Affinity chromatography of putrescine oxidase from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens. Putrescine oxidase [EC 1.4.3.4], putrescine : oxygen oxidoreductase (deaminating) (flavin-containing), from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens were adsorbed on amine-Sepharose 4B in which one of the terminal amino groups of diamine or triamine was covalently bound to Sepharose 4B leaving the other terminal amino group(s) free. The affinities of these enzymes for the amine-Sepharose 4B increased on increasing the chain length of the methylene groups in the immobilized amines and fell upon addition of the substrate. The affinity of putrescine oxidase modified with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC) was reduced in comparison with that of the native enzyme so far as 1,12-diaminododecane-Sepharose 4B was concerned. From these results, it can be concluded that the interactions between the enzyme and the amine-Sepharose result from specific affinities mediated through the active sites of the enzymes. It is suggested that spermidine dehydrogenase as well as putrescine oxidase has as anionic point and a hydrophobic region in the active site. On the basis of these results, the applicability of the enzyme affinities to purification procedures was examined. When partially purified enzymes were subjected to affinity chromatography, the following results were obtained. Putrescine oxidase gave a purification factor of 40-fold with about 100% recovery on a 1,12-diaminododecane-Sepharose column. In the case of spermidine dehydrogenase, the purification factor and recovery on a 1,8-diaminooctane-Sepharose column were about 1,200-fold and 86%, respectively. By introducing affinity chromatography as a purification step, each enzyme could be purified more simply and with higher recovery.


Substrate binding characteristics of the active site of spermidine dehydrogenase from Serratia marcescens. The substrate specificity of spermidine dehydrogenase from Serratia marcescens was studied using many kinds of naturally occurring and synthetic polyamines. Diamines inhibited the enzyme competitively and their inhibitor constants tended to decrease with increasing methylene chain length in the diamines. All of the triamines and tetramines examined were active as substrates, and the amines containing a 4-aminobutylimino moiety (NH2(CH2)4NH-) in their structures were more active. N-Alkylputrescine was also oxidized by the enzyme. All of the amines containing a 4-aminobutylimino group were oxidized to form 1-pyrroline stoichiometrically as one of the products. Tetramines containing a 3-aminopropylimino group (NH2(CH2)3NH-) were oxidized to form 1,3-diaminopropane. However, in the case of an amine containing both 4-aminobutylimino and 3-aminopropylimino groups, the imino moiety of the former was preferentially oxidized by the enzyme. On the basis of the substrate specificity, the binding characteristics of the enzyme are discussed and a subsite model for the binding site is proposed.


Studies on bacterial chemotaxis. III. Effect of methyl esters on the chemotactic response of Escherichia coli. The methylation-demethylation reaction of methyl-accepting chemotaxis protein (MCP) is tightly coupled to the appearance of the chemotactic response in Escherichia coli. The bacteria might therefore show a unique response upon the addition of a compound containing a methyl group. We selected methyl N-methyl anthranilate (NMMA) and its analogs for examination. When NMMA was added to a suspension of E. coli (wild type), the bacteria tumbled as it does in the presence of a repellent. NMMA caused tumbling of wild-type bacteria for at least 20 min, while a conventional repellent makes the bacteria tumble for at most one min. The effect of NMMA requires functional MCP, cheA gene product, cheB gene product, and possibly cheX gene product. A positive signal of NMMA (i.e. sudden dilution) was detected by cheZ mutants with much higher sensitivity than that of a conventional repellent, indole, while both signals were rather poorly but equally detected by cheB mutants. These results suggest that the drug is related to the function of cheB gene product, a possible demethylating enzyme of MCP.


Chemical modification of lactose repressor protein using N-substituted maleimides. Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.


Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae. A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.


Role of the spoT gene product and manganese ion in the metabolism of guanosine 5'-diphosphate 3'-diphosphate in Escherichia coli. Addition of divalent ion chelating agents picolinic acid, 1,10-phenanthroline, or quinoline-2-carboxylic acid to wild type, relA, or relX, but not spoT strains of Escherichia coli increases the levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Poorly chelating analogs of these agents and a larger and more highly charged chelating agent, ethylene glycol bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid are ineffective. Mn2+ reverses the increase in ppGpp. The increase in ppGpp in wild type cells can be explained by an inhibition of degradation. In spoT cells the response is more complex; ppGpp does not increase although degradation is completely inhibited. The lack of increase in spoT cells suggests a role for spoT in synthesis of ppGpp in addition to its known role in degradation. Growth of both spoT+ and spoT cells is inhibited following chelator addition. This suggests that growth inhibition is through a mechanism not directly involving ppGpp. The results of this study provide evidence in intact cells for a role for Mn2+ and the spoT gene product in ppGpp degradation, and provide further evidence for an involvement of spoT and possibly divalent ions in ppGpp synthesis.


Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter. Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.


Identification of regions of pRZ2 which have delayed elution behavior on RPC-5 column chromatography. DNA restriction fragments generally elute from RPC-5 in order of their size. However, some fragments elute later than predicted. Chromatographic studies were performed on five different restriction digests (Hae III, Hha I, Alu I, Taq I, and Hae III + HindII) of pRZ2 DNA in an effort to localize the regions which have the delayed properties. Also, the magnitude of the delay was quantitated in each case. Most of the delayed fragments were localized in one major (931 bp) and one minor (approximately 210 bp) region of the genome. The fragments exhibiting a greater extent of delay were in the major region. The results described herein and in the following paper show that, in most cases, this effect can be explained by the base composition, or sequence of the fragments, or both.


Influence of A-T content on the fractionation of DNA restriction fragments by RPC-5 column chromatography. The properties of the fractionated Hae III fragments of pRZ2 DNA (Patient, R.K., Hardies, S.C., and Wells, R.D. (1979) J. Biol. Chem. 254, 5542-5547) were studied in an effort to determine why several of the fragments bind more tightly to RPC-5 than expected on the basis of their length. The purified fragments were analyzed for their nucleotide composition by direct determination of their constituent mononucleotides and by analytical CsCl and Cs2SO4 density gradient analyses. A-T-rich fragments elute at higher salt concentrations than fragments of equivalent size which are not A-T-rich. In addition, denaturation mapping studies by electron microscopy indicate that an A-T-rich run within an otherwise G-C-rich fragment can give rise to delayed elution. At least one other factor influences the separation of DNA restriction fragments by RPC-5 chromatography. Some of the fragments in this digest which elute later than predicted from their size either contain known genetic regulatory sites or bind regulatory proteins.


In vivo transcription of rRNA operons in Escherichia coli initiates with purine nucleoside triphosphates at the first promoter and with CTP at the second promoter. 32P-labeled RNA was isolated from growing Escherichia coli and the 5' end nucleoside triphosphates of rRNA were analyzed after hybridization to various DNA fragments derived from one of the rRNA operons, rrnE. The results show that there are two transcription start sites for each rRNA operon in vivo, one initiating with ATP (or GTP), and the second initiating with CTP. Transcription from the first site was observed to be stronger than that from the second site. These two sites correspond to the two promoters identified in the previous in vitro studies, indicating that the two promoters are used in vivo. These results also demonstrate previously unrecognized transcription initiation with CTP in growing E. coli cells.


A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase. We describe a new method for quantitatively assaying the omega subunit of Escherichia coli RNA polymerase. The assay is based on the ability of RNA polymerase holoenzyme to catalyze the continuous synthesis of the dinucleotide pApU on a poly[d(A-T)] . poly[d(A-T)] template when supplied with AMP and UTP as substrates. Core enzyme, lacking omega subunit, catalyzed this reaction at a rate less than 1% that of holoenzyme. The omega subunit was not released from the enzyme/DNA complex during dinucleotide synthesis. Using this assay, a titration of a fixed concentration of core enzyme was observed with increasing concentrations of added omega subunit. Below a 1:1 omega:core ratio the measured activity increased linearly with omega concentration, whereas above a 1:1 ratio the activity remained constant. An immediate application of the assay is in determining the concentration of active omega, or equivalently of active holoenzyme, in any RNA polymerase preparation.


Total synthesis of a tyrosine suppressor transfer RNA gene. XIV. Chemical synthesis of oligonucleotide segments corresponding to the terminal regions. Chemical syntheses of the two dodecanucleotides d(T-C-A-A-C-G-T-A-A-C-A-C) and d(A-C-G-T-T-G-A-G-A-A-A-G), the two undecanucleotides d(T-T-T-A-C-A-G-C-G-G-C) and d(T-G-T-A-A-A-G-T-G-T-T), the decanucleotide d(A-G-T-C-C-G-A-A-A-G), and the nonanucleotide d(A-A-T-T-C-T-T-T-C) are described. These deoxyribo-oligonucleotide segments, excluding the decanucleotide, represent the DNA duplex corresponding to the previously determined nucleotide sequence -30 to -51 of the promoter region of the gene for the tyrosine suppressor tRNA (Sekiya, T., Gait, M.J., Norris, K., Ramamoorthy, B., and Khorana, H.G. (1976) J. Biol. Chem. 251, 4481-4489) and include the EcoRI restriction endonuclease sequence at the appropriate 5'-end. The nona- and decanucleotide along with the previously synthesized deoxyribo-oligonucleotide segments 25 to 27 (Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G. (1976) J. Biol. Chem. 251, 676-694) together represent the DNA duplex corresponding to the natural nucleotide sequence 121 to 142 of the region adjoining the C-C-A end of the tyrosine tRNA gene and, in addition, a run of nine nucleotides which include the EcoRI restriction enzyme sequence at the 5'-end. The syntheses used protected mono- and oligonucleotides and stepwise condensation methods. A noteworthy feature of the present syntheses was the use of reverse phase high pressure liquid chromatography for the rapid and efficient separation of synthetic reaction mixtures.


Total synthesis of a tyrosine suppressor tRNA gene. XV. Synthesis of the promoter region. By use of polynucleotide kinase and polynucleotide ligase, the 10 deoxyoligonucleotide segments, whose syntheses have been described in accompanying papers, have been joined to form the 62-nucleotide-long DNA corresponding to the promoter region of an Escherichia coli suppressor tRNA gene. The following sequence in the joining reactions was used to obtain error-free and optimal yields of the products: 1) joining of Segment P-1 to P-3 in the presence of Segment P-2; 2) joining of Segments P-4 to P-7 to form Duplex [P4-7]; 3) joining of Segments P-8 to P-10 to Duplex [P4-7] to form Duplex [P4-10]; and finally, 4) joining of P-(1 + 3) and P-2 to Duplex [P4-10] to form the total promoter Duplex [P].


Total synthesis of a tyrosine suppressor tRNA gene. XVIII. Biological activity and transcription, in vitro, of the cloned gene. The chemically synthesized gene for Escherichia coli tyrosine suppressor tRNA has been joined to both plasmid (ColE1 ampr) and bacteriophage (Charon 3A) vector chromosomes after the latter had been digested with the restriction endonuclease EcoRI. Suppression of both bacterial (trpA, his, lacZ) and bacteriophage lambda amber mutations (Aam32, Bam1) has been demonstrated after transformation of E. coli with the recombinant DNA molecules carrying the synthetic suppressor tRNA gene. The cloned synthetic gene has been reisolated from the vector chromosomes after digestion of the latter with EcoRI restriction endonuclease and characterized in regard to its size and its ability to serve as a source of suppressor activity in further transformation experiments. This synthetic gene has also been shown to suppress bacterial amber mutations after it had been incorporated into the E. coli chromosome as part of a lambda prophage. Transcription, in vitro, of the cloned synthetic suppressor gene gave a product which, on treatment with a crude E. coli extract, afforded the tyrosine suppressor tRNA precursor. The latter was characterized by two-dimensional fingerprinting after digestion with T1-RNase. Exposure of the in vitro transcript to RNase P Selectively released the 41-nucleotide-long fragment characteristic of the 5'-end of the tRNA precursor. Thus, the nucleotide sequence of the cloned gene is accurate and its expression is controlled by its promoter.


Purification of hog liver isomerase. Mechanism of isomerization of 3-alkenyl and 3-alkynyl thioesters. A hog liver enzyme that catalyzes the reversible conversion of 3-acetylenic fatty acyl thioester to (+)-2,3-dienoyl fatty acyl thioester has been purified to homogeneity. The enzyme is not inhibited by the allenic product that it generates. The same homogenous enzyme catalyzes the conversions of 3-cis- or 3-trans-acyl Coenzyme A derivatives to 2-trans-acyl-CoA derivatives. Four forms of the isomerase differing in charge (pI = 6.57, 6.83, 7.01, and 7.27) have been separated by isoelectric focusing. Ultracentrifugation and sodium dodecyl sulfate-gel electrophoresis indicate that each of these enzyme forms is dimeric and composed of two 45,000-dalton subunits. With 3-acetylenic substrates, all enzyme forms exhibit broad specificity for chain length (C6 to C12) and for the thioester moiety (N-acetylcysteamine (NAC), pantetheine, or CoA). The 3-cis and 3-trans olefinic substrates are active only in the form of their coenzyme A derivatives; their NAC thioesters inhibit competitively. Mechanistic studies favor an isomerization pathway by way of carbanion intermediates. The acetylene-allene isomerase described here and the reported crotonase-catalyzed hydration of allenic thioesters (Branchini, B.R., Miesowicz, F.M., and Bloch, K. (1977) Bioorg. Chem. 6, 49-52) may be responsible for the degradation of naturally occurring acetylenic and allenic acids.


The structure of lipopolysaccharide from an Escherichia coli heptose-less mutant. III. Two fatty acyl amidases from Dictyostelium discoideum and their action on lipopolysaccharide derivatives. Two fatty acyl amidases have been partially purified from the slime mold, Dictyostelium discoideum. Their action on lipopolysaccharide derivatives, especially Compound I, has been studied. Amidase I removes specifically the beta-hydroxymyristyl group, which is present on the amino group adjacent to the C-1 phosphate. The product, Compound V, is then a substrate for Amidase II, which removes the remaining beta-hydroxymyristyl group from the amino group in the distal glucosamine ring to give Compound VI. Compound I itself is resistant to Amidase II. Thus, the two enzymes show a high degree of structural specificity. The structure of lipopolysaccharide from the E. coli K-12 mutant is concluded in the light of studies reported in this and the accompanying papers, and this structure is discussed in relation to other bacterial lipopolysaccharides.


DNA replication with bacteriophage T4 proteins. Purification of the proteins encoded by T4 genes 41, 45, 44, and 62 using a complementation assay. The proteins encoded by bacteriophage T4 genes 41, 45, 44, and 62 are known from the genetic studies of Epstein et al. ((1963) Cold Spring Harbor Symp. Quant. Biol. 28, 375--394) to be required for viral DNA synthesis. A convenient assay for each of these proteins is described which is based on the specific stimulation by each protein of DNA synthesis in extracts of Escherichia coli infected with mutants of bacteriophage T4 unable to make that protein. The T4 41 protein, 45 protein, and the complex of the 44 and 62 proteins have been highly purified. For each protein there is co-chromatography during the final purification step of (i) activity in the complementation assay, (ii) activity required for DNA synthesis with other purified T4 proteins, and (iii) a subunit of the size previously identified as that of the corresponding gene product.


DNA replication by bacteriophage T4 proteins. The T4 43, 32, 44--62, And 45 proteins are required for strand displacement synthesis at nicks in duplex DNA. The T4 bacteriophage gene 43 (T4 DNA polymerase), 32 (DNA helix-destabilizing protein), and 45 proteins and the complex of the gene 44 and 62 proteins are all required for DNA synthesis beginning at single-stranded breaks in duplex DNA. This synthesis occurs by strand displacement and is not dependent on ribonucleotides, the T4 gene 41 protein, or the T4 initiating protein, each of which is required to begin new chains on single-stranded templates. Electron microscopic analysis shows that duplex molecules with long single-stranded branches are the predominant products of this strand displacement synthesis.


R plasmid dihydrofolate reductase with subunit structure. Dihydrofolate reductase, specified by the type II plasmid of a trimethoprim-resistant Escherichia coli, was purified 40-fold to homogeneity using a combination of gel filtration, DEAE-Sephacel chromatography, and hydrophobic chromatography. The final product shows a single protein band on polyacrylamide gel electrophoresis and has a specific activity of 1.0 unit/mg. The molecular weight of the purified enzyme is 36,000 as determined both by gel filtration and Ferguson analysis of polyacrylamide gel electrophoresis. In contrast, a single polypeptide with a molecular weight of 8,500 was observed on sodium dodecyl sulfate-gel electrophoresis. These experiments suggest that, unlike any bacteria or vertebrate dihydrofolate reductase previously examined, the type II R plasmid reductase is a tetramer composed of four identical subunits. A partial amino acid sequence determination shows no heterogeneity of the subunits and also no clear homology with any reductase sequence previously reported.


Biosynthesis of hyaluronic acid by Streptococcus. Synthesis of hyaluronic acid was investigated in a cell-free system derived from a strain of Group A streptococci. Preparative procedures were improved so that an enzyme system 70 times more active than that previously reported was obtained. The hyaluronic acid synthesized could be separated into trichloroacetic acid-soluble and -insoluble fractions. On the basis of pulse-chase experiments, it was shown that the trichloroacetic acid-insoluble fraction is a precursor of the soluble fraction. The release of the trichloroacetic acid-insoluble hyaluronic acid is specifically blocked with p-chloromercuribenzoate, without inhibition of chain elongation. The addition of butanol to trichloroacetic acid resulted in solubilization of all of the hyaluronic acid. No detectable difference in molecular size was observed between the two hyaluronic acid fractions, both of which were estimated to be more than one million daltons in size. Testicular hyaluronidase digestion of either one of the two types of hyaluronic acid yielded no high molecular weight fragments, indicating that hyaluronic acid is not bound covalently to protein. However, following incubation of enzyme assay mixtures with UDP-[14C]GlcUA, even in the absence of UDP-GlcNAc, radioactive high molecular weight hyaluronic acid was obtained which suggests that the enzyme system elongates rather than initiates hyaluronic acid chains. Tunicamycin did not inhibit hyaluronic acid synthesis, indicating lack of participation of an intermediate of pyrophosphorylpolyisoprenol type. The results obtained are consistent with the hypothesis that chain elongation of hyaluronic acid proceeds by alternate addition of monosaccharides from UDP-sugars by a membrane-bound synthesizing system followed by release of completed hyaluronic acid chains.


Effect of anthraquinone dyes and evaluation of the kinetic mechanism of yeast phosphoglycerate kinase. Cibacron Blue 3G-A (CB3G-A, I) was investigated as a ternary complex analogue of lactate dehydrogenase and phosphoglycerate kinase as had been suggested earlier (Stellwagen, E. (1977) Accts. Chem. Res. 10, 92-98). CB3G-A and Procion Brilliant Blue (PBB, II), a structural isomer of the Cibacron dye without the sulfonated benzoyl moiety, were attached covalently to Sepharose CL-6B. The two enzymes were adsorbed to the columns and then eluted by substrates in various combinations. CB3G-A and PBB interact similarly with the two enzymes in spite of the structural differences between the dyes indicating that the specific structure of CB3G-A is not a ternary complex analogue. Inhibition studies of phosphoglycerate kinase by CB3G-A suggest that 2 molecules of dye bind per monomer and are consistent with multiple substrate binding sites. It is suggested that the kinetic mechanism of phosphoglycerate kinase is best described as steady state random.


Synthesis of a peptide form of N-delta-(phosphonoacetyl)-L-ornithine. Its antibacterial effect through the specific inhibition of Escherichia coli L-ornithine carbamoyltransferase. N-delta-(Phosphonoacetyl)-L-ornithine is a potent inhibitor of the Escherichia coli L-ornithine carbamoyltransferase (Ki = 0.77 microM, pH 8.0, 37 degrees C). Nevertheless, the analog does not cross the bacterial membrane. Therefore we have designed a tripeptide, glycylglycyl-N-delta-(phosphonoacetyl)-L-ornithine, to take advantage of the broad specificity of the oligopeptide permease system of the bacterium. A lag effect, related to the tripeptide concentration, was observed in the growth of the wild type P4X strain. At high concentration (greater than or equal to 0.75 mM) the peptide appears to be bacteriostatic and the cells which escape this action were characterized gentically as mutants devoid of the oligopeptide transport system. It was shown that the in vivo cellular target of the toxic tripeptide is solely restricted to L-ornithine carbamoyl-transferase and that the tripeptide is probably split in the cell to permit an effective inhibition by N-delta-(phosphonoacetyl)-L-ornithine. Resistance of the wild type cells to moderate levels (less than 0.75 mM) of the phosphonic analog is accompanied by a derepression of the L-ornithine carbamoyltransferase activity. The P4XB2 strain, which is an arg R regulatory mutant, has a reduced lag effect in the presence of the tripeptide and appears to react to the intoxication by a further adjustment of the L-ornithine carbamoyltransferase cellular level.


Relative efficiency of anticodons in reading the valine codons during protein synthesis in vitro. Using a protein synthesizing in vitro system programmed with MS 2-RNA, the relative efficiency (in the presence of each other) of valine tRNAs with the anticodons U*AC (U* represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC to read the valine codons was investigated. An anticodon which can read all three positions of the codon according to the rules of Watson-Crick base-pairing and the wobble hypothesis is an order of magnitude more efficient than an anticodon which misreads the codon by reading only the first two positions and presumably disregards the third nucleotide of the codon. There are two seeming exceptions to this behavior: the anticodon U*AC reads the codon GUU quite efficiently and IAC is as effective as U*AC in reading the codon GUG. The significance of these exceptions is evaluated with respect to the organization and evolution of the genetic code.


A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding. 70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions. The resulting ribosomes were fully active. 30 S subunits isolated from these particles were also fully active. They contain approximately 0.7 eq of fluorescent dye. Nearly all of it is attached to protein S18. Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein. The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles. Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome. The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra. Thus it affords a simple way to quantitate mRNA binding directly. In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid.


Purification of the gene 43, 44, 45, and 62 proteins of the bacteriophage T4 DNA replication apparatus. A procedure has been developed which allows the T4 bacteriophage proteins corresponding to the products of genes 43, 44, 45, and 62 to be purified to near homogeneity from a single T4-infected cell lysate (greater than 90% single species as judged by sodium dodecyl sulfate polyacrylamide elctrophoresis). In these preparations, the major problem of removing all contaminating nucleases has been overcome. Each of the above proteins is known from genetic analysis to be essential for phage DNA replication. The protein product of gene 43 is T4 DNA polymerase, and its recovery can be monitored using a standard DNA polymerase assay. The other three gene products have been designated as "polymerase accessory proteins," since they directly enhance polymerase function on both single- and double-stranded DNA templates. Their activities were monitored by an "in vitro complementation assay," which measures the stimulation of DNA synthesis observed in a concentrated lysate of T4 mutant-infected Escherichia coli cells when the missing T4 wild type protein is added. Starting from 300 g of infected cell paste, we obtained 9.3 mg of gene 43 protein, 21 mg of gene 45 protein, and 70 mg of a tight complex made up of 44 and 62 proteins; final yields were estimated at 30%, 14%, and 28%, respectively, of the initial activity present in the lysate. When the above purified proteins are incubated with preparations of two other T4 DNA replication proteins (gene 41 and gene 32 proteins) plus deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single- and double-stranded DNA templates. As reported elsewhere, this synthesis mimicks that catalyzed by the T4 DNA replication apparatus in vivo.


Purification of gene 41 protein of bacteriophage T4. The T4 bacteriophage gene 41 protein is known from genetic analysis to be essential for phage DNA replication in vivo. It became possible to monitor the activity of this protein during purification after development of an "in vitro complementation assay," which measures its stimulation of DNA synthesis in a concentrated crude lysate prepared from Escherichia coli cells infected with a T4 bacteriophage mutant in gene 41 (L. Moran and B. Alberts, manuscript in preparation). In this report, a purification procedure involving three chromatographic steps is described which reproducibly yields a 90% homogeneous preparation of this rather unstable protein. The major polypeptide chain present (58,000 daltons) is shown to cosediment with a DNA-dependent GTPase (and ATPase) activity, and to induce extensive in vitro DNA synthesis on both single- and double-stranded DNA templates when incubated with our preparations of five other purified T4 DNA replication proteins (plus deoxyribonucleoside and ribonucleoside triphosphates).


Activation of human inactive ("pro-") renin by cathepsin D and pepsin. Inactive human renin is found in amniotic fluid, plasma, and kidney and may be a renin precursor ("prorenin"). The mechanism of activation of inactive renin in vivo is not known. The present study examined the hypothesis that cathepsin D, a lysosomal pepsin-like endopeptidase may be capable of eliciting activation. Cathepsin D was incubated with inactive renin in human amniotic fluid at pH 4.8 and 22 C for 0-5 h. Marked activation occurred and the reaction displayed first order kinetics with respect to the concentration of cathepsin D. The initial velocity of conversion of inactive renin to active renin by cathepsin D was 0.007%/min/microgram cathepsin D. Under identical conditions, the initial velocity of conversion by pepsin was 0.18%/min/microgram pepsin. The 25-fold higher potency of pepsin compared with cathepsin D is in accordance with the recognized relative substrate affinities and catalytic efficiencies of the two enzymes. Inactive renin in human amniotic fluid seems to be similar to that found in human kidney and since cathepsin D is present in juxtaglomerular cells, this activation process may have physiological importance.


The prevention of myocardial ischaemia by the method of profound selective myocardial hypothermia during open heart surgery. A new method of myocardial protection, profound selective myocardial hypothermia has been employed in order to avoid the effects of cardiac ischaemia during aortic cross clamping in the course of operations undertaken under extracorporeal circulation. It consists of the irrigation of the aortic root immediately after cross clamping with 4 liters of Ringer Lactate at 4 degrees C which perfuses the entire coronary bed and induces complete uniform profound coling of the intrapericardial mass to a temperature of approximately 10 degrees C with resultant flat electrocardiogram and cessation of all electrical activity of the myocardium. This inactivity remains complete until the aorta is unclamped permitting reoxygenation and rewarming of the heart with resumption of its function. This method has been used in the course of 250 cardiac procedures involving the use of extracorporal circulation including 177 valve replacements, either aortic or mitral and aortic, 54 saphenous vein bypass procedures, either simple or with valve replacement and 19 other assorted procedures including cardiac transplantation and complex congenital malformations. Thermal, electrocardiographic, hemodynamic, histologic, histochemical and enzymatic observations have proven the minimal significance of the ischemic lesions produced in hearts protected in this manner. The total mortality was 6.4% which proves the value of this method and its superiority over the 2 techniques most often used at the present time, coronary perfusion or topical myocardial hypothermia according to Shumway's method.


The mechanism of ovulation inhibition by triamcinolone acetonide. A single dose of 25 mg triamcinolone acetonide, when given on day 1 or 2 of the menstrual cycle, inhibits ovulation. To examine the mechanism of this action, daily determinations of plasm FSH, LH, estrone plus estradiol (E1 + E2), and progestins were performed. Some subjects also received a single dose of LH-RH or hCG on cycle day 15 or clomiphene citrate on days 5-9. Triamcinolone acetonide itself caused variable suppression of plasma estrogens, loss of the mid-cycle gonadotropin surge, and a deficient or absent rise in plasma progestins. Impaired secretion of estrogen did not seem to be due to low gonadotropin levels. FSH and LH responses to LH-RH were adequate in relation to prevailing estrogen levels. Four of six women treated with clomiphene responded with plasma progestin levels which exceed 8 ng/ml. Triamcinolone acetonide seems to affect the hypothalamic-pituitary-ovarian axis mainly by hypothalamic suppression and possibly by a direct effect on the ovary as well.


Gonadotropin insufficiency in patients with thalassemia major. To elucidate whether the cause of sexual maturational arrest in thalassemia major is of hypothalamic or pituitary etiology, three female and two male patients were extensively studied. After the iv administration of 150 micrograms gonadotropin-releasing hormone (GnRH) and 500 micrograms of TRH, all patients demonstrated a minimal LH and no FSH response, with variable PRL and TSH responses. The test was repeated after the patients received 100 micrograms GnRH im daily for 7 days. The LH, FSH, PRL, and TSH responses were similar to those obtained previously. The female patients were then retested twice, after priming with 17 beta-estradiol (2 mg daily for 7 days) and again after treatment with human menopausal gonadotropins. The male patients were treated with hCG and, after testosterone reached normal adult male values, the GnRH-TRH stimulation test was repeated. In both the female and male patients, the pituitary responses remained unchanged. These results demonstrate the presence of primary gonadotropin insufficiency as well as the lack of positive estrogen feedback mechanism in patients with thalassemia major. The site of this abnormality has been demonstrated to be the pituitary gland, since hemosiderosis of the pituitary without hypothalamic involvement has been found at autopsy in one patient.


Unusual prolactin response to luteinizing hormone-releasing hormone in some anovulatory women. We studied PRL, FSH, and LH response to LRH in 82 anovulatory and 4 normally ovulating women. Ten anovulatory patients who were basally hyperprolactinemic showed no significant change in PRL concentration after LRH. Of the remaining 72 anovulatory patients with basal PRL levels in the normal range, 59 showed no PRL modification after LRH (as in normals) whereas in 13 patients, a prompt and significant rise of PRL concentration above basal levels in response to LRH was observed. In these 13 patients, the basal PRL levels were significantly higher than those of the other 59 normoprolactinemic women. No significant differences in gonadotropin concentrations were detected among the three groups. The unusual rise in PRL levels after LRH in these 13 patients can be interpreted as a paradoxical response of the pituitary to a specific stimulus, as seen in other clinical conditions. It is suggested that this phasic hyperprolactinemia might represent an intermediate phase between true normoprolactinemia and chronic hyperprolactinemia.


The functional relationship between priming and releasing actions of luteinizing hormone-releasing hormone. To examine the relationship between the priming and releasing actions of LRF on LH secretion, 14 normal cycling women received 4 different rates of LRF infusion (0.005, 0.01, 0.05, and 0.1 microgram/m2.min for 4 h). The releasing action of the infusion was measured as the area under the curve and the priming effect was assessed by the acute LH increment in response to a test pulse of LRF (10 microgram) at the end of infusion. At the lower 2 infusion rates, there were only minor changes in releasing function, but it increased exponentially (r = 0.986) with higher rates of infusion. In contrast, the priming effect of the lower 2 doses of infusion increased markedly as a function of infusion rate, but no additional priming was found with the higher rates of infusion. Thus, over the range of infusion rates employed, the releasing and priming functions of LRF appear to be dose dependent. These results indicate that the interdependent releasing and priming actions of LRF on LH secretion are functionally dissociable and that large elevations of LRF tend to favor release, while small LRF increments seem to promote priming preferentially.


Shortening of the bleeding time in rabbits by hydrocortisone caused by inhibition of prostacyclin generation by the vessel wall. The effect of hydrocortisone on thrombocytopenic bleeding has been studied in rabbits using a jugular vein bleeding-time technique and a microvascular bleeding-time technique. An inverse relationship was found between the bleeding time and platelet count with both techniques in rabbits made thrombocytopenic by either X-irradiation or injection of heterologous platelet antiserum. Hydrocortisone shortened both bleeding times in thrombocytopenic animals when given in single large doses intravenously (25-100 mg/kg), in daily doses (6 mg/kg) intramuscularly, and shortened the jugular bleeding time when applied to the outside of the jugular vein or instilled intraluminally into the vein. This effect was also noted in normal animals. The effect on thrombocytopenic bleeding was dose related. When given daily, the effect was greater when hydrocortisone was given for 10 d than for 5 d. Both indomethacin and tranylcypromine also reduced the jugular vein bleeding time when instilled intraluminally into the jugular vein, whereas exogenously provided arachidonic acid reversed the effect of hydrocortisone but did not reverse the effect of indomethacin or tranylcypromine. Exogenously provided linoleic acid did not have any effect. Perfusion of the vessel segment with prostacyclin (PGI(2)) reversed the effect of intraluminally administered hydrocortisone, indomethacin, and tranylcypromine. Similarly, hydrocortisone, indomethacin, and tranylcypromine all reduced the rate of loss of fluid from a standard wound in isolated vessels emptied of blood and perfused with saline under constant pressure. PGI(2) reversed the action of these three agents, however, arachidonic acid reversed only the effect of hydrocortisone and did not reverse the effect of indomethacin and tranylcypromine. The generation of PGI(2)-like material and 6-keto-prostaglandinF(1) alpha from jugular vein strips was prevented by prior exposure of the animals or vessel wall to hydrocortisone. These results are compatible with the hypothesis that the vessel wall releases smooth muscle-relaxing prostaglandins when injured and that inhibition of prostaglandin formation by hydrocortisone enhances hemostasis by allowing vasoconstriction to be maintained.


Inhibition of prostacyclin by treatment of endothelium with aspirin. Correlation with platelet adherence. Aspirin treatment of cultured endothelial cells from the umbilical vein increased the adherence of 51Cr-platelets when thrombin was present. If the cyclooxygenase activity of endothelium was inhibited by aspirin, as it is in the platelet, reduction of endogenous prostacyclin (PGI2) production could have been responsible. By correlating thrombin-induced adherence of platelets to endothelial monolayers with PGI2 release (as measured by radioimmunoassay for 6-keto-prostaglandin FI1 alpha [6-keto-PGF1 alpha]), we have demonstrated an inverse relationship between platelet adherence and PGI2 levels. Untreated endothelial monolayers exposed to thrombin and platelets resulted in 4% platelet adherence and 107 nM 6-keto-PGF1 alpha. With 0.1 mM aspirin treatment, which is known to block platelet cyclooxygenase, adherence was 5% and 6-keto-PGF1 alpha decreased to 45 nM. Increasing the aspirin concentration to 1 mM resulted in 44% adherence and less than 3 nM 6-keto-PGF1 alpha. When 25 nM exogenous PGI2 was added to 1 mM aspirin-treated endothelium, adherence returned to 5%. The increase in thrombin-induced platelet adherence to 1 mM aspirin-treated monolayers was reversed 2 h after removal of the aspirin solution. 6-Keto-PGF1 alpha returned to 37% of the untreated monolayer value. Recovery from the aspirin effect did not occur when cycloheximide, an inhibitor of protein synthesis, was present during the 2-h period.


Use of a human plaque-forming cell assay to study peripheral blood bursa-equivalent cell activation and excessive suppressor cell activity in humoral immunodeficiency. A plaque assay that detects human mononuclear blood cells producing immunoglobulin (Ig)M antibody to sheep erythrocytes was investigated for its usefulness in studying B-cell activation and regulation in 24 patients with humoral immunodeficiency. Cells from 3 of 15 patients with common variable agammaglobulinemia produced some plaques (range 40--160/10(6) cells; normal range 80--1240/10(6)), but those from the other 12, from all 7 with x-linked agammaglobulinemia and from the 2 with x-linked immunodeficiency with hyper-IgM failed to produce any detectable plaques. In co-cultures of patient and normal cells a very good correlation was seen between results of the plaque assay and an IgM biosynthesis assay in detecting excessive suppressor cell activity. Cells from 7 of 15 common variable agammaglobulinemics, from 3 of 7 x-linked agammaglobulinemics, and from both patients with hyper-IgM caused significant suppression of IgM biosynthesis and(or) plaque formation by normal cells. The observations in the last two groups and discordance for excess suppressor activity in identical twins with common variable agammaglobulinemia suggest that the activity develops secondarily to whatever their primary defects may be. Culturing non-T cells from common variable agammaglobulinemics exhibiting excessive suppressor cell activity with normal T cells resulted in plaque formation in four of five patients so studied; in all five the suppressor activity was found in the T-cell population. The availability of a plaque assay for the study of blood cells from immunodeficient patients provides a new probe to examine the cellular nature of such defects.


Role of Ia-like products of the main histocompatibility complex in conditioning skin allograft survival in man. This report correlates the survival time of 93 intrafamilial skin allografts performed under conditions of main histocompatibility complex (HLA) haploidentity with donor-recipient compatibility for products of the HLA-A, -B, -C, and -DR, as well as C3 proactivator, Glyoxalase I, and P loci located on the human 6th chromosome. Incompatibilities for HLA-A and -B (and to a lesser extent for HLA-C) and(or) for HLA-DR products exerted a strong influence upon the fate of skin allografts. When HLA-A and -B were considered alone, the most compatible group of grafts had a mean survival time of 15.8 d, as compared with 11.3 d for the most incompatible transplants. HLA-DR compatibility alone was associated with a mean survival time of 15.3 d, whereas HLA-DR-incompatible grafts had a mean survival time of 11.5 d. Incompatibilities for C3 proactivator, Glyoxalase I, and P did not have a significant effect upon graft survival. There was no evidence of an association between donor-recipient incompatibility at HLA-A, -B, or -C or at HLA-DR; such incompatibilities occurred independently of each other, in spite of the state of linkage disequilibrium known to exist between HLA-B and -DR. Incompatibilities for HLA-A, -B, and for HLA-DR exerted a potent additive effect upon graft survival. Skin grafts bearing one, two, or three incompatibilities had a mean survival time of 16.2, 13.7, and 10.7 d, respectively (P <0.0005).The results point to the important role played by the Ia-like products of the HLA complex (HLA-DR) in conditioning skin allograft survival in man. This consideration may be of direct relevance to the potential clinical usefulness of in vitro serological techniques for the detection of donor-recipient compatibility for HLA-DR.


Deficient serum bactericidal activity against Escherichia coli in patients with cirrhosis of the liver. The serum bactericidal activity (SBA) of cirrhotic patients was compared with that of normal individuals using the release of (51)Cr from radiolabeled Escherichia coli as the assay method. 80% (22/27) of patients were found to have deficient SBA against at least one of three smooth, serum-sensitive test strains of E. coli. Cirrhotic patients were found to have normal levels of serum lysozyme. Although some patients were mildly hypocomplementemic, this abnormality did not correlate with the presence of a bactericidal defect. Bactericidal antibody in normal and cirrhotics' sera was limited to the immunoglobulin (Ig)M class. Purified IgM from patients with deficient SBA against E. coli 0111 had lower concentrations of bactericidal antibody for that E. coli than did IgM from normal sera; the calculated bactericidal activity of total serum IgM was also lower. The bactericidal defect in cirrhotic serum could be completely corrected by either human antiserum to the homologous strain of E. coli or by purified, normal human IgM. However, because higher concentrations of IgM were required to restore normal SBA to a cirrhotic's serum than to agammaglobulinemic serum, there may be an inhibitor of bactericidal antibody in addition to a deficiency of bactericidal IgM antibody to E. coli in the serum of patients with cirrhosis. The bactericidal activity of the alternative complement pathway was also assessed. Sera from cirrhotic patients had no deficit in SBA attributable to the alternative complement pathway. In fact, in some, the activity of the alternative complement pathway was supernormal, compensating in part for the deficit in IgM-mediated SBA.


Insulin sensitivity and insulin binding to monocytes in maturity-onset diabetes. Tissue sensitive to insulin and insulin binding to monocytes were evaluated in 15 nonobese maturity-onset diabetics and in 16 healthy controls. Insulin sensitivity was determined by the insulin clamp technique in which the plasma insulin is acutely raised and maintained 100 muU/ml above the fasting level and plasma glucose is held constant at fasting levels by a variable glucose infusion. The amount of glucose infused is a measure of overall tissue sensitivity to insulin. In the diabetic group, the fasting plasma glucose concentration (168+/-4 mg/dl) was 85% greater than controls (P < 0.01) whereas the plasma insulin level (15+/-1 muU/ml) was similar to controls. During the insulin clamp study, comparable plasma insulin levels were achieved in the diabetics (118+/-5) and the controls (114+/-5 muU/ml). However, the glucose infusion rate in the diabetics (4.7+/-0.4 mg/kg.min) was 30% below controls (P < 0.01). Among the diabetics, the glucose infusion rate correlated directly with the fasting plasma glucose level (r = 0.57, P < 0.05). In five diabetic subjects, glucose metabolism was similar to controls, and these diabetics had the highest fasting glucose levels. When they were restudied after prior normalization (with insulin) of the fasting plasma glucose (100+/-1 mg/dl), the glucose infusion rate during the insulin clamp was 30% lower than observed in association with hyperglycemia (P < 0.01). Studies that employed tritiated glucose to measure endogenous glucose production indicated comparable 90-95% inhibition of hepatic glucose production during hyperinsulinemia in the diabetic and control subjects.(125)I-insulin binding to monocytes in the diabetics (5.5+/-0.6%) was 30% below that in controls (P < 0.01). Insulin binding to monocytes and insulin action as determined with the insulin clamp were highly correlated in both control (r = 0.67, P < 0.01), and diabetic subjects (r = 0.88, P < 0.001). We conclude that (a) tissue sensitivity to physiologic hyperinsulinemia is reduced in most maturity-onset diabetics; (b) this decrease in sensitivity is located, at least in part, in extrahepatic tissues; (c) the resistance to insulin may be mediated by a reduction in insulin binding; and (d) in maturity-onset diabetics with normal tissue sensitivity to insulin, hyperglycemia may be a contributing factor to the normal rates of insulin-mediated glucose uptake.


Insulin secretion and metabolism during the perinatal period in the rat. Evidence for a placental role in fetal hyperinsulinemia. To better understand why plasma immunoreactive insulin (IRI) concentration is high in the rat fetus during the last 3 d of gestation and why fetal hyperinsulinemia abruptly subsides after birth, insulin secretion and metabolic clearance rates were estimated in fetuses and nursed pups. Intravenously injected [(125)I]monoiodoinsulin was cleared from the plasma of prematurely delivered pups at least as rapidly as from that of 7- to 10-d-old pups, suggesting that fetal hyperinsulinemia is not a result of slow clearance of the hormone. The fetal liver bound 35% of the injected label within 3 min, and binding was saturable. The uptake of radioactivity by the fetal kidney was nonsaturable and much lower than that of adult rat kidney. Isolated fetal islets were already reactive to glucose on the 19th d of gestation. Pancreatic insulin secretory capacity was estimated by measuring (a) the insulin release of isolated islets incubated in the presence of 2.8 mM glucose, (b) the insulin content of the same islets, and (c) the total insulin extracted from the pancreas, using the formula (a x c)/b. 2 d before birth, the pancreatic insulin secretory capacity was high, accounting for fetal hyperinsulinemia. It was even higher after birth, not accounting for the postnatal decrease in plasma IRI concentration. Pups delivered by caesarian section 1 d before term exhibited a brisk decrease in plasma IRI concentration when the cord was cut. By contrast, if the feto-placental unit was removed from the dam, maintaining fetal blood circulation through the placenta, fetal plasma IRI concentration remained as high as in utero. These experiments suggest that a placental factor stimulates fetal insulin secretion. After birth, when the cord is cut, insulin secretion is rapidly turned off, and the pups switch from a state of unlimited fuel supply by the mother to a state of fuel saving.


Intravascular clearance of parasitized erythrocytes in rodent malaria. Little is known about host defense mechanisms responsible for protective immunity in malaria. The intravascular location of the infection suggested that removal of parasitized erythrocytes by reticuloendothelial organs might be important. To study this possibility, we examined the clearance of (51)Crlabeled Plasmodium berghei-infected erythrocytes in rats. Infected erythrocytes were removed more rapidly from circulation than homologous uninfected erythrocytes. The rate of clearance of infected cells during the 1st hour after inoculation was approximately three times greater in rats rendered immune by prior infection than in control rats. This accelerated clearance resulted from greater splenic uptake in immune rats and appeared to correlate with spleen size. Since the clearance pattern of infected erythrocytes more closely resembled the clearance of Heinz body-containing uninfected erythrocytes than of antibody-coated (immunoglobulin G) uninfected erythrocytes, rheologic alterations of parasitized erythrocytes might be a more important determinant of clearance than an antibody-dependent process. During the phase of malaria infection in which increasing parasitemia is observed, organ uptake of infected erythrocytes did not increase despite splenic and hepatic enlargement. However during the spontaneous onset of resolution of malaria infection characterized by decreasing parasitemia, a marked enhancement of splenic clearance was noted. These observations suggest that sudden alteration in splenic clearance of parasitized erythrocytes might be important in the resolution of acute malaria.


Thrombin-induced platelet secretion. Further evidence for a specific pathway. We have studied the interaction between thrombin and washed, human platelets using prostacyclin, a reversible inhibitor of platelet secretion. The effect of thrombin is limited to those reactions that are not inhibited by an increased concentration of platelet cyclic adenosine 3',5'-monophosphate, because prostacyclin is a potent inducer of the latter. Prostacyclin-treated platelets were briefly (15-30 s) exposed to low concentrations of human thrombin (0.01-0.2 U/ml). After removal of the prostacyclin and thrombin, the platelets were incubated with fresh thrombin. Although they had not undergone the release reaction after the first thrombin incubation, these platelets had a diminished capacity to secrete [(3)H]serotonin when exposed to thrombin the second time. Refractoriness was concentration dependent: the higher the initial thrombin concentration, the greater the degree of inhibition of serotonin secretion on subsequent thrombin exposure. Inhibition was closely related to the ability of thrombin to induce platelet secretion and not to its esterase or fibrinogen clotting activity. Diisopropyl fluorophosphate-inactive thrombin did not induce refractoriness. Refractoriness to thrombin did not increase when the time of the initial incubation with thrombin was lengthened, nor was it reversible.INHIBITION WAS THROMBIN SPECIFIC: serotonin secretion induced by collagen, wheat germ agglutinin, and the ionophore A23187 was minimally affected. For an equivalent amount of thrombin bound, a decrease was observed in serotonin secretion by thrombin-pretreated platelets compared to control platelets. Thus, there is at least one step in the secretory pathway between thrombin binding and regulation of adenylate cyclase. This step appears to transmit the signal that leads to extrusion of intracellular granular contents.


Interference with the mannose binding and epithelial cell adherence of Escherichia coli by sublethal concentrations of streptomycin. When Escherichia coli was grown in sublethal concentrations of streptomycin, mannose binding activity and epithelial cell adherence of the E. coli cultures at stationary phase were significantly reduced in the drug-grown organisms. In a strain whose minimal inhibitory concentrations was 30 mug/ml, the percentage of reduction in mannose binding activity was dose related over a range of concentrations between 0.5 and 10 mug/ml streptomycin. Concomitant with the drug-induced suppression of mannose binding activity, antigenic and ultrastructural alterations on the surface of the drug-grown organisms were observed by agglutination tests and electron microscopy, respectively. The streptomycin effect was reversible, required actively growing organisms, and was most apparent in the early log-phase of growth. High doses of antibiotic were ineffective when added to cultures which had acquired mannose binding activity. An isogenic derivative with high-level resistance to streptomycin was obtained as a single-step mutation from the test E. coli strain. Whereas the isogenic mutant possessed mannose binding activity and adhering ability similar to the parent strain, it was resistant to the streptomycin-induced suppression of the two activities at enormous concentrations (up to 10,000 mug/ml) of streptomycin. Taken together the results suggest that the suppression of epithelial cell adherence and mannose binding activity of E. coli grown in sublethal concentrations of streptomycin is a result of classic mechanisms of drug action upon the bacterial ribosome. The results support the possibility that antibiotics may act through mechanisms other than inhibition of growth and bacterial killing to eradicate bacteria from mucosal surfaces.


Role of microtubules in the synthesis, conversion, and release of (pro)insulin. A biochemical and radioautographic study in rat islets. In the pancreatic B cell, microtubules are thought to be involved in the process of insulin release. Their possible participation in the sequence of events leading from the biosynthesis and conversion of proinsulin to the release of newly synthesized insulin was investigated in rat isolated islets exposed to colchicine (0.1 mM). When the islets were preincubated for 30 min with colchicine and [3H]-leucine and, thereafter, incubated for two successive periods of 90 min each, still in the presence of colchicine, the release of preformed insulin was progressively inhibited and that of newly synthesized hormone delayed. When the islets were preincubated for 120 min with colchicine, subsequently pulse-labeled with [3H]leucine, and eventually examined by ultrastructural autoradiography, the export of newly synthesized proinsulin out of the rough endoplasmic reticulum, its transit through the Golgi complex, and its eventual packaging in secretory granules were all retarded. This situation was associated with a delayed conversion of proinsulin to insulin. Under the same experimental conditions, colchicine failed to affect the oxidation of glucose and adenylate charge in the islets. The effect of colchicine upon the release of preformed and newly synthesized insulin was not reproduced by lumicolchicine. It is concluded that colchicine interferes with the system controlling the intracellular transfer of secretory material from site of synthesis to site of release. This interference is likely to be linked to the effect of colchicine on microtubules.


Response of variant hereditary angioedema phenotypes to danazol therapy. Genetic implications. Hereditary angioedema (HAE), an auto-somal dominant disorder characterized by attacks of episodic edema is associated with decreased functional levels of the C1 esterase inhibitor. Approximately 85% of patients have lowered antigen levels of a normal inhibitor protein. 15% of patients have normal or elevated antigenic levels of functionless protein. We have examined the response to danazol therapy of patients with the variant HAE phenotypes possessing the abnormal protein in an effort to determine if these patients possess a normal structural C1 inhibitor allele. Four patients with a variant HAE phenotype were treated successfully with danazol. In two patients, distinguished by the presence of a functionless, albumin-bound, C1 inhibitor (phenotype 2), phenotypic analysis of the danazol response by bidirectional immunoelectrophoresis revealed the appearance of the normal C1 inhibitor gene product during danazol therapy. This relatively cathodal C1 inhibitor peak appears in conjunction with the development of nearly normal functional activity. All of the functional C1 inhibitory activity which appeared in the phenotype 2 treatment serum was associated with the electrophoretically normal inhibitor. This normal protein could be separated from the functionless inhibitor protein by immunoadsorption and molecular sieve chromatography. Danazol therapy of the two patients with an electrophoretically normal, functionless C1 inhibitor (phenotype 3) also resulted in a clinical remission associated with development of a significant increment in functional serum C1 inhibitory activity and C1 inhibitor protein. These findings demonstrate that these two HAE phenotypic variants are heterozygous for the normal serum C1 inhibitor, a finding which was not apparent before phenotypic analysis of this serum during danazol therapy. These data provide strong evidence for a basic similarity between the common form of HAE and its phenotypic variants. They also suggest that a structural gene lesion may result in the abnormalities of serum C1 inhibitor function and disease expression in all three of these HAE phenotypes.


Antigenic modulation and turnover in human neutrophils. Turnover of membrane constituents appears important in many biologic processes. We studied this process in neutrophils by immunologic methods. The capacity of neutrophils to recognize other neutrophils coated with antibodies against membrane antigens was used to determine the changes that occur after attachment of the antibody to the neutrophil membrane. Neutrophils were sensitized for 30 min at 22 degrees C with antibodies from three patients with antineutrophil autoantibodies. The sensitized neutrophils were recognized by normal neutrophils, which responded with an increase in glucose oxidation. If, after sensitization, the sensitized neutrophils were not immediately exposed to normal neutrophils, but instead were incubated at 37 degrees C for varying times, the capacity to elicit a recognition response decayed and was gone by 30 min. Additionally, the capacity of the cells to be resensitized by reexposure to antibody also decayed during this period. However, after further incubation at 37 degrees C, the neutrophils recovered the capacity to become sensitized; and this recovery was not inhibited by the addition of cycloheximide. Control incubations with normal immunoglobulin (Ig)G did not elicit a recognition response. The decay in recognition response was temperature dependent. Direct immunofluorescent studies with fluorescein-conjugated antineutrophil IgG revealed that the antibodies were cleared by aggregation and endocytosis. We conclude that: (a) neutrophils clear antibody from the cell surface by a temperature-dependent mechanism; (b) antigenicity is cleared concomitantly; (c) the mechanism of clearance involves internalization; and (d) with time, antigenicity reappears on the cell surface.


Antipili antibody affords protection against experimental ascending pyelonephritis. The ability of antipili antibody to prevent ascending urinary tract infection was investigated in rats. One group of rats was immunized passively with rabbit antisera to purified pili and challenged by intravesicular inoculation of 5 x 10(7) heavily piliated Escherichia coli. Only 2 of 14 immunized animals developed cortical abscesses as compared to 13 of 15 control rats given normal rabbit serum (P equals 0.0001). The mean log titer of bacteria in the kidneys of the immunized rats was 0.85 vs. 6.08 in the controls (P less than 0.005). A second group was actively immunized with pili. 3 of 16 immunized animals became infected as compared to 10 of 15 controls (P equals 0.01). The mean log titers were 2.13 and 4.54, respectively (P less than 0.01). A third group was passively immunized and challenged with a strain that had different O, K, and H antigens but shared pili antigens. Abscesses occurred in 4 of 15 immunized animals as compared to 13 of 15 controls (P equals 0.001). The mean log titers were 2.37 and 5.63, respectively (P less than 0.005). These results indicate that antipili antibody protects rats against ascending urinary tract infections.


Particle size distribution analysis for the rapid detection of microbial infection of urine. The accuracy and practicality of particle size distribution analysis for rapid screening of urine specimens are assessed. Six hundred urines were subjected simultaneously to routine bacteriological examinations and particle size distribution analysis using a Coulter Counter (ZBI) linked to a C1000 Channelyzer. There was complete agreement in the results of 593 (98.8%) specimens. Characteristic profiles of various bacterial species in infected specimens were consistently obtained. This system can easily be linked to any existing computer reporting in a district hospital laboratory, and the results of negative specimens (70--80%) can be obtained within 5--10 minutes.


The chloride corrosion behavior of silver-base casting alloys. The chloride corrosion behavior of five silver-palladium dental casting alloys was investigated in a 1% NaCl solution through the use of electrochemical hysteresis technique. The corrosion mechanism has been interpreted in terms of the microstructure of these alloys and their possible electrochemical reactions.


A hydrophilic denture base resin.

A hydrophilic denture base resin. A Denture Base Resin is marketed with hydrophilic characteristics which, it is claimed, improve retention. The claim has been questioned, but this article is concerned with the physico-chemical properties of the product.


Studies on the Sm-Co magnet as a dental material. The Sm-Co magnet has superior and convenient magnetic properties as compared with conventional ones used hitherto in prosthesis, and even very small pieces can provide the force necessary in dentistry. Also, the magnet has high corrosion resistance and is innocuous to tissues. Therefore, it can be used as a dental material, overcoming the limitations in past applications.


A method for detection of complement cleavage in gingival fluid. A sensitive method is described for detection of C3 cleavage in gingival fluid taken from sites of both normal and inflamed gingiva. Preliminary data suggest a direct relationship between C3 conversion and the presence of periodontal disease.


A comparison of the effects of endotoxin upon fibroblast proliferation and macromolecular syntheses. The effects of Escherichia coli endotoxin upon mouse L929 cell proliferation, DNA synthesis, protein synthesis, and proline incorporation were determined. It was found that a level of endotoxin which inhibited cell proliferation prompted a similar inhibition of DNA synthesis and overall cell protein synthesis. In contrast, endotoxin was shown to inhibit incorporation of proline into cell protein to a significantly greater extent.


A thermographic study of heat distribution during ultra-speed cavity preparation. Thermography was employed to determine the pattern of heat generation, distribution and dissipation during ultra-high speed cavity preparation. Results indicate an increase in intrapulpal temperature during cutting procedures. No significant differences in the cooling effectiveness between air-water spray and air alone were found. Thermography appears to be an effective research tool for determining thermal changes.


Comparison of four weeks' treatment with fenoterol and terbutaline aerosols in adult asthmatics. A double-blind crossover study. Fenoterol and terbutaline, two long-acting beta 2-adrenoceptor agonists in aerosol form, were compared in an 8-wk randomized double-blind crossover study in 22 mild to moderately severe asthmatics. Patients completed diary cards, recorded peak expiratory flow rate (PEFR) twice daily, and attended a clinic for measurement of PEFR, 1-sec forced expiratory volume (FEV1), and forced vital capacity (FVC) twice during each treatment period. Fifteen patients completed the study; 5 dropped out while using fenoterol, and 2 while using terbutaline. At clinic attendances, the patients had a significantly higher mean PEFR after 4 wk on terbutaline (385 L/min) than after fenoterol (316 L/min) (p less than 0.001). Similar results were found on analysis of the morning and evening PEFR recordings. On comparing each individual's PEFR recordings during the 2 treatments, it was found that there was no significant difference among the treatments in 3 patients, while 9 patients had a better response to terbutaline, and 3 patients had a better response to fenoterol. While similar number expressed a subjective preference for each treatment, the lung function data suggested that the effectiveness of fenoterol appeared to decline during the trial period.


Immunosuppressive activity associated with a reticulum cell sarcoma of aged mice. Aging BC3F1 hybrid mice frequently develop a reticulum cell sarcoma (RCS) causing marked hypercellularity of lymphoid tissues and pronounced suppression of humoral immunity. Cells of malignant spleens gave feeble responses in vitro to thymus-dependent and -independent antigens; moreover, they suppressed normal spleen cell responses to the antigens. The RCS was passed, serially, in young mice and the spleen and ascites cells of these recipients suppressed normal cell responses in vitro. Malignancy resulted in a substantial increase in the proportion of spleen cells of relatively lower density as determined by BSA gradient centrifugation; the cells of lower density were strongly immunosuppressive. The suppressive cells appeared to be neither typical T cells nor macrophages. Humoral suppressive substances associated with the RCS were demonstrated in vivo. The suppressive influence of the RCS may account for the hostile milieu in old BC3F1 mice with respect to lymphohematopoietic cells.


Gerovital-H3: a clinical trial as an antidepressant. Twenty-five volunteers, all in their fifth decade or beyond, all with mild to moderate, nonpsychotic depression of at least several months' duration participated in a double-blind study of Gerovital vs placebo. There was no significant difference between the Gerovital and placebo groups; both groups showed significant improvement on self-rating as well as observer rating scales.


A bibliography of doctoral dissertations on aging from American institutions of higher learning, 1975-1977. This bibliography is the seventh supplement to the original title which covered 1934-1969. The supplements include all titles of earlier years which were found after the original bibliography was published, Journal of Gerontology, 1971, 26, 391-422. Included in this year's supplement are 15 titles identified in a search of the Datrix data base which covered the years 1856-1934. Due to the nature of bibliographic control in regard to doctoral dissertation, each supplement will try to cover the academic year rather than the physical year. This means that a dissertation issued in 1970 may be either in the original bibliography or in the supplements. The arrangement of the supplement is similar to the original bibliography.


Current publications in Gerontology and Geriatrics. The subject categories are those in A Classified Bibliography of Gerontology and Geriatrics by Nathan W. Shock, published by Stanford University Press, Stanford, California (1951). Only major headings are used and the Roman numerals correspond to those given in the bibliography. Insofar as possible, references are classified according to organ systems. Thus, most of the material on Geriatrics will be found under the organ system involved in the disease. Cross references are indicated by numbers at the end of each section. When available, abstract references are given (B.A.-Biological Abstracts. P.A.-Psychological Abstracts, and P.I.-Population Index). Abbreviations for journals are those in A World List of Scientific Periodicals Published in the years 1900-1933, 2nd Edition. For journals not listed, abbreviations were devised to the general rules used in the above source. It is impossible to cover all journals and list all papers concerned with aging and the aged. Authors and publishers are requested to call attention to publications or to send reprints to the Gerontology Research Center, Baltimore City Hospitals, Baltimore, Maryland 21224.


A bibliography of doctoral dissertations on aging from American institutions of higher learning, 1976-1978. This bibliography is the eighth supplement to the original title which covered 1934--1969. The supplements include all titles of earlier years which were found after the original bibliography was published, Journal of Gerontology, 1971,26, 391--422. Due to the nature of bibliographic control in regard to doctoral dissertations, each supplement will try to cover the academic year rather than the physical year. This means that a dissertation issued in 1970 may be either in the original bibliography or in the supplements. The arrangements of the supplement is similar to the original bibliography.


On death, dying, and terminality: today, yesterday, and tomorrow. Not long ago, America viewed death and dying with a familiar fear and hostility. But today the subject has become fashionable, part of the transient pop culture, and as a consequence, trivialized and sentimentalized. When lifespans were shorter, deaths quicker, and man felt helpless before nature, death and dying were repeated intruders into the daily routine, usually occurring at home. But with the birth of the modern clinic in late eighteenth century Europe, the focus shifted from the patient to his affliction. Medicine became more scientific and effective as a result; yet the patient has suffered from this change, too, often feeling profoundly alienated from the entire medical apparatus. For terminally ill patients, the problem is especially severe; but the hospice approach (patient-centered like the old medieval hospital, and yet owing much to modern specialization and technology) promises physical and emotional amelioration of suffering in an imperfect but valuable departure from recent tradition.


Horseradish peroxidase labeled antisera--a diagnostic method for evaluating the percentages of B lymphocytes in peripheral blood. Horseradish peroxidase labeled antisera supplied commercially was used to evaluate B lymphocytes in peripheral blood. This technique not only gave comparable results to the conventional fluorescein method, but also proved to be more advantageous.


Catalase in skeletal muscle fibers.

Catalase in skeletal muscle fibers. Catalase has been localized immunocytochemically with anti-bovine catalase in long thin filament structures in aerobic type I fibers in the skeletal muscles of normal and genetically dystrophic hamsters. The filaments range in length from 1 to 60 micron, are orientated regularly along the long axis of the fibers, and also seem to surround and project from muscle nuclei. The enzyme thus appears to be more prominent in the sarcoplasmic reticulum than in peroxisomes, and in this situation is suitably placed for destroying toxic hydrogen peroxide which may be continously generated in aerobic fibers.


Alpha-bungarotoxin-horseradish peroxidase conjugate: preparation, properties and utilization for the histochemical detection of acetylcholine receptors. A method is presented for the efficient conjugation of horseradish peroxidase to alpha-bungarotoxin. The 1:1 molar conjugate obtained is purified to completion by gel filtration on Sephadex G-100, followed by ion exchange chromatography on CM-Sephadex. The conjugate retains half of the activity of unmodified horseradish peroxidase and binds effectively to the nicotinic acetylcholine receptor of muscle. The conjugate is proven to be useful reagent for the histochemical staining of the receptor on muscle fibers for light and electron microscopy.


Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques. Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.


An instrument for measuring bacterial penetration through fabrics used for barrier clothing. A new instrument has been designed to measure the penetration by rubbing of bacteria from cloth contaminated in the nursing of burn patients through fabrics designed for barrier garments. Most fabrics tested dry reduced the transfer of bacteria from the source cloth to about 10%, irrespective of the results of air filter tests, which agrees with mock nursing results. When the fabrics were tested against a wet surface, the transfer of bacteria rapidly reached 100% if the fabrics had a high wettability, but was slower for fabrics with a low wettability. Through closely woven waterproofed cotton, transfer was 5--25%, but increased three- to four-fold after ten launderings, in line with the water absorption. Transfer through plastic-laminated material was less than 1%. The results suggest that barrier garments should be made either of plastic or of recently waterproofed closely woven cotton at points of contact between nurse and patient where the clothes may be wetted by bacteria-containing wound secretions.


Differentiation of Mycoplasma mycoides subsp. mycoides from certain closely related caprine mycoplasmas by mycoplasmaemia and cross-protection tests in mice. In recent years, mycoplasma taxonomists have found that numerous mycoplasma strains from goats are serologically indistinguishable from Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia (CBPP), by routinely used tests, e.g. the metabolism- and growth-inhibition tests. As a result, such organisms are now openly referred to as M. mycoides subsp. mycoides. Seven of these so-called M. mycoides subsp. mycoides strains from goats were compared with two strains of M. mycoides subsp. mycoides from CBPP, and with one strain of M. mycoides subsp. capri, by means of two in-vivo tests, namely, (1) a test of the ability of each strain, injected intraperitoneally into mice, to produce mycoplasmaemia, and (2) a cross-protection test in mice. Of the seven strains, only one ('O goat') was indistinguishable from genuine M. mycoides subsp. mycoides; it also had small colonies resembling those of genuine M. mycoides subsp. mycoides. The other six were easily distinguished from genuine M. mycoides subsp. mycoides, and they produced large colonies. These six strains and others like them should no longer be given a name that fails to distinguish them from the causative agent of CBPP. Cross-protection tests showed that the seven goat strains referred to above differed from M. mycoides subsp. capri.


Four bills of health issued from Malta by the Order of St John. The specimens here described enlarge our knowledge of the bills of health of the Order of St John issued from Malta, an aspect of public health that had hitherto remained unexplored. These lone survivors from a vanished by-way of Maltese medical history formed, in their age, an integral part of the daily social, commercial and sanitary pattern of life in Malta. The earliest met so far, and described in the present series, dates from 1713; two belong to 1766 and another dates from 1794. A further bill, issued in 1782, has been published elsewhere. The chance preservation of these documents in unlikely places, as here indicated, makes it probable that additional ones may eventually come to light.


Serotypes of antibiotic resistant Escherichia coli isolated from the sewage of Palmerston North (New Zealand). Antibiotic resistant strains of Escherichia coli were isolated from the domestic sewage of Palmerston North. The ability of these strains to transfer the drug resistance markers to two different recipient E. coli strains was assessed. The 'O' and 'H' serotypes of all strains were determined and attempts at correlation with the drug resistance studies were made. Some typically 'human' serotypes were found as well as a number of rarely described ones. No correlation between serotype and resistance pattern was observed.


An investigation into the incidence and sources of klebsiella infections in hospital patients. Coliforms isolated from infections over a period of 22 months were examined and 16% were shown to be klebsiellas. The biochemical reactions and serotypes of these klebsiellas were determined and the relationship between species, serotype and type of infection investigated. Although no obvious outbreaks of infection occurred during this period a number of clusters of isolations of the same serotype of klebsiella were found. Using an enrichment method for the isolation of klebsiellas from faeces, sero and bacteriocin typing, and the examination of ten colonies, it was demonstrated that half of the patients carried a klebsiella of the same type in the bowel as caused the infection.


A survey of trimethoprim resistance in the enteric bacterial flora of farm animals. For 29 months the Veterinary Investigation Centres, covering the whole of Great Britain, forwarded trimethoprim-resistant gram negative enteric bacteria to the Wellcome Research Laboratories. These were examined for degree of resistance, presence and type of R factors. Trimethoprim resistance was found in 0.6% of the total number of strains examined by the Veterinary Investigation Centres. Trimethoprim R factors were demonstrated in one quarter of the resistant strains, and R factors were found in two strains of Salmonella typhimurium. It was concluded that while the incidence of trimethoprim resistance revealed by the survey gave no cause for alarm, the detection of resistant strains, and particularly R factors, indicated that the drug should continue to be used only for specific therapeutic purposes.


Immunoregulation of Heymann's nephritis. I. Induction of suppressor cells. Pretreatment of Lewis rats with a series of injections of a renal tubular antigen (RTA) in IFA prevented induction of Heymann's nephritis (HN) when the rats were challenged with RTA in FCA. This absence of disease was confirmed by immunofluorescent staining for rat IgG and histologic examination of the kidneys as well as by lack of development of significant proteinuria. Passive transfer of spleen and lymph node cells from rats receiving such pretreatment into syngeneic recipients prevented induction of HN when these recipients were challenged with RTA in FCA. Passive transfer of serum obtained from pretreated rats was without effect. These results suggest that one of the mechanisms involved in preventing HN by this pretreatment regimen was the induction of suppressor cells. The results of spleen cell transformation indicated that the suppressor cells were specific for RTA as the immune response to a second antigen, PPD, was unaffected. When rats already had active early HN, the diseas course was unaffected by transfer of suppressor cells.


T cell helper defect in patients with chronic lymphocytic leukemia. Purified peripheral blood T lymphocytes from normal donors were shown to help allogeneic tonsillar B cells to differentiate and secrete specific anti-SRBC antibody in vitro in a plaque-forming assay. Utilizing this system, a comparison was made between the allogeneic helper activity generated by the T cells of normal individuals and patients with various disease states. Allogeneic helper activity was absent when T lymphocytes from patients with CLL were used. Conversely, relatively normal allogeneic helper function was provided by T cells of patients with a variety of other disorders studied. Thus, a functional deficiency was identified in CLL patients in the subpopulation of regulatory T cells responsible for providing helper activity in allogeneic interactions.


The immunochemistry of antibodies sharing concanavalin A's anti-mannosyl binding specificity. Procedures were developed for the synthesis of the disaccharide hapten, p-isothiocyanatophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside, and for its conjugation to hemocyanin. The synthetic carbohydrate: protein antigen was then emulsified in complete Freund's adjuvant and injected into the footpads of New Zealand White rabbits. A population of the resulting anti-conjugate antibodies displayed some binding properties analogous to concanavalin A, the carbohydrate-binding protein of the jack bean. The antisera weakly percipitated mannans from Saccharomyces rouxii, S. cerevisiae, and an alpha-(1 leads to 3)-mannopyranosyl transferase-deficient mutant from Kluyveromyces lactis Y58a. These polysaccharides, possessing side chains containing terminal alpha-(1 leads to 2)-mannobiosyl residues, produce strong percipitation reactions with concanavalin A. In addition, various saccharides were tested for their ability to inhibit the interaction of anti-conjugate antisera with alpha-(1 leads to 2)-mannobiosyl-containing polymers. p-Nitrophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside showed a strong complementarity for the binding sites of both the anti-conjugate antisera and concanavalin A. However, the antibody failed to bind a concanavalin A-reactive mouse fibrosarcoma or to stimulate mitogenesis of human peripheral lymphocytes.


Immunoadjuvant treatment of primary grafted and spontaneous AKR-leukemia. I. Treatment efficiency correlated to autoimmune reactivity. Young AKR mice grafted i.v. with 10(1) or 10(3) cells from spontaneous AKR thymomas were treated with repeated i.v. injections of BCG or subcutaneous injections of irradiated AKR thymoma cells. BCG often cured mice from graft leukemia, whereas the effect of irradiated thymoma cells was less effective. Mice that did not develop graft-leukemia after graft of 10(1) leukemia cells and BCG treatment showed a spontaneous leukemia in 30% of the cases later. Ninety percent of nongrafted mice developed spontaneous leukemia whether BCG-treated or not. General immune reactivity as assessed in individual mice by T and B lymphocyte mitogen tests as well as the hemolytic plaque-forming cell assay had no clear correlation to the effects of immune adjuvants in respect to survival. In contrast, occurrence of self-directed immune reactions were clearly correlated to survival and cure of grafted and BCG-treated mice as revealed by assays both in vitro and in vivo. However, 13 to 25% of the mice apparently cured of leukemia developed a wasting-like syndrome that sometimes terminated in death. The immplications of self-directed immune reactions as mediators of the anti-neoplastic effects of immunoadjuvants.


Secretory component: interactions with intracellular and surface immunoglobulins of human lymphoid cells. Unstimulated and PWM-stimulated lymphocytes from normal human peripheral blood, cord blood, peripheral blood of patients with panhypogammaglobulinemia and selective IgA deficiency, as well as human lymphoblastoid cell lines were examined for their ability to bind secretory component (SC) on the surface and in the cytoplasm. SC binding was not detected on the cell surface at any stage of differentiation in these cells. However, binding of SC was detected in the cytoplasm of 2.3% of normal peripheral blood lymphocytes cultured in the presence of PWM for 6 to 7 days, and in two IgA producing lymphoblastoid cell lines. The capability of lymphoid cells to bind SC was not concurrent with J chain production. Although IgA was detected in the cytoplasm of PWM-stimulated lymphocytes from IgA-deficient patients, these cells did not bind SC. The failure to detect surface receptors indicates that SC is not a probable factor determining the homing of IgA precursor cells into exocrine tissues.


Lack of responsiveness of C3H/HeJ macrophages to lipopolysaccharide: the cellular basis of LPS-stimulated metabolism. The rate of glucose utilization has been used as a measure of LPS-induced activation of cultures of C3H/HeN and C3H/HeJ spleen cells, peritoneal cells, and purified peritoneal adherent cells. Peritoneal cells utilized 40 to 60 times more glucose than did spleen cells and purified adherent monolayers were more active than mixed peritoneal cells, suggesting that only macrophage metabolism was being measured. The cell preparations for C3H/HeJ mice were not activated by Escherichia coli K235 LPS prepared by extensive phenol extraction, whereas C3H/HeN cells were activated by the LPS. Cells from both strains were activated by a commercially obtained E. coli 0111:B4 LPS and butanol-extracted K235 LPS. The addition of 10% C3H/HeN spleen cells to C3H/HeJ peritoneal cells resulted in a marked enhancement of glucose utilization. These findings suggest that LPS-induced enhancement of macrophage metabolism occurs both by direct action of LPS on macrophages as well as indirectly through activated lymphocytes.


Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. IV. Role of macrophage and its soluble factor in antigen-induced MIF production of immune T lymphocytes. Histocompatibility-linked restriction of macrophage-T lymphocyte interaction in antigen-induced MIF production by sensitized lymphocytes was examined, by using combinations of inbred strain 2, strain 13, and JY-1 guinea pigs. The effective interaction of the antigen-bearing macrophages with the immune T lymphocytes was observed when the donor of the antigen-bearing macrophages and that of the immune lymphocytes shared Ia antigens of the major histocompatibility complex. Identities of B antigens and S antigens were not important for this cooperation. It was further demonstrated that the previously reported soluble factor derived from LPS-stimulated peritoneal adherent cells (macrophages) could help antigenic activation of the immune lymphocytes across the strain barrier provided a small number of macrophages (0.01%) from syngeneic strain were present. These results show that the presence of macrophages is absolutely required to present antigen to immune T lymphocytes in a genetically restricted manner and the soluble factor from macrophages appears to give a nonspecific effect on the lymphocyte activation in addition to or in collaboration with antigenic stimulation.


Kinetic studies of cell migration in a modified Boyden chamber: dependence on cell concentration and effects of the chymotrypsin-like cationic protein of human granulocytes. Migration of PMN cells was measure kinetically by the "leading front" method in a modified Boyden chamber. In some experiments, release of lactoferrin and of chymotrypsin-like cationic protein were measured simultaneously. Migration, both directed and random, was increased by increased cell concentration. Kinetically, directed migration consisted of two phases. The second phase was more influenced by the cell concentration. Preincubation of the cells with low concentrations of chymotrypsin-like cationic protein stimulated cell migration in the same way as did high cell concentrations, i.e., by primarily affecting the second phase. The effect of chymotrypsin-like cationic protein was time and dose dependent and dependent on the chymotrypsin-like activity of the protein. At high concentrations of protein, the cells were immobilized. Release of granular proteins did not take place during the first phase of migration but was parallel to the second phase. It is concluded that chymotrypsin-like cationic protein might be one of the substances responsible for the effect of cell concentration seen on migration in vitro.


Cell differentiation in the presence of cytochalasin B: studies on the "switch" to IgG secretion after polyclonal B cell activation. Mouse spleen cells were cultured with lipopolysaccharide in conditions that activate both IgM and IgG secretion. Addition of cytochalasin B (CB), an inhibitor of cytokinesis, lead to a high degree of polynucleation, with little effect on Ig secretion. Using cytoplasmic staining with fluorochrome conjugated antisera, we determined the numbers of IgG-containing cells that also contained IgM in their cytoplasm. Such double staining cells were relatively more frequent at early times of the cultures, but at all times single producing cells were in the majority. Addition of CB over the period when the IgG producing cells first appear, lead to a marked increased frequency of double staining, polynucleated cells. This characteristic was stable over a period of at least 42 hr, suggesting that each double staining cell actively synthesized both isotypes. When CB was added after IgG production had started, little increase in the numbers of double staining cells were observed, although polynucleation remained extensive. These data confirm previous findings that the lineage of one cell can produce both IgM and IgG. Furthermore, the results suggest that cells in the process of switching from IgM to IgG go through an asymmetric division leading to one IgM-producing and one IgG-producing daughter cell.


Lipid synthesis: an indicator of antigen-induced signal transduction in antigen-binding cells. A biochemical parameter of lymphocyte activation, lipid synthesis, has been measured in a purified specific antigen-binding cell population (ABC). ABC isolated form immune and nonimmune animals by sequential centrifugation on buoyant density and sedimentation velocity gradients have a 2- to 7-fold higher rate of 14-C choline incorporation into phospholipid than either unfractionated spleen cells or cells depleted of ABC. Aslo ABC from immune animals were shown to have a 4- to 7-fold higher rate of 14C-acetate incorporation into their neutral lipids than nonbinding controls. The elevated lipid synthesis seen in both nonimmune SRBC-ABC and TNP-SRBC ABC indicates that antigenic contact via the B cell immunoglobulin receptor results in signal transduction and activation of the specific receptor-bearing lymphocyte population. Binding of the same particle (SRBC) to B cells via their Fc receptors did not regularly result in activation of lipid synthesis. The magnitude of the increased lipid synthesis in ABC populations approached that seen in LPS-stimulated spleen cells. We propose that the measurement of early activation events in purified ABC may be a more appropriate criterion for antigen-induced signals that later events such as thymidine incorporation or antibody secretion.


Further studies on the activation of lymphocytes by endotoxin protein. Endotoxin protein, an outer membrane protein of Gram-negative bacteria associated with lipopolysaccharide endotoxin, has been found to be a potent activator of lymphocytes. In the absence of T lymphocytes and macrophages, endotoxin protein can stimulate murine B lymphocytes to synthesize DNA and produce antibodies of diverse specificities. This stimulation is greater than that obtained with two well known murine B cell activators, lipopolysaccharide endotoxin or PPD-tuberculin. Splenic lymphocytes from other species, such as rats, rabbits, and guinea pigs also proliferate when cultured with endotoxin protein. Of particular significance is the finding that endotoxin protein is an activator of human peripheral blood lymphocytes.