Enzymatic defenses against the toxicity of oxygen and of streptonigrin in Escherichia coli. Anaerobically grown Escherichia coli K-12 contain only one superoxide dismutase and that is the iron-containing isozyme found in the periplasmic space. Exposure to oxygen caused the induction of a manganese-containing superoxide dismutase and of another, previously undescribed, superoxide dismutase, as well as of catalase and peroxidase. These inductions differed in their responsiveness towards oxygen. Thus the very low levels of oxygen present in deep, static, aerobic cultures were enough for nearly maximal induction of the manganese-superoxide dismutase. In contrast, induction of the new superoxide dismutase, catalase, and peroxidase required the much higher levels of oxygen achieved in vigorously agitated aerobic cultures. Anaerobically grown cells showed a much greater oxygen enhancement of the lethality of streptonigrin than did aerobically grown cells, in accord with the proposal that streptonigrin can serve as an intracellular source of superoxide. Anaerobically grown cells in which enzyme inductions were prevented by puromycin were damaged by exposure to air. This damage was evidenced both as a decline in viable cell count and as structural abnormalities evident under an electron microscope.


Regulation of murein biosynthesis and septum formation in filamentous cells of Escherichia coli PAT 84. Both the beta-lactam antibiotic, cephalexin, and the deoxyribonucleic acid synthesis inhibitor, nalidixic acid, are known to inhibit cell division in Escherichia coli and induce the formation of filaments. The biosynthesis of murein was investigated in these filaments and compared with the murein synthesized by the normally dividing rods of E. coli PAT 84. Differences were found in the extent of peptide side-chain cross-linkage. Filamentous cells had higher extents of cross-linkages in their newly synthesized murein. Quantitative analyses of the D-alanine carboxypeptidase and transpeptidase reactions in the different cells revealed that the carboxypeptidase activity of the filamentous cells was partially inhibited. These results were similar to those previously found with filaments that were obtained after growth of the thermosensitive division mutant at its restrictive temperature. We conclude that the formation of new cell ends (septa) depends on the proper balance between the activities of the D-alanine carboxypeptidase that regulates the availability of precursor doners and the transpeptidase, which catalyzes cross-linking and attachment of newly synthesized murein.


Physical characterization of a plasmid cointegrate containing an F'his gnd element and the Salmonella typhimurium LT2 cryptic plasmid. A recombinant plasmid (pAS19) isolated from a derivative of Salmonella typhimurium LT2, containing the strain LT2 cryptic plasmid and an F'his gnd element, has been physically characterized. The pAS19 plasmid contour length equals the sum of the contour lengths of the cryptic plasmid and F'his gnd element. Deoxyribonucleic acid (DNA)-DNA hybridization experiments demonstrated that whereas the pAS19 plasmid exhibits extensive DNA homology with both the cryptic plasmid and the F'his gnd element, there is little DNA homology between these latter two plasmids. The DNA fragmentation pattern of the pAS19 plasmid produced by the restriction endonuclease R-EcoRI is consistent with that expected for a composite plasmid cointegrate containing most, if not all, of the DNA sequences present in its two component plasmids.


Identification of a membrane protein associated with expression of the surface exclusion region of the F transfer operon. Membrane preparations from radioactively labeled male and female strains of Escherichia coli K-12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. An intensely labeled band corresponding to a protein of molecular weight of 24,000 was readily apparent in preparations from Hfr and F-prime strains but not in those from female strains. When preparations from a series of Hfr strains containing transfer operon deletions were examined, presence of the band was found to be associated with retention of the region of the F transfer operon between ilzA and traD. Thus, the band ("protein S") appears to be the product of an F tra operon activity corresponding to traS (the gene for surface or entry exclusion), or an unknown gene in its vicinity. As predicted, protein S was subject to Fin+ control; only a faint band was detectable if the repressed plasmid R100 was also present in the F lac strain. A 24,000-dalton protein was also found in membrane preparations from strains carrying the derepressed plasmids R100-1 and R1-19 but not in those from strains carrying the repressed plasmids R100 or R1. Thus, the appearance of protein S in the membrane may be a general phenomenon resulting from transfer operon expression of F-like plasmids.


Tn402: a new transposable element determining trimethoprim resistance that inserts in bacteriophage lambda. We have found that the trimethoprim resistance determinant of the IncP plasmid R751 (Jacob et al., 1977; Jobanputra and Datta, 1974) transposes to bacteriophage lambda. We call this transposable element Tn402.


Action of a major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. Protein II, a major outer cell envelope membrane protein, was found together with lipopolysaccharide to stoichiometrically inhibit conjugation in Escherichia coli K12.


I-protein a new regulatory protein from vertebrate skeletal muscle. II. Localization. Rabbit antiserum raised agains I-protein was used for immunofluorescent staining of chicken myofibrils. The FITC-conjugated anti-I-protein antibody stained A-band regions except at their middle regions. According to the conditions used, the myofibrils stained by their fluorescent antibody showed slightly different patterns, i.e., the nonstained regions in the center of the A-bands were wider. On fixing with glutaraldehyde, myofibrils were stained in the A-band regions except at their middle regions. Therefore, I-protein may be localized at A-bands except for the center.


Effects of polyamines on the activities of Escherichia coli ribonuclease I and II. The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by E. coli ribonuclease I [ribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.23] and ribonuclease II [EC 3.1.4.1] have been studied. The degradation of poly(C) by RNase II was stimulated by spermine and spermidine, while that of poly(A) by RNase II was not affected by polyamines. Under our standard experimental conditions, the breakdown of poly(U) by RNase II was inhibited slightly by polyamines. The stimulatory effect of spermine and spermidine on the breakdown of poly(C) occurred in the absence of monovalent cations but not in the absence of divalent cations. When polyamines were used as a stimulant of RNase II, the ratio of poly(C) degradation to poly(U) degradation was greater in the presence of inhibitors such as poly(G) than in their absence. Although the breakdown of all synthetic polynucleotides by RNase I was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)greater than poly(A)(see text)poly(U). However, the difference in degree of stimulation among polynucleotides decreased as monovalent cation concentration was increased.


Further studies on the reactions of phenylglyoxal and related reagents with proteins. 1. The reactivities of phenylglyoxal (PGO), glyoxal (GO), and/or methylglyoxal (MGO) with several proteins, including ribonuclease A [EC 3.1.4.22] and its derivatives, alpha-chymotrypsin [EC 3.4.21.1], trypsin [EC 3.4.21.4], lysozyme [EC 3.2.1.17], pepsin [EC 3.4.23.1], rennin [EC 3.4.23.4], thermolysin, and insulin and its B chain, have been examined. From analyses of the reaction products, PGO was shown to be the most specific for arginine residues. GO and MGO also reacted rapidly with arginine residues, but they also reacted with lysine residues to a significant extent. A side reaction with N-terminal alpha-amino groups was observed with each of these reagents. 2. Two arginine residues out of four in ribonuclease A, two out of three in alpha-chymotrypsin, one out of two in trypsin, one out of two in pepsin, and one out of five in rennin appeared to react with PGO fairly rapidly, indicating a difference in the relative accessibility of these residues by the reagent. Extensive modification of the arginine residues by PGO occurred with RCM-derivatives of ribonuclease A and insulin B chain. The N-terminal isoleucine residues of alpha-chymotrypsin and trypsin appeared to be unreactive with PGO because of salt bridge formation with an aspartyl residue. The activity of alpha-chymotrypsin toward N-benzoyl-L-tyrosine ethyl ester and the lytic activity of lysozyme were lost rapidly on treatment with PGO, as in the case of ribonuclease A. Pepsin and rennin were only partially inactivated by reaction with PGO.


[Arrest of dental development in the osteopetrotic rat and the healing effect of normal bone marrow]. The development of the osteopetrotic "op" rat's mandible was examined from birth to 4 months of age. Before birth the growth of incisors is disturbed and after birth the molar growth stops. Bone resorption is severely reduced and enamel formation ceases soon on the inferior aspect of the incisor, due to degeneration of ameloblasts and lack of vascularisation. A single injection or normal bone marrow cured the disorder at the long bone level within 2-3 months and had a beneficial effect in re-establishing the growth of dental tissue, yet without leading to tooth eruption.


The ovalbumin gene. Insertion of ovalbumin gene sequences in chimeric bacterial plasmids. Double-stranded ovalbumin DNA was amplified and purified by the cloning of bacterial transformants. The double-stranded DNA was synthesized from a complete complementary DNA transcript of ovalbumin mRNA using Escherichia coli DNA polymerase I and the self-priming ability of the initial transcript. After S. nuclease treatment, poly(dA) was added to the 3' termini with terminal deoxynucleotidyltransferase and the ovalbumin gene was hybridized to a linear plasmid DNA, pMB9, containing 3'-poly(dT) termini. This hybrid molecule was used to transform the E. coli strain X1849. The cloned transformants contained from 30 to 53% of the complete ovalbumin DNA as determined by hybridization with full length cDNA. The length of the inserts was confirmed by treatment of the isolated plasmids with the restriction enzyme Hha I. Separation of the fragments by agarose gel electrophoresis showed that the amount of inserted DNA in clones tested varied from 680 to 1090 base pairs.


Effect of CrATP on the association of the reacting forms of yeast hexokinase. Reacting enzyme sedimentation studies have been performed with yeast hexokinase isozymes A and B in the presence and absence of chromium ATP at pH 6.75. Preincubation of either isozyme with CrATP causes a shift in the monomer-dimer equilibrium toward the monomeric form. The results are consistent with the observed increase in inhibition caused by CrATP (Danenberg, K.D., and Cleland, W.W. (1975) Biochemistry 14, 28-39) being due to a conformational change in the protein which causes a decrease in the association constant for the monomer.


Monovalent cation activation of tryptophanase. The interaction of monovalent cations with holotryptophanase has been examined by spectral and kinetic methods. Using S-orthonitrophenyl-L-cysteine as a substrate, activation by the following monovalent cations was demonstrated; values of KA (mM, in italics) and Vmax (mumol min-1 mg) aare given in parentheses: Li+ (54 +/- 11.6, 4.3 +/- 0.28), Na+ (40 +/- 0.03, 18) K+ (1.44 +/- 0.06, 41.1 +/- 3.5), Tl+ (0.95 +/- 0.1, 39 +/- 4.4), NH4+ (0.23 +/- 0.01, 57.9 +/- 2.6), Rb+ (3.5 +/- 0.3, 33.5 +/- 1.8), Cs+ (14.6 +/- 2.6, 21 +/- 2.3). It was demonstrated by circular dichroic spectra that the competitive inhibitor, ethionine, interacts with the holoenzyme in the absence of activating monovalent cations, although it does not undergo labilization of the alpha proton. On addition of monovalent cation to the holoenzyme-ethionine complex, a marked increase occurs in absorption of 508 nm resulting from labilization of the alpha proton with formation of the quinoid form of the pyridoxal phosphate moiety of the enzyme-substrate complex at the catalytic center (Morino, Y., and Snell, E.E. (1967) J. Biol. Chem; 242, 2800-2809. The extent of formation of this quinoid intermediate was linearly related to the maximum velocity observed with each cation except NH4+, which was anomalously active. When measured at 500 nm, the change in absorption ranged from deltaA = 0.45 mg-1 of tryptophanase for NH4+ to 0.06 mg-1 for Li+. Two moles of thallium (I) were bound per mole of subunit. The data are most consistent with the interaction of monovalent cation at or near the catalytic center in such a way that it either participates directly in the reaction or is required for the critical alignment of one or more functional groups necessary for catalysis.


Interaction of AMP-aminohydrolase with myosin and its subfragments. We have shown that purified rabbit skeletal muscle AMP-aminohydrolase binds to rabbit muscle myosin, heavy meromyosin, and Subfragment 2 but does not bind to light meromyosin nor to Subfragment 1. The dissociation constant for binding to myosin was determined to be 0.14 muM. A new sedimentation boundary, presumably reflecting formation of a complex between AMP-aminohydrolase and heavy meromyosin or Subfragment 2, can be observed using the analytical ultracentrifuge. Binding of AMP-aminohydrolase to myosin, heavy meromyosin, or Subfragment 2 is abolished by phosphate (less than 10 mM), an inhibitor of AMP-aminohydrolase. No other rabbit muscle enzyme tested showed any interaction with myosin under the same conditions and there was no indication of complex formation between AMP-aminohydrolase and phosphofructokinase or phosphocreatine kinase in the analytical ultracentrifuge.


DNA polymerases from bakers' yeast.

DNA polymerases from bakers' yeast. Two DNA polymerases are present in extracts of commercial bakers' yeast and wild type Saccharomyces cerevisiae grown aerobically to late log phase. Yeast DNA polymerase I and yeast DNA polymerase II can be separated by DEAE-cellulose, hydroxylapatite, and denatured DNA-cellulose chromatography from the postmitochondrial supernatants of yeast lysates. The yeast polymerases are both of high molecular weight (greater than 100,000) but are clearly separate species by the lack of immunological cross-reactivity. Analysis of associated enzyme activities and other reaction properties of yeast DNA polymerases provides additional evidence for distinguishing the two species. Enzyme I has no associated nuclease activity but does carry out pyrophosphate exchange and pyrophosphorolysis reactions, and has an associated 3'-exonuclease activity. Enzyme I does not degrade deoxynucleoside triphosphates and cannot utilize a mismatched template. Enzyme II does carry out a template-dependent deoxynucleoside triphosphate degradation reaction and can excise mismatched 3'-nucleotides from suitable template systems. Earlier studies have shown that both Enzyme I and Enzyme II are inhibited by N-ethylmaleimide. The yeast enzymes are not identical to any known eukaryotic or prokaryotic DNA polymerases. In general, Enzyme I appears to be most similar to eukaryotic DNA polymerase alpha and Ezyme II exhibits properties of prokaryotic DNA polymerases II and III.


Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates. Studies of manganese complexes by NMR relaxation rates of water protons. The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The binding of Mn(II) to the enzyme was investigated by EPR. The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8. The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP. PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not at all to (E-P)-Mn. Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement. No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement. However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA. An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively. The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon. Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.


Structure of peptide from active site region of Escherichia coli L-asparaginase. The L-asparagine analogue 5-diazo-4-oxo-L-[5-14C]norvaline binds irreversibly to the active site of Escherichia coli L-asparaginase. Conditions for optimal labeling in buffers containing 50% dimethylsulfoxide have been developed and kinetic parameters of the inactivation have been determined. After reduction, alkylation and subsequent degradation of the modified enzyme with alpha-chymotrypsin, the principal radioactive decapeptide of sequence Val-Gly-Ala-Met-Arg-Pro-Ser-Thr-Ser-Met was isolated. A second radioactive hexapeptide Arg-Pro-Ser-Thr-Ser-Met resulting from chymotryptic digestion of the decapeptide was also isolated. Evidence is presented for the attachment of the 5-diazo-4-oxo-L-norvaline residue to serine-9 in the decapeptide via an acid-labile linkage.


The elongation factor Tu coded by the tufA gene of Escherichia coli K-12 is almost identical to that coded by the tufB gene. Radioactive elongation factor Tu coded by either the tufA or the tufB gene of Escherichia coli K-12 was isolated from cells incubated with a mixture of radioactive amino acids after infection with the defective lambda phage particles that carry either of these genes. Two-dimensional chromatographic analyses of tryptic digests of the tufB gene product revealed about 50 radioactive spots. These same spots plus an additional one were also found in tryptic digests of the tufA gene product. Furthermore, these peptide maps are qualitatively the same as those of the elongation factor Tu obtained from two separate isolates of uninfected E. coli K-12 or from rel+ and relA strains of E. coli B. Because the number of spots recovered is consistent with the number of trypsin-sensitive sites, these analyses indicate that the tufA and tufB genes have not significantly diverged from each other.


Studies on Limulus amoebocyte lysate. Isolation of pro-clotting enzyme. A pro-clotting enzyme capable of causing the gelation of clottable proteins in Limulus polyphemus (horseshoe crab) has been purified to apparent homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The activation of the pro-clotting enzyme depended on the presence of both Ca+ and endotoxin. It contained gamma-carboxyglutamic acids and gave a single NH2-terminal lysine. The enzyme was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and soy bean trypsin inhibitor, indicating that it is a serine protease. The molecular weight of the proclotting enzyme was determined to be at least 150,000 by sodium dodecyl sulfate-gel electrophoresis under reducing and denaturing conditions. The protein appears to consist of a single peptide chain, since exposure of the reduced and carboxymethylated enzyme to 6 M guanidine hydrochloride failed to dissociate it into any subunits.


Subunit structure of biodegradative threonine deaminase. The molecule weight of the biodegradative threonine deaminase from Escherichia coli was determined to be approximately 147,000 by sedimentation equilibrium ultracentrifugation. Similar experiments using 5 M guanidinium chloride gave a value of 39,000 for the molecular weight of the enzyme subunit. On sodium dodecyl sulfate-gel electrophoresis the enzyme also dissociated into a single subunit with an estimated molecular weight of 38,000. The NH2 terminus of the enzyme was determined to be methionine by the dinitrophenylation procedure. Quantitative analysis revealed that 3.6 mol of methionine were detected per 147,000 g of enzyme. The selective tritium labeling method established alanine as the COOH-terminal residue. The sequence of residues at the NH2 terminus, determined using an automated sequence analyzer, was: (formula: see text). The fact that a single amino acid was released at each degradation step in the above experiment strongly suggests that the subunits in the enzyme contain the same amino acid sequence. Therefore, the native enzyme with a molecular weight of 147,000 appears to be composed of four identical polypeptide subunits.


Cloning and amplification of rabbit alpha- and beta-globin gene sequences into Escherichia coli plasmids. cDNA, synthesized from rabbit globin mRNA, was used in a self-priming reaction, with avian myeloblastosis virus DNA polymerase, for the synthesis of double-stranded DNA. Globin DNA ranging from about 400 to 650 base pairs was elongated with dG tails using deoxypolynucleotide transferase and was annealed to linear Escherichia coli plasmic pCR1, elongated with dC tails. Preparation of the plasmid DNA involved an enzymatic reconstruction of one EcoRI-specific site on each side of the molecule. After transformation of E. coli cells to kanamycin resistance with the hybrid molecules, bacterial clones harboring recombinant plasmids were studied for the presence of globin-specific DNA. Plasmids containing either alpha or beta rabbit globin gene sequences were obtained. There was a 4-fold excess of recombinant plasmids containing beta-globin sequences over those with alpha-globin DNA. The longest beta-globin sequences found in plasmids were about 550 to 600 pairs long, and correspond therefore to the entire beta-globin structural gene and to some of the untranslated regions. The alpha-globin sequences were 400 to 450 base pairs long. Treatment of clone pCR1betarG 19 with EcoRI endonuclease released two DNA fragments (410 and 210 base pairs) resulting from cleavage at two reconstructed external EcoRI sites and at one internal EcoRI site within the rabbit globin gene. The same treatment of pCR1alpharG 11 released one fragment. In most other recombinant plasmids studied however, no fragment was released by EcoRI digestion.


Automated sequencing of insoluble peptides using detergent. Bacteriophage fl coat protein. Peptides which are highly nonpolar and insoluble under moderate conditions of pH and ionic strength cannot be subjected to automated sequence analysis. We report a method for solubilization of one such peptide, bacteriophage fl coat protein, by chemical modification in the presence of sodium dodecyl sulfate. Following this treatment the 50-residue peptide was degraded stepwise in an automated sequenator using a single cleavage Quadrol program with high repetitive yield through residue 47. We also report a modified program using detergent incorporated into dimethylallylamine buffer which permitted sequencing with high repetitive yields for at least the first 18 residues of the unmodified and otherwise highly insoluble coat protein. The presence of detergent caused no observable difficulties in detection of residues by gas chromatography, thin layer chromatography, or amino acid analysis.


Isolation of Escherichia coli precursor tRNAs containing modified nucleoside Q. Affinity chromatography based on the complex formation of the modified nucleoside Q with boronic acid has been applied to the isolation of specific tRNA precursors containing this modified nucleoside. When [32P]RNA isolated from an Escherichia coli strain containing a thermolabile ribonuclease P was chromatographed on dihydroxyboryl-substituted cellulose, the precursors for asparagine, aspartate, histidine, and tyrosine tRNA were specifically retained. All precursors were monomeric. The nucleotide sequences of four asparagine tRNA precursors were determined.


A new form of structural lipoprotein of outer membrane of Escherichia coli. Among the membrane proteins synthesized in toluene-treated cells of Escherichia coli were two distinct membrane proteins of different molecular weights, which were cross-reactive with antiserum against a structural lipoprotein of the outer membrane. One was thought to be the known membrane lipoprotein since it migrated to the same position as that of the lipoprotein (Mr = 7,200) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, the other protein migrated slower than the lipoprotein. No protein corresponding to the slower-migrating species was detected in the membrane proteins synthesized in vivo. The apparent molecular weight of the protein at the new peak was estimated to be between 10,000 and 15,000. Both the new protein and the lipoprotein were found to be synthesized from stable mRNA(s) in the toluene-treated cells. The synthesis of the new protein as well as the lipoprotein was sensitive to chloramphenicol, indicating that both proteins were synthesized on ribosomes. Peptides mapping of the new protein revealed the same COOH-terminal sequence as in the lipoprotein. This indicates that the new protein has an extra sequence at the NH2-terminal end. This hypothesis is supported by the finding that the NH2 terminus of the new lipoprotein is methionine, while that of the lipoprotein is a substituted cysteine. From double label experiments with each of 17 different amino acids and arginine, the amino acid composition of the extra region was deduced. The new protein was found to contain at least 18 to 19 extra amino acid residues over the lipoprotein, if it is assumed that the new protein has no extra arginine residues. It was found that 4 out of the 5 amino acids which were deficient in the lipoprotein (phenylalanine, tryptophan, proline, and histidine) were also deficient in the new protein, but the fifth one, glycine, was present in the new protein. From these results, it seems possible that this new form of the lipoprotine is a precursor of the lipoprotein (prolipoprotein) in the process of biosynthesis and assembly of the lipoprotein in the outer membrane.


Effect of estrogen on gene expression in the chick oviduct. Mercurated UTP was used as a substrate for RNA polymerases in the in vitro transcription of chromatin so that newly synthesized RNA could be efficiently separated from endogenous chromatin RNA by means of sulfhydryl-Sepharose affinity chromatography. Utilizing this technique, it was possible to examine the effect of varying enzyme to DNA ratios on the transcription of specific genes from chromatin. For both Escherichia coli RNA polymerase and wheat germ RNA polymerase II, lowering the enzyme to DNA ration resulted in an increase in the percentage of ovalbumin mRNA sequences transcribed from chick oviduct chromatin. Similar results were also obtained for the transcription of the globin gene from chick reticulocyte chromatin. On the other hand, transcription of the globin gene from oviduct chromatin or the ovalbumin gene from reticulocyte chromatin or deproteinized chick DNA was not significantly affected by varying enzyme to DNA ratios. These results indicate that preferential transcription of certain chromatin genes relative to total RNA synthesis can occur and that this process is dependent on the presence of chromosomal proteins. Utilizing a cDNA probe complementary to the anticoding strand of the ovalbumin gene, the degree of asymmetry of the in vitro transcription of this gene was also examined. The percentage of ovalbumin RNA sequences homologous to the anticoding strand was not significantly affected when the enzyme to DNA ratio was lowered 16-fold. Since the percentage of coding ovalbumin mRNA sequences increased more than 6-fold over the same range, the percentage of asymmetric transcription of this gene increased. At the lowest enzyme to DNA ratio tested, the transcription of the ovalbumin gene from oviduct chromatin was almost totally asymmetric and, thus, closely resembled the pattern of gene transcription characteristic of the in vivo state.


Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process. Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.


Non-union of carpal scaphoid fractures in children. Fracture of the carpal scaphoid is uncommon in children, but does occur and may fail to unite. Eight patients with established non-union have been reiewed, with an average follow-up of almost four years. All non-unions were grafted with autogenous bone. Excellent clinical and radiological results have been obtained. It is concluded that non-union in children is best managed by bone grafting through the anterior approach. Possible aetiological factors concerned in non-union of scaphoid fractures in this age group are discussed.


[65 orthotopic transplantations of the liver in the pig. Clinical and laboratory results (author's transl)]. The authors report the clinical and laboratory results of 65 orthotopic allotransplantations of the liver in the pig. The operative mortality (8 cases) was nil in the last 23 transplantations. Vascular filling without blood transfusion, blood alkalinisation and precautions to avoid hypothermia, seem essential to reduce early mortality. The various causes of secondary mortality are studied, using certain preventive measures. Thus, deaths due to gallbladder or hepatic ischemia have become rare by conserving end-to-end anastomosis on the hepatic artery and taking certain precautions. The grafts were rejected only in incompatible pigs in the SLA system, but were not rare in this group (13/43) and sufficient to cause death in one case out of two. Gastric ulcers were frequent, even after vagotomy, but vagotomy protects fairly well against ulcer hemorrhage. However, ulcers almost always accompany a disease which is alone sufficient to cause death. Cholangitis appears less frequent after cholecysto-jejunal anastomosis on an excluded omega loop. The mortality from extra-hepatic causes was severe (12 cases), in particular due to mechanical complications at the level of the small intestine. Laboratory analyses showed a definite rise in SGOT transaminase levels and, above all, alkaline prosphatase levels in cases of rejection compared with cases of biliary obstruction or hepatic necrosis.


[Continuous and interrupted intestinal sutures. Experimental and clinical study (author's transl)]. The authors report a clinical study of 101 cases of continuous digestive suture and an experiment comparing intestinal healing after continuous and interrupted sutures. A certain discrepancy exists between the excellent clinical results and the pathological and microscopic findings with continuous sutures, and in particular the presence of pseudo-diverticular pouches in 20% of cases developing at the expense of a mucosal evagination within the suture. If carefully performed, continuous suture seems excellent, preventing by its watertightness, the onset of early fistula.


Characteristics of antibody produced during chronic treatment with LHRH. In a patient with hypogonadotropic hypogonadism treated with luteinizing hormone releasing hormone (LHRH), secondary failure of both subjective and hormone responses occurred at the time of appearance of binding of 125I-LHRH by the patient's serum. On electrophoresis of the patient's serum with 125I-LHRH, label was found only in the gamma globulin region. 125I-LHRH added to the patient's serum was precipitated by sheep anti-human immunoglobulin G (anti-IgG) but not by sheep anti-human immunoglobulin M (anti-IgM). Competitive displacement of 125I-LHRH by unlabeled LHRH was demonstrated while TSH releasing hormone (TRH), somatostatin and rat pituitary hormones showed no displacement when tested at concentrations 5 X 10(6) greater than that of LHRH. Studies using 14 different analogs of LHRH revealed that those with changes at the carboxy terminus showed binding similar to LHRH. It is concluded that IgG antibody to LHRH was produced in this patient by repeated administration of synthetic LHRH. It is further concluded that antibody specificity is directed toward the N terminus region.


Serotyping of Serratia marcescens: evaluation of Le Minor's H-immobilization test and description of three new flagellar H antigens. The H-immobilization test of Le Minor for determining flagellar (H) antigens was evaluated and compared with tube and slide H-agglutination tests. The test proved specific and easy to perform, and titration end points were clearly discernible. The degree of serological cross-reactivity between H antigen reference strains of Serratia marcescens was low. Consequently, this test was adopted for routine serological analysis of H antigens, using unabsorbed rabbit immune anti-H sera. As a result of using this procedure, three new provisional H antignes, designated H14, H15, and H16, are proposed.


Effect of storage conditions of the performance of bismuth sulfite agar. Refrigerated storage of bismuth sulfite agar plates for up to 4 days did not adversely affect growth and colonial characteristics of selected Salmonella strains. Incubation of inoculated plates for 48 h favored the development of more salmonellae with typical morphology. Inoculated plates of freshly poured medium incubated for 48 h gave recoveries similar to those on refrigerated plates and showed a high selectivity against Citrobacter freundii and Proteus vulgaris, organisms which mimic the colonial characteristics of Salmonella on this medium. The use of bismuth sulfite plates stored at room temperature for more than 2 days should be avoided.


Rapid speciation of Haemophilus with the porphyrin production test versus the satellite test for X. The detection of porphyrins produced from omega-aminolevulinic acid was evaluated for use in speciation of the Haemophilus. Two hundred and forty-eight clinical isolates of nonhemolytic Haemophilus were tested concurrently for porphyrin production and for X and V requirements. The porphyrin production test was more rapid (4 h) and more accurate than the satellite test.


Fever of unknown origin: attempts to isolate L-forms and other aberrant bacterial forms. An investigation was conducted with 65 selected febrile patients, 33 of whom fulfilled in all respects the classic criteria of "fever of unknown origin." Clinical evaluation included extensive radiological and immunological testing. Multiple blood cultures were examined by conventional methods in control studies. In addition, venous blood was cultured in a variety of hyperosmolar media using the special techniques used to detect L-forms and other cell wall-defective bacterial forms. By the extensive methods used, no bacterial forms were isolated. The use of media containing osmotic stabilizers did not detect L-forms or other aberrant bacterial forms, nor did it contribute to the determination of the etiology of fever of unknown origin in these patients.


New culture medium for the presumptive identificaion of Candida albicans and Cryptococcus neoformans. A new medium composed of Tween 80, oxgall, caffeic acid, and Davis agar (TOC) that provides for the rapid presumptive identification of Candida albicans and Cryptococcus neoformans is described herein. C. albicans is differentiated from other yeasts by the sequential production of germ tubes and chlamydospores. In a comparison with cormeal agar control plates, there was an increase of chlamydospore-forming strains of C. albicans (97.1% versus 87.2%) and a decrease in the time required for chlamydospore formation (24 h versus 48 h). C. neoformans produced a brown pigment of TOC, which is specific for its identification, thus differentiating it from the other yeasts. A comparison of 24-h pigment production by C. neoformans on TOC with that of birdseed agar showed a dark, coffee brown color in the former cultures and a light brown color in the latter. The change in pigmentation of C. neoformans, as well as morphological changes in C. albicans, can be induced within 3 to 12 h and in not more than 24 h on the TOC medium.


Rapid fluorescent-antibody method with bromelase for identification of group A streptococci. This is a preliminary report on a rapid fluorescent-antibody staining method utilizing the enzyme Bromelase that effectively eliminates nonspecific staining of staphylococci. By this simple method, group A streptococci were accurately identified from 2- to 4-h broth cultures.


A subpopulation of normal human peripheral B lymphcytes that bind IgE. Isolated normal human peripheral lymphocytes were analyzed for their ability to bind IgE as shown by rosette formation with aldehydefixed ox red cells coated with an IgE myeloma protein (Eo'-IgE) as indicator cells. An average of 4.3% of the cells in the lymphocyte preparations of 12 donors formed Eo'-IgE rosettes. The lymphocyte preparations contained an average of 0.36% basophils which also formed Eo'-IgE' rosettes, suggestiing that about 4% of the lymphocytes bound IgE. The rosette formation was inhibited by IgE myeloma protiens or IgE Fc fragments but not by IgE Fab fragments or Ig of the other classes. On the average, the lymphocytes of the 12 donors contained 70.5% cells forming spontaneous rosettes with sheep erythrocytes (E), 10.6% cells having surface immunoglobulin (SIg), and 15.5% bunding IgG as shown by rosette formation with IgG-coated ox red cells (EoA). Fractionation of the lymphocytes into populations rich in spontaneously E-rosetting cells and cells with SIg indicated that the majority of the lymphocytes forming Eo'-IgE rosettes belonged to the SIg-positive lymphocytes. Analyses of lymphocyte populations lacking cells forming EoA rosettes and of rosetting with mixed indicator cells both demonstrated that over 90% of the lymphocytes forming Eo'-IgE rosettes did not form EoA rosettes and apparently had no Fc receptors for IgG. Pretreatment of the lymphocytes with trypsin in amounts that did not alter the number of EoA-rosetting cells abolished the Eo'-IgE rosette formation. These data indicate that  a subpopulation of normal human peripheral lymphocytes, probably belonging to the B-cell type, binds IgE presumably via trypsin-sensitive receptors specific for the Fc fragment of IgE. The surface markers of these lymphocytes resemble those of cultured human lymphoblastoid cells that have recently been shown to bind IgE.


A shorter immunoperoxidase technique for the demonstration of carcinoembryonic antigen and other cell products. A reliable immunoperoxidase schedule for the cellular demonstration of tumour and normal cell products which can be completed in well under two hours is described. The preparations are as clear and readable as those stained by routine histological techniques and should form a valuable adjunct in research and diagnostic studies.


An evaluation of the Modified R/B Enteric Differential System for the identification of the Enterobacteriaceae. Two hundred and twenty-two organisms were identified by conventional methods and by the Modified R/B Enteric Differential System. Two hundred and eighteeen (98-2%) of these organisms were correctly identified by the R/B system. The results of individual tests were also compared. The convenience and general ease of handling of the R/B system are discussed.


Improving the performance of anaerobic bacteriology in a hospital laboratory. A comparison was made of the performance of a newly established anaerobic section of a clinical laboratory and the routine diagnostic section in terms of isolation and identification of anaerobic bacteria. Both sections attempted to isolate obligate anaerobes from the same clinical specimens which were not transported in anaerobic containers. Anaerobic and diagnostic sections isolated anaerobes from 35% and 6% respectively of clinical specimens. The use of antibiograms greatly improved the identification of anaerobic organisms.


Automation of the rapid plasma reagin test using the Autotape System. The paper describes the Autotape System, a new piece of apparatus which is automated slide testing equipment, and its successful application to the rapid plasma reagin test.


Drug resistance in relation to use of silver sulphadiazine cream in a burns unit. Topical chemoprophylaxis of extensive burns with silver sulphadiazine cream led to a large increase in the proportion of sulphadiazine-resistant Gram-negative bacilli in a burns unit. When all sulphonamide treatment in the ward was stopped; the incidence of sulphonamide-resistant strains fell back to levels similar to those recorded when silver sulphadiazine treatment was introduced. This was associated with a large reduction in the incidence of resistance of certain Gram-negative bacilli (especially Klebstella sp) to several antibiotics. Transferable resistance to sulphadiazine, shown by conjugation experiments with Escherichia coli K12, was found in a majority of the strains of Klebsiella sp tested, and in other species. A pattern of transferable resistance to tetracycline, cephaloridine, chloramphenicol, ampicillin, carbenicillin, and sulphadiazine (T Ce Cl A Ca S) was found in four of the 22 strains of Klebsiella tested, and closely related patterns were transferred by five other strains. These patterns of resistance were commonly found in Klebsiella sp isolated from burns in the period before the withdrawal of sulphonamides from the ward but were found in none of the Klebsiella strains isolated in the first six months after that period. Strains of Acinetobacter and Proteus, in which transferable resistance was not found, showed no appreciable fall or rise in sulphadiazine resistance; there was no fall in resistance of these organisms to tetracycline, cephaloridine, chloramphenicol, ampicillin or carbenicillin on withdrawal of sulphonamides from the ward, but there were substantial falls in resistance of Acinetobacter to kanamycin, gentamicin, trimethoprim, and tetracycline which were probably not caused by the withdrawal of sulphonamides.


Screening of blood donors for the detection of antitetanus antibodies suitable for the production of human antitetanus immunoglobulin. During a 12-month period, 40  146 blood donors were tested for the presence of high titre antitetanus antibodies. An attempt has been made to define the most suitable donors, those with antitetanus antibody titres of 4 IU/ml or above, by virtue of titre, sex, location, and length of time since most recent immunisation. Particular note is made of those donors from university sessions. Towards the end of the series, donors who had an initially acceptable level of antitetanus  antibody were being tested a second time.


Zinc and di-iodohydroxyquinoline therapy in acrodermatitis enteropathica. A 25-year-old patient with acrodermatitis enteropathica who had been treated with di-iodohydroxyquinoline for 20 years was changed to zinc sulphate therapy and studied under full metabolic balance control for zinc, calcium, magnesium, and inorganic phosphorus. The results obtained indicate that there is only a small overall deficit of body zinc stores in this disease and that the function of di-iodohydroxyquinoline is to increase the amount of zinc absorbed and retained by the body.


Radiographs in periodontics.

Radiographs in periodontics. Intraoral radiographs are widely used in periodontal diagnosis and research. However, accurate radiographic interpretation is only possible with high quality images. Some of the technical and geometric variables to consider have been presented. Early periodontal lesions are not detected in radiographs. The amount of periodontal destruction in more advanced disease is generally underestimated. The accurate topography of periodontal pockets and the buccal and lingual aspects of the teeth cannot be visualized. Clinical probing is therefore a prerequisite for a complete periodontal diagnosis. However, radiographs are a valuable adjunct for the periodontal diagnosis and the diagnosis of trauma from occlusion. With standardized systems, radiographs may furnish additional quantitative data in clinical research.


Influence of "time of day", pocket depth and scaling on gingival fluid flow. Gingival fluid was sampled from the orifices of the gingival crevices in five male subjects with clinically healthy gingiva. The sampling was performed at 0915 h, 1215 h and 1515 h of the same day. Variation in "time of day" did not seem to influence the rate of gingival fluid flow. A total of 144 pockets (2-11 mm) in 19 patients were sampled for gingival fluid at the orifices. The adjacent gingiva was evaluated accordingly to the Gingival Index (GI). Gingival fluid flow was much more influenced by the degree of gingival inflammation than by pocket depth. Scaling with curettes was performed in five patients. Gingival fluid was sampled at the orifices immediately before scaling and on days 1, 14 and 28 after scaling. The flow rate decreased to a minimum on day 14 after scaling for all pocket depths and remained at almost the same level until day 28. Pockets less than 5 mm deep were more frequently without measurable amounts of fluid than pockets deeper than 5 mm on days 14 and 28.


The influence of experimental gingivitis on plaque formation. The rate of plaque growth was assessed using daily Plaque Index measurements over a 7-day period in eleven dental students with an initial mean Gingival Indes of 1.21 (+/- 0.05). The mean Gingival Index was then reduced to 0.27 (+/- 0.04) and the rate of plaque growth assessed in the same way. Bacterial smears were taken from the teeth at intervals during both experimental periods and percentage counts of various morphological types of bacteria made. The rate of plaque growth was found to be significantly greater when the Gingival Index was high compared with when the Gingival Index was low (P less than 0.001). There was also an earlier development of a complex bacterial flora when the Gingival Index was high. The hypothesis is presented that gingival exudate has no inhibitory action on the formation of dental plaque, but contains substances which enhance bacterial aggregation and the colonization of the tooth surfaces by these aggregates.


Some antibacterial properties of periodontal dressings. Three commercial periodontal dressings were tested for antibacterial activity against salivary microorganisms after having been stored dry or in liquid for 1 and 2 days. Inhibition of growth of mixed salivary flora was evaluated on blood agar dishes. Also, the ability of Strep. mutans to colonize the surface of the dressings was tested. The surface contact test indicated antibacterial activity against salivary microorganisms by freshly made Coe-pak and Wondrpak. Some activity could also be observed after 2 days' storage in about half the experiments. The initial bacteriostatic effect of Peripac was limited to the setting phase. The plaque accumulation test revealed single cells only on the surfaces of Coe-pak and Wondrpak whereas Peripac seemed to be colonized by the test bacteria.


Application of nonparametric procedure for bioassay data to the evaluation of analgesics in man. Parametric tests for bioassay data are commonly applied to scores of pain intensity and relief for the assessment of potency ratios of analgesic drugs. It has been demonstrated, however, that scores derived from semiquantitative scales often deviate from normal distribution. In addition, when scores decrease as a consequence of analgesic treatment, the variances may be nonhomogenous. Both parametric and nonparametric procedures have been employed in this study for the evaluation of results of a double-blind multicenter trial of the analgesic effect of indoprofen and ASA (both drugs at three dose levels) and placebo in episiotomy pain. There was a good agreement between potency ratios obtained with the two assays. Peak PID appeared a less efficient means of estimating potency ratio than other measurements such as SPID and TOTPAR. The nonparametric test for quantitative bioassay appears to be a valid statistical procedure for evaluating results of clinical trials, and it does not imply any assumptions as to the type of distribution of the data.


Comparative effectiveness of nine hypnotic drugs: sleep laboratory studies. The effectiveness of nine hypnotic drugs was compared using a standard protocol in separate sleep laboratory drug evaluation studies. All of these drugs were relatively effective in improving sleep with initial and short-term use. However, with intermediate-term use (two weeks), most of the drugs showed a marked decrease in their effectiveness. Following withdrawal, sleep was similar to baseline with most of the drugs, continued to be improved with flurazepam (Dalmane), 30 mg, and worsened beyond baseline levels with triazolam (U33030), 0.5 mg. The determination of a drug's effectiveness with continued use is most important clinically in enabling the physician to rationally and effectively use hypnotic drugs in the adjunctive treatment of insomnia.


Pregastric esterase and other oral lipases--a review. The secretion of pregastric esterase and other oral lipases has been detected in 13 species. Research on secretion by the human, calf, kid goat, lamb, and rat of pregastric esterase has been significant. Secretion by calves is little affected by age or diet but is greater when calves are nipple fed than when pail fed. Whole milk sham-fed to calves exhibits immediate, sharp decreases in pH and rennet coagulation time resulting from liberation of free fatty acids by pregastric esterase. Bacterial counts in sham-fed products are higher than in control (nonfed) products, but during subsequent incubation bacterial numbers increase less rapidly in sham-fed products. Calf pregastric esterase is a major fat digestive enzyme in young calves but gradually becomes subsidiary to pancreatic lipase as secretion of the latter develops with age. Calf, kid goat, and lamb pregastric esterase exhibits optimum activity on milk fat but is capable of splitting other dietary fats. Data on oral and "gastric" lipases in calves, humans, and rats suggests that gastric lipase is oral lipase. Data on pH and temperature optima as well as activation and inhibition of oral lipases is contradictory but appears to vary considerably between species. Calf pregastric esterase exhibits a unique specificity for fatty acids 4:0 to 10:0 and preferentially hydrolyzes the primary ester position of glycerin. Preparations of calf, kid goat, and lamb pregastric esterase are used commercially to impar typical flavors to Italian-type and Feta cheeses and to accelerate flavor development in other cheeses and cheese-like products. Butterfat modified by pregastric esterase is utilized to impart dairy flavor character to a wide range of processed foods. Treatment with pregastric esterase of calf scours and human malabsorption of syndrome also has been reported.


The effects of a modeling film on social isolation in preschool children: a methodological investigation. Children in Headstart program (N=113) were assessed on two occasions using a naturalistic observation system and peer sociometric measures. Thirty-two children were selected by O'Connor's (1969, 1972) convergent criteria of teacher rankings and being below 15% peer interaction frequency. Half of the children saw an experimental modeling film designed to teach children how to initiate entry into peer groups and half of the children saw a control film. The present investigation included methodological elements left uncontrolled in previous investigations. Results indicated no significant multivariate F ratios for treatment main effects, sociometric main effects, or treatment by sociometric interactions. This failure to replicate previous results with the O'Connor film raises serious methodological criticisms of previous work with socially withdrawn children.


Comparative study of selected alginate materials and devices. Four different brands of alginate impression materials were mixed by hand and ten impressions of each were made of a block for detail reproductions. In addition, two different mechanical devices were used to prepare one of these alginate impression materials. Thus a total of six groups was tested, with ten impressions per group. Stone casts of these 60 impressions were compared by the line study method and the number of surface defects. A statistical analysis was performed on the collected data. The results indicated that the Whip-Mix vacuum mechanical spatulator produced significantly superior results to the Columbus system centrifugal mechanical spatulator, and that among the four alginates hand mixed, the Jeltrate impression material gave better results.


Dose-response effects of albuterol aerosol compared with isoproterenol and placebo aerosols: response to albuterol, isoproterenol, and placebo aerosols. Albuterol aerosol was an effective bronchodilator as reflected by indices of pulmonary function obtained from spirometry and flow volume curves; Compared with isoproterenol, there were minimal side effects even at the highest doses, and bronchodilation lasted significantly longer. In addition albuterol was successfully used by a patient with idiopathic hypertrophic subaortic stenosis who was unable to tolerate isoproterenol, as well as by some patients with idiosyncratic responses to isoproterenol; A few patients appeared to derive little benefit from either albuterol or isoproterenol.


A graded dose assessment of the efficacy of beclomethasone dipropionate aerosol for severe chronic asthma. In a 26-wk double-blind controlled study of 34 patients whose asthma had been poorly controlled despite oral steroids, valuable clinical and pulmonary function improvement was derived by adding beclomethasone aerosol to the prednisone regimen. The amount of improvement correlated linearly with beclomethasone dosage over the range 200 to 1,600 microng/day. These patients required relatively high dosage. Success in achieving asymptomatic status was only 26% with the conventional 400 microng/day and 60% at 1,600 microng/day. Oropharyngeal candidiasis was also dose-related but did not prohibit the use of high-dosage beclomethasone. Respiratory infections, physical signs, blood glucose, and electrolytes were unaffected by the drug. A dose-related suppression of cortisol secretion was demonstrated, but about 1/4 of the group had normal plasma cortisol even at 1,600 microng/day plus the oral prednisone. An individualized risk-benefit assessment seems a better basis for choosing an optimal beclomethasone regimen for each patient than adherence to a conventionalized fixed dosage of 400 microng/day. This requires definition of: (1) a specific goal of treatment in the individual patient and the beclomethasone dosage required to achieve it; (2) the adrenocortical functional response of that particular patient to the desired dose of beclomethasone; and (3) the presence and degree of any dose-limiting constraints such as preexisting complications of steroid use.


Nutrient intake and stimulant drugs in hyperactive children. Recent studies have demonstrated suppressed growth of height and weight in children receiving stimulant drugs for hyperactivity. For approximately twelve months, growth data and food records were collected on two subjects receiving different types and dosages of stimulant drugs. The two cases demonstrated that dextroamphetamine levels of 10 mg. or more and methylphenidate levels of 30 mg. or more decreased caloric intake significantly. This decrease may be limiting for long-term growth. Both subjects had a variety of feeding problems due to poor appetite. Careful nutritional evaluation and planning are important to insure optimal energy and nutrient intake in these children receiving stimulant drugs.


Computer-simulated clinical encounters. I. Development, utilization, and evaluation of a program. Positive findings from the two-year pilot study indicated no significant difference in academic learning and clinical performance when students substituted computer-simulated experiences for hospital-based clinical experiences. There was also strong evidence that the time spent by students in fewer hospital-based clinical experiences was more decisively monitored by faculty. Based on these positive findings, the computer-simulated clinical encounters are being continued as part of the established curriculum in the coordinated undergraduate program in dietetics at The Ohio State University.


Computer-simulated clinical encounter. II. Case flow and program content. The completed computer-simulated case study provides the student with a written model of all components and steps involved in providing nutritional care to a patient. The completed study provides the instructor with a written record of the student's knowledge, her/his response to the patient's inquiries, and her/his organization of the clinical encounter and patient counseling. It is through this written record that student and instructor can analyze and strengthen the student's clinical skills.


The climacteric disease.

The climacteric disease. Evidence is provided to show that the account of "The Climacteric Disease" given in 1813 by Sir Henry Halford, Physician to King George III, was his interpretation that the King's illness was due to aging as a disease. This laid foundations upon which are placed the unfavorable attitudes toward Geriatric Medicine today, and which may effect the provision of medical care for the elderly and aged tomorrow.


A memory assessment technique for use in geriatric psychopharmacology: drug efficacy trial with naftidrofuryl. This study was conducted to compare an automated test of paced stimulus material (Sperling's Perceptual Trace, SPT) with several other standard memory scales. Sixty patients with mild senile organic brain syndrome were divided into two groups of 30; one group was treated for 90 days with 300 mg daily of naftidrofuryl (Praxilene), and the other group received placebo. The patients were tested before treatment and at 30, 60 and 90 days after the beginning of treatment. The data indicated that the SPT indirectly assesses short-term memory, is resistant to practice effects, and is drug-sensitive. It is suggested that the SPT might become the test of choice in the indirect assessment of short-term memory in the elderly.


Nocardia asteroides thenar space infection: a case report. Nocardia commonly is thought of as producing a chronic pulmonary or granulomatous skin infection, but this report illustrates that it may begin as an acute inflammatory process. In view of the tenosynovitis which followed exploration of the tendon sheath in the patient presented and which required a second operation, it may be better to aspirate the sheath at a distant site to look for pus. Actinomycetes should be suspected and looked for with appropriate methods in any suppurative lesion, especially one not responding to conventional antibiotics.


Reconstructive surgery of roller injuries of the hand. Eleven patients with roller injuries to the upper extremity are described. Nine were treated initially; two were referred for treatment within 3 weeks after injury. Six patients were injured by kneading machines in bakeries, two by hot dry-cleaning mangles, two by industrial rolling machines, and one by a transmission belt which acted as a roller. The tissue damage depends on (1) the space between the rollers, (2) the speed of the rollers, (3) the hardness of the rollers, (4) the temperature of the rollers, and (5) how violently the patient attempts to withdraw the entrapped part. Lesions may be closed, consisting of a compression of the soft tissues, which may result in skin necrosis, or a tearing and separation of the skin and soft tissues away from the deep fascia (more common on the dorsal surface of the hand), or destruction of skin and deep tissue because of burns. Closed injuries usually respond to conservative care, although decompression sometimes is necessary. Experience gained from treating these 11 patients indicates that the inexperienced surgeon often tries to replace distally based flaps. Such flaps usually die and predispose to deep infection. If the condition of the wound permits, primary skin grafts should be applied; but if the bed is of poor quality, skin grafting can be delayed for several days. If it is necessary to protect exposed deep structures, they should be covered with immediate pedicle flaps. It is better to use skin from an uninjured area for a free skin graft than to use the avulsed skin as a graft. Distant flaps should be used if secondary reconstruction is anticipated. In circumferential skin loss, a combination of pedicle skin and free skin grafts is better than encircling the part with a pedicle flap. The thumb and radial fingers should be preserved, but the ulnar fingers are expendible in severe injuries. By amputating parts of fingers, reconstruction often is simplified.


The germicidal effect of the open air in different parts of The Netherlands. Using the microthread technique the survival of Escherichia coli MRE 162 in open air was measured in different parts of The Netherlands. The presence of bactericidal compounds (open air factor = OAF) could be demonstrated on several days and quantitated in relative units of OAF concentration. In the absence of ozone the OAF concentration was always low. In the presence of ozone the OAF concentration was dependent on wind direction. At the selected microthread exposure sites air from areas with high traffic intensity contributed more to OAF production than air from industrial areas. OAF production is probably related to the nature of hydrocarbons in the air.


Bacteriostasis of Escherichia coli by milk. II. Effect of bicarbonate and transferrin on the activity of infant feeds. Fresh human and bovine milk are bacteriostatic in vitro for only some (milk-sensitive) strains of E. coli. The addition of bicarbonate to the test system potentiates the bacteriostasis so that otherwise milk-resistant strains are inhibited. By titration of the bicarbonate in the milk, it is possible to determine the minimum concentration that will activate milk aganinst a milk resistant strain but be ineffective in boiled milk, i.e. it potentiates a heat-labile system in milk and does not merely exert a direct toxic effect. This concentration is lower for human milk than for cow's milk and can be reduced even further by the addition of more iron-binding protein. Lactoferrin and bicarbonate may be present in the gut of the newborn. In an attempt to imitate conditions in the infant gut, we therefore reinvestigated, in vitro and in the presence of added bicarbonate and transferrin, the bacteriostatic activity against E. coli of fresh breast-milk, commercial bottle-milk, and mixtures of these as fed to infants in the study. The results, and information about events in vivo deduced from the ratio of milk-sensitive to milk-resistant strains of E. coli isolated from babies' stool suggest that neonatal intestinal secretions may contribute to the bacteriostatic activity of their feeds so that (1) in fully breastfed babies all strains of E. coli are inhibited to the same extent; there is no selection on the basis of milk sensitivity and equal numbers of strains resistant and sensitive to milk are found in the stools; (2) in fully bottle-fed babies E. coli is not inhibited since the milk is non-bacteriostatic and again there is no selection; (3) in babies fed at the breast but bottle-milk supplemented, only milk-sensitive strains are inhibited; milk-resistant strains are not, and preferentially colonize the large intestines.


Serological characterization of group-A streptococci associated with skin sepsis in meat handlers. A series of outbreaks of skin sepsis among meat handlers in England during 1974 and 1975 afforded an opportunity to study the group-A streptococci commonly isolated from the lesions. Few of these streptococci could be M typed with existing antisera. Intensive study of strains from two outbreaks of sepsis  in one abattoir in Shropshire led to the recognition of three new provisional M types. The streptococci were first sorted according to T-typing pattern and ability to produce opacity factor. Opacity-factor producing strains with the same T pattern were then screened for inhibition of opacity production by the sera of convalescents from the same outbreak. Finally, M antisera were made in rabbits against representative cultures. Sera against the three new provisional types were used to re-examine streptococci from 20 other outbreaks or incidents of sporadic infection among meat handlers. This increased the proportion of typable strains from 3% to 55%. Two of the new provisional types (nos. 2015 and 1658; both T25/Imp 19, opacity-factor positive) were confined to the Shropshire outbreak, but the third (no. 2681; T14, opacity-factor negative) was found among strains from meat workers in eight other geographically distinct areas. In all, 31% of 131 distinct strains from meat workers, but less than 1% of 2816 strains from other British sources, belonged to provisional type 2681. Thus, in Britain, one M-type of group-A streptococcus appears at present to be almost exclusively associated with sepsis in meat workers.


Bacteriophage-typing designations of Salmonella typhimurium. The phage-typing scheme of Callow (1959) has been extended. The original number of types was 34; this has now risen to 207. Tables are presented which show the provisional type designations and the definitive designations now being introduced.


Properties of tRNA-specific antibodies from NZB/NZW mice. Antibodies that bind tRNA are produced spontaneously in New Zealand Black/New Zealand White (NZB/NZW) F1 hybrid female mice. An assay for the detection of these antibodies has been developed by using gel filtration and radioactive tRNA. This assay was found superior to the widely used ammonium sulfate precipitation assay because of the nature of the interaction between the protein and the tRNA. The ant-bodies bound native tRNA preferentially to tRNA denatured by cross-linking with formaldehyde. This conformational specificity was confirmed in competition experiments. The antibodies to native tRNA had an average association constant of 5 x 10(7) leter/mole at 4 degrees C and could bind to more than one site per tRNA molecule. Experiments with immunoglobulin class-specific anti-mouse antisera, in solution and by radioimmunoelectrophoresis, showed that the antibodies were heterogeneous, but were predominantly of the IgG class. These antibodies may be useful for detection, localization, and conformational analysis of tRNA in solution as well as for understanding the pathogenesis of the lupus-like syndrome in these mice.


A simple one-step hemolytic assay for C2 with C2-deficient human serum. A simple one-step procedure has been developed for the molecular titration of C2 by utilizing the ability of the test material to restore the hemolytic activity of human serum selectively deficient in C2 (C2D serum). In this assay, equal volumes of EA (10(8) cells/ml), C2D serum (1/20), and a suitable dilution of a source of C2 were incubated at 37 degrees C for 60 min and the fraction of cells lysed was used to calculate the effective molecules of C2/ml test material. The assay can be used to titrate C2 in human, guinea pig, rat, mouse and rabbit sera, but not C2 in dog serum. The assay is simple and reproducible, and comparable in sensitivity to the conventional two-step assay with EAC14 cells and Cgp-EDTA.


Immune response to immunization via the anterior chamber of the eye. I. F. lymphocyte-induced immune deviation. The immunizing abilities of alloantigens placed within the anterior chamber of the eye have been studied in inbred rats. Although intracameral inoculation of F1 hybrid lymphocytes into parental strain recipients elicited both cell- and antibody-mediated immunity, a delimited interval was identified postinoculation during which the systemic cell-mediated immune response was suppressed as indicated by prolonged acceptance of orthotopic skin allografts. The prompt appearance of hemagglutinating antibodies in the serum of immunized rats followed a time course which coincided with the suppression of cell-mediated immunity and suggested that the two events are casually related. Since exposure to allogeneic antigens on lymphoid cells via the anterior chamber elicits a transient suppression in cell-mediated immunity, where humoral immunity is preserved, the phenomenon resembles immune deviation.


Mitogen induced cellular cytotoxicity (MICC) in multiple myeloma. Mitogen induced cellular cytotoxicity (MICC) was noted to be markedly increased in patients with multiple myeloma as compared to normal controls and to patients with chronic lymphocytic leukemia (CLL). Enhanced MICC was present at various effector-to-target cell ratios and at several mitogen concentrations. Removal of adherent, phagocytic cells by carbonyl iron, glass wool, or rayon columns abolished the MICC response from the peripheral blood of both multiple myeloma patients and normal controls. Thus, the effector cell mediating MICC may be monocytic in origin and closely resembles the suppressor cell for immunoglobulin synthesis described in patients with multiple myeloma. Our data suggest that the MICC assay with chicken red blood cells as targets may provide a convenient method for identifying pathologic conditions where this cytotoxic effector cell population plays an active role.


Activity of tumor-associated lymphoid cells at short intervals after administration of irradiated syngeneic and allogeneic tumor cells. After a single intraperitoneal injection of irradiated tumor cells, host cells capable of responding against syngeneic tumors were detected in peritoneal exudates of mice. Although irradiation of the injected tumor prevented its overgrowth, it did not significantly alter the antigenicity of the tumor. Immunologic activities of tumor-associated host cells in the peritoneal cavity were continuously monitored, starting 48 hr after tumor administration. In vitro cell-mediated lysis of syngeneic tumors appeared as early as 3 days after irradiated tumor administration. In addition, peritoneal exudate cells from inoculated mice were capable of adoptively transferring immunity. Purification of these peritoneal exudate cells on nylon wool columns yielded a nonadherent Ig-negative lymphocyte fraction whose cytolysis was tumor-specific and T cell-associated. The macrophage-free lymphocyte fraction exhibited a higher in vitro activity against tumors than unpurified peritoneal exudates. This tumor-host system allowed the study of cells which directly interact with the tumor cells in vivo, starting shortly after tumor administration. The results reported in this paper show that tumor-associated lymphoid cells capable of mounting anti-tumor response in vivo and in vitro can be demonstrated as early as 3 days after tumor inoculation.


Human bone marrow lymphocytes. III. Polyclonal activation of B lymphocytes in human bone marrow measured by a direct plaque-forming cell assay. Lymphocytes from the bone marrow and peripheral blood of the same normal individuals were assayed simultaneously for blast transformation as well as polyclonal activation with differentiation to antibody-forming cells after stimulation with pokeweed mitogen. Blastogenic responses were measured by tritiated thymidine incorporation and antibody-forming cell assay. There was no significant difference between the blastogenic responses of lymphocytes in the peripheral blood compared to the bone marrow of the same individuals. However, differentiation to antibody-forming cells measured by the plaque-forming cell response was significantly greater in lymphocytes in the bone marrow as compared to peripheral blood of the same individuals. These studies demonstrate that the lymphocytes in human bone marrow are at a stage of differentiation whereby they can be readily induced to differentiation toward antibody production by polyclonal activation, even more so than peripheral blood lymphocytes. This supports the concept that the bone marrow is a major source of immunoglobulin production in man.


Cyclic antibody formation to polyglycerophosphate in normal and injected rats. Normal adult Sprague-Dawley rats were bled serially over a 30-week period and their sera were examined for antibodies to polyglycerophosphate (PGP) by a standardized passive hemolysis test. Levels of "natural" antibodies to PGP fluctuated during this period with a majority of animals exhibiting pronounced cycling of serum antibody levels, however, the individual cycles were not synchronized with each other. Feeding of radiolabeled Gram-positive bacilli to these animals and examination of lymphoid tissue by liquid scintillation counting revealed that the antigen persisted mainly in the mesenteric lymph nodes. A second group of rats was injected i.v. with radiolabeled Gram-positive bacilli and tissues were examined for plaque-forming cells (PFC) of PGP specificity, and the sera were examined by passive hemolysis. Cycling of both anti-PGP antibodies and PFC became synchronized in the injected animals with peaks of serum antibody evident at 16 and 28 days post-injection and splenic PFC peaks at 4 and 16 days post-injection. Cycling was also observed in the mesenteric lymph nodes and bone marrow Examination of lymphoid tissue from the rats injected i.v. revealed that antigen introduced by this route also perisisted mainly in the mesenteric lymph nodes, This report demonstrates individual cycling of natural responses to environmental antigen and to the same determinant in secondary responses, indicating its importance as a regulatory mechanism.


Immunologic release of heparin from purified rat peritoneal mast cells. High m.w. [35S]heparin, labeled in vivo or in vitro, was released from purified rat mast cells by challenge with rabbit anti-rat F(ab')2, guinea pig anti-rat IgE, or calcium ionophore. The released and the residual heparin were isolated by Dowex 1 chromatography and were of comparable size by Sepharose 4B gel filtration. The majority of the released heparin was found by differential centrifugation to be granule-associated. Net percentage of mast cell heparin release, quantitated by metachromasia after isolation on Dowex 1 chromatography, correlated in a linear fashion with net percentage of histamine release, with heparin exhibiting a threshold requirement for onset of release. The correlation of histamine and high m.w. heparin release provides chemical support for the conclusion of others from ultrastructural studies that mast cell activation by immunologic means or by the calcium ionophore results in secretion of the whole granule.


A microtiter solid phase radioimmunoassay  for detection of the human reovirus-like agent in stools. The development of a microtiter solid-phase radioimmunoassay for detection of the human reovirus-like (RVL) agent is described. The test is simple to perform and uses small amounts of reagents; a large number of specimens can rapidly be tested in a single test. Both filtered and unfiltered stool suspensions can be employed. The test was as sensitive as immune electron microscopy, and with an appropriate blocking test, was specific for the human RVL agent.


Immunity to sporozoite-induced malaria infeciton in mice. I. The effect of immunization of T and B cell-deficient mice. The cellular basis of immunity to sporozoites was investigated by examing the effect of immunization of T and B cell-deficient C57BL/6N X BALB/c AnN F1 (BLCF1) mice compared to immunocompetent controls. Immunization of T cell-deficient (ATX-BM-ATS) BLCF1 mice with x-irradiated sporozoites did not result in the generation of protective immunity. The same immunization protocols protected all immunocompetent controls. In contrast, B cell-deficient (micron-suppressed) BLCF1 mice were protected by immunization in the majority of cases. The absence of detectable serum circumsporozoite precipitins or sporozoite neutralizing activity in the micron-suppressed mice that resisted a sporozoite challenge suggests a minor role for these humoral factors in protection. These data demonstrate a preeminent role for T cells in the induction of protective immunity in BLCF 1 mice against a P. berghei sporozoite infection.


Inhibition of polymorphonuclear leukocyte capping by a chemotactic factor. Incubation of human polymorphonuclear leukocytes with colchicine and fluorescein-concanavalin A leads to the formation of a polarized cap of fluorescence not seen if cells are incubated with fluorescein-Con A along. When cells are preincubated with a chemotactic factor before colchicine treatment, the capping is inhibited in a dose-related manner. Studies with alpha-methylmannoside indicate that the caps represent extracellular fluorescein-Con A and are not areas of Con A internalization. Experiments utilizing an irreversible inhibitor of serine esterases suggest that a chemotactic factor-activated enzyme is involved in the inhibition of cap formation in the human neutrophil.


The effect of cyclophosphamide on the splecific unresponsiveness to skin allografts induced in ALS-treated mice infused with donor bone marrow. Specific unresponsiveness to skin allografts can be induced in ALS-treated mice by the injection of bone marrow from the graft-donor strain. Mice bearing long-term grafts in perfect condition have evidence of cell-mediated immunity against donor antigens and also serum-blocking factors. The effect of cyclophosphamide on graft prolongation was investigated in this model. Cyclophosphamide was given either before or after marrow. Cyclophosphamide given before marrow abrogated the enhancing effect of marrow possibly due to a depletion of antibody-forming cells. Cyclophosphamide given after marrow potentiated the effect of marrow probably due to the destruction of immunocompetent cells responding to the challenge of marrow.


Detection of plaque-forming cells in the peripheral blood of actively immunized humans. Ten adult human volunteers were immunized with Salmonella typhi and their peripheral blood leukocytes were collected for 14 days after immunization. These peripheral blood leukocyted, rich in lymphocytes, were plaqued in a modified Jerne assay against sheep erythrocytes coated with either Salmonella or Escherichia lipopolysaccharide. A specific direct and indirect PFC response developed in immunized individuals by day 7 and peaked at day 10. This vigorous PFC response rapidly declined to normal levels by day 14. This marked and specific PFC response of human peripheral blood leukocytes may be developed as a useful tool for monitoring the humoral immune response of patients with Gram-negative bacterial infections.


Drugs as ligands of immunogenic molecules in parasites: an approach to the isolation of target-antigens. Schistosomicide drugs are used as ligands to isolate the target antigens of schistosome by affinity chromatography. Anti-target antigens immune sera produced in rabbit permit their localization on the parasite using immunofluorescence. Target antigens of some drugs determine a relatively high immunoprotection in rat or mouse schistosomiasis infection.


A solid phase radioimmunoassay for detection of serum autoantibodies in systemic lupus erythematosus. Solid phase radioimmunoassay (RIA) methods for measuring autoantibodies in systemic lupus erythematosus (SLE) patients' serum were developed to improve the sensitivity and quantitative precision of these determinations. Two mechanical systems were studied: (1) acetone fixed cell monolayers in glass tubes and (2) antigen coated plastic beads. Both systems were sensitive and reproducible, giving serum dilution end-points between two and four orders of magnitude (100-10,000 times) greater than those obtained by fluorescence microscopy. The most sensitive, versatile system involves the coating of plastic beads with nuclear antigen(s), incubation overnight with sera and labelling with 125I conjugated antihuman globulin. Linear binding of this radioactive tag is obtained over a wide range of SLE serum dilutions and the slopes of the serum dilution titration curves are almost identical for all SLE patients' sera we have tested. Therefore, a standard titration curve can be constructed from the results with a positive serum, and end-point dilutions of unknown sera estimated from results obtained with a single serum dilution. Alternatively, binding ratios of unknown sera can be usefully compared at fixed dilutions with standard positive and negative sera. For example, high binding ratios (greater than 3.0) were obtained with 19/20 SLE sera and 0/20 control sera. Antigens used in these systems include crude, whole-cell lysates and lysates from purified nuclei. These RIA methods appear to provide certain advantages over existing autoantibody assay methods because they are relatively simple, sensitive, reproducible and potentially capable of measuring a variety of autoantibody specificities.


Improved methods for separation and purification by affinity chromatography. Defined conditions for simplified Affinity Chromatography suitable for instructional and research purposes, were developed. The standard method of activation of polysaccharide gels using BrCN was modified and improved using a highly buffered reaction solution. A batchwise protocol for the immunoadsorbent isolation of anti-BSA was used (with commonly employed dissociation agents) and comparative data were obtained.


Human lymphocyte subpopulations: giant human red blood cell rosettes. Human thymus-derived (T) lymphocytes form rosettes with sheep red blood cells (SRBC) and human red blood cells (HRBC) in vitro. Twenty-four hours after inactivation by the mitogenic enzymes sodium periodate (NaIO4) and neuraminidase plus galactose oxidase (NGAO), rosette-forming cells appeared which could bind greater than or equal to 36 SRBC and greater than or equal to 21 HRBC. These were defined as giant SRBC and giant HRBC rosettes respectively. They were absent in control samples. When lymphocyte responses were assayed by the rates of DNA synthesis, they were almost eliminated by the absence of monocytes. However, the generation of giant SRBC rosettes was unaffected. The generation of giant HRBC rosettes was very significantly depressed. It was concluded that lymphocytes could still be significantly activated in the absence of monocytes. The activated lymphocytes could be recognized by their ability to form giant SRBC rosettes. In the presence of monocytes, the activated lymphocytes have the additional characteristics of being able to form giant HRBC rosettes.


Procedure for the embedment and ultrastructural visualization of cells cultured on plastic microtest plates. The polystyrene Microtest plate has served as an excellent means of quantitation in the interaction of immunological, chemotherapeutic and radiobiological treatments with cultured cells. In order to assess the accompanying ultrastructural changes it was necessary to develop a technique which allowed cells grown in the wells of the plates to be embedded for electron microscopy. Conventional epoxy resin embedding techniques require the use of propylene oxide as a clearing agent. Unfortunately propylene oxide is a solvent of the polystyrene plates. By the substitution of absolute ethanol for propylene oxide, toluidine blue staining during the procedure and other preparative techniques it was feasible to prepare reacted cells grown in Microtest plates for electron microscopy.


Immune lysis of spin label loaded liposomes incorporating cardiolipin; a new sensitive method for detecting anticardiolipin antibodies in syphilis serology. Liposomes prepared from a mixture of the pure lipids cholesterol, lecithin and cardiolipin (molar ratio 50/45/5), are able to bind antibodies directed against Treponema pallidum. When the liposomes are loaded with the water soluble spin label tempocholine chloride, the release of spin label from the liposomes can be monitored directly by observing changes in the paramagnetic resonance (ESR) spectrum from the spin label. The method offers a convenient technique for monitoring the complement-mediated lysis of liposomes, and may be applied in the serological diagnosis of syphilis, and a method for quantitative measurement of complement.


Pronase pretreatment of tissue sections enhances sensitivity of the unlabelled antibody-enzyme (PAP) technique. Treatment of dewaxed sections of formalin-fixed paraffin embedded material with 0.1% pronase for 15 min at 37 degrees C prior to the performance of the unlabelled antibody-enzyme (PAP) procedure greatly enhanced its sensitivity. This was shown by detection of immunoglobulin within plasma cells in chronic gastritis specimens.


Fractionation on lymphocyte surface antigens. I. Rapid method for eliminating labeled lipid from cell surface antigens iodinated by the lactoperoxidase catalysed reaction. Fractionation of lactoperoxidase iodinated cell surface material on miniature DEAE-cellulose columns provided a rapid method for separating labeled lipid from cell surface antigens. The procedure also removed poorly solubilized aggregates yielding a labeled preparation which demonstrated stable, reproducible immunoprecipitation results. Using these fractionated antigens components tentatively designated as human 'T' cell specific antigens have been identified.


The human cutaneous basement membrane--anchoring fibril complex: preparation and ultrastructure. The human cutaneous basement membrane (CBM) has been isolated together with the anchoring fibrils (AF) of the dermis. These structures are removed from the dermis as a single complex, the CBM-AF complex. The epidermis is first removed from the dermis by immersion of whole, defatted skin in cold 2 n NaSCN for 5 to 6 hr. Subsequently, removal of the CBM-AF complex is effected by focusing high-intensity ultrasonic energy against the dermal surface. Purification of the preparation is achieved by low-speed sedimentation. All stages of the process were monitored by electron microscopy, which demonstrated that the morphology of the CBM-AF complex was well preserved and that this complex represents the principal mass component in the system. In addition, our report describes an ultrasound-induced variation in collagen fibril morphology, which we have called 'disordered collagen.' This altered type of fibril lacks periodicity or recognizable banding and has a frayed appearance. Finally, the experiments provide the basis for an analysis of the relative strengths of certain bonds at the dermal--epidermal junction. The strongest of these appears to be the bond between basement membrane and anchoring fibril.


Vaginal colonization with group B streptococcus: a study in college women. Vaginal specimens for culture of group B Streptococcus and anonymous questionnaires were obtained from 499 college women. Group B Streptococcus was isolated from 90 (18.0%) of the participants. A selective broth medium was more sensitive for detection of vaginal isolates (85 of 493; 17.2%) than was direct inoculation of blood agar plates (44 of 466; 9.4%). The most prevalent serotypes among the isolates were type III (37.9%) and type II (25.3%). Logit analysis identified four factors associated with a higher prevalence of vaginal colonization with group B Streptococcus. These organisms were isolated significantly more often from (1) women who had an intrauterine device (50% vs. 18.6%; P less than 0.001), (2) sexually experienced women (20% vs. 7.1%; P less than 0.02), (3) women studied during the first half of the menstrual cycle (26.5% vs. 14.5%; P less than 0.01), and (4) women 20 years of age or younger (21.4% vs. 14.8%; P less than 0.05). The prevalence of colonization with group B Streptococcus was not related to sexual practices, history of venereal disease, use of oral contraceptives, presence of gynecologic symptoms, use of antibiotics, race, educational level, marital status, or history of pregnancy.


Diarrhea due to Escherichia coli in the rabbit: a novel mechanism. A strain of Escherichia coli O15 (RDEC-1) isolated from several rabbits with diarrhea was examined to determine (1) whether the strain could produce diarrhea when administered by the orogastric route to other rabbits and (2) whether this strain was invasive or enterotoxigenic. Strain RDEC-1 produced diarrhea in 48 of 62 rabbits when given by the orogastric route in doses that ranged from 1.5 X 10(2) to 4 X 10(10) bacteria. The organism did not give a positive result in the 18-hr ileal loop or Serény tests and did not invade HeLa cells. Culture supernatants of strain RDEC-1 did not give positive results in the 6- or 18-hr rabbit ileal loop, suckling mouse, Y-1 adrenal cell, or Chinese hamster ovary cell assays. Fluorescent antibody staining of sections of intestine prepared in a cryostat revealed great numbers of E. coli strain RDEC-1 that adhered to the epithelial surface. It is evident that E. coli can produce diarrhea without being able to invade the mucosa or synthesize enterotoxin. Strains of E. coli similar to RDEC-1 may account for some of the E. coli-associated diarrhea that occurs in humans.


Hepatitis B immune globulin in prevention of hepatitis B among hospital staff members. In a double-blind trial of hospital staff members at risk of contracting hepatitis B, 110 subjects received hepatitis B immune globulin or immune serum globulin prophylactically. An additional 125 individuals working in the same locations were unwilling to participate in the trial and received no prophylaxis. During the study period of 28 months, 13 cases of clinical hepatitis B occurred in the untreated group, whereas only three cases occurred among the subjects who received prophylaxis.


Enterotoxigenic Escherichia coli and diarrheal disease in Mexican children. Enterotoxigenic strains of Escherichia coli were isolated from eight (16%) of 50 Mexican children admitted to the hospital with diarrhea and from one of 50 children hospitalized for nonenteric disorders. All of the toxigenic strains tested elaborated a heat-labile enterotoxin, and in seven of nine patients no E. coli capable of concomitant production of heat-stable enterotoxin were found. None of the strains of E. coli with classical enteropathogenic serotypes isolated from nine patients with diarrhea produced either heat-labile or heat-stable enterotoxin. Although the results of this study strongly suggest that enterotoxigenic strains of E. coli are probably responsible for a significant number of cases of diarrhea in an indigenous Mexican pediatric population, further proof will require demonstration of in vivo production of enterotoxin and/or antitoxin.


Bile acids, diarrhea, and antibiotics: data, speculation, and a unifying hypothesis. The primary bile acid, chenodeoxycholic acid, and the secondary bile acid, deoxycholic acid, when present at a concentration of greater than 3 mM, induce salt and water secretion from the human colon and cause a marked increase in the permeability of the human colon to molecules of a molecular weight of 200-500 daltons. Scanning electron microscopy indicates that this action may be associated with tissue damage in some species. In the healthy individual, the primary bile acids, cholic and chenodeoxycholic acid, are dehydroxylated in the colon and are simultaneously precipitated from solution; at pH less than 7, deoxycholic and lithocholic acid are insoluble. In patients with bile acid diarrhea resulting from bile acid malabsorption, dehydroxylation is decreased, and the concentration of bile acids in the colon is markedly elevated. The major secretory bile acid in solution is chenodeoxycholic acid. Administration of cholestyramine, a resin that binds bile acid, reduces the elevated concentration of chenodeoxycholic acid and abolishes the diarrhea. These facts can be used to develop a unifying hypothesis which proposes that elevated concentrations of primary bile acids in the colon play a role in diarrhea and pseudomembranous colitis induced by clindamycin.


Therapy for experimental intraabdominal sepsis: comparison of four cephalosporins with clindamycin plus gentamicin. An experimental model of intraabdominal sepsis in rats was used for evaluation of treatment with four cephalosporins: cephalothin, cefazolin, cefoxitin, and cefamandole. Doses were 115 mg of each of the cephalosporins/kg and, in additional experiments, 350 mg of cephalothin or cefazolin/kg. Response to treatment was evaluated by mortality rate and the incidence of intraabdominal abscess. All four cephalosporins and gentamicin produced a similar reduction in mortality rates. There was a marked variation in incidence of abscess dependent upon the cephalosporin selected and the dosage tested. Regimens most effective in reducing abscesses were clindamycin, low-dose cefoxitin, or high-dose cephalothin.


Therapy for infections due to anaerobic bacteria: an overview. Several classes of antimicrobial agents (e.g., penicillins, cephalosporins, tetracyclines, chloramphenicol, and clindamycin) are useful in treatment of infections due to anaerobic bacteria. However, certain anaerobic bacteria have shown a striking resistance to antimicrobial agents. In vitro susceptibility tests are useful for selection of optimal therapy. The choice of agent depends, to some extent, on the organisms responsible for the infection. Bacteroides fragilis is the most commonly encountered anaerobe, and it is also the most resistant to antimicrobial agents. Other factors influencing the selection of therapy include pharmacologic characteristics, degree of bactericidal activity, and toxicity. Proper therapy for anaerobic infections often requires intensive antimicrobial therapy for a prolonged period. Surgical intervention, including drainage of abscesses and excision of necrotic tissue, is important.