assay was strongly associated with strains isolated during acute diarrheal illness when compared with strains isolated in convalescence (p less than 10(-5)) or with strains from control infants without diarrhea (p less than 10(-5)). This study raises the possibility of an outbreak of disease caused by a transmissible plasmid responsible for a cholera-like enterotoxin production in several enteric bacterial strains.


Interaction of E. coli strains with human serum: lack of relationship to K1 antigen. Twenty-eight strains of E. coli isolated from infants were compared with respect to opsonic requirements, sensitivity to serum, and ability to activate serum chemotactic factors. Six of the strains were isolated from stools of healthy newborn infants; 22 were isolated from the cerebrospinal fluid or blood of infants with meningitis and/or septicemia. Eighteen of the strains had K1 polysaccharide antigen. Fourteen of the strains (seven with K1 antigen) activated complement via the alternative pathway and all of these strains were well opsonized in 4% pooled human serum. A higher concentration of serum was necessary to opsonize 12 of the 14 strains that did not activate the alternative pathway. A wide variation was also found in opsonic requirements of E. coli strains isolated from healthy and sick infants. There was no relationship of the K1 antigen to opsonic requirements, to capacity to activate complement via the alternative pathway, to generation of chemotactic factors, or to sensitivity to serum cidal activity. Therefore, the association of E. coli with K1 antigen and neonatal meningitis did not appear to be related to these bacteria-serum interactions.


Midline hamartomas masquerading as meningomyeloceles or teratomas in the newborn infant. Congenital miline dorsal malformations in infants most commonly represent spina bifida cystica or teratoma. The five patients reported here are a distinct clinicopathologic entity. They presented at birth with a dorsal midline mass, with no neurologic impairment, and no hydrocephalus developed subsequently. The lesions were removed in all infants without significant mobidity. The masses were comprised prinicipally of mesodermal elements, i.e., bone, cartilage, fat, muscle, and fibrous tissue. The well-differentiated cellular components, forming mature structures and the lack of primitive organoid structures as well as the absence of neoplastic characteristics, favors a diagnosis of hamartoma rather than of teratoma.