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PubMed1956

Transformation reactions with two non-allelic R mutants of the same strain of Pneumococcus type VIII.

Two distinct R mutants of pneumococcus type VIIIS have been described in which the mutations leading to loss of type VIII polysaccharide production are non-allelic. A DNA extract prepared from either R strain restores the capacity to produce type VIII polysaccharide in the other strain. Autologous extracts are inert in this respect. DNA extracts from pneumococcus type IIS when applied to the two R mutants consistently yielded a high proportion of cells transformed to type VIIIS and relatively small numbers of type IIS cells. When extracts prepared from other encapsulated pneumococci, types IS, VIIS, XIVS, and XVIIIS, were applied to R mutant VIII-R1, a mixture of type VIIIS cells and cells corresponding to the type of the donor appeared, although the proportion of type VIIIS cells was not uniformly in excess. The same extracts applied to the second R mutant, VIII-R13, yielded transformed cells of the type of the donor only, or else transformation did not occur. Transforming extracts of a strain of pneumococcus type IIIS gave rise only to type IIIS pneumococci when applied to either strain. Type VIIIS pneumococci were never recovered from such interactions.

Mutation↗

PubMed1957

Transformation reactions between Pneumococcus and three strains of Streptococci.

Reciprocal transformation reactions to streptomycin resistance have been demonstrated between pneumococcus and 2 strains of viridans streptococci, which by the usual bacteriological criteria are distantly related to pneumococcus. Desoxyribonucleic acid (DNA) extracts of a strain of streptomycin-resistant Streptococcus salivarius have been found capable of transforming pneumococcus and two strains of viridans streptococci but the strain itself was not transformed by autologous or heterologous DNA extracts. The two strains of viridans streptococci were more efficient than pneumococcus as receptors of both autologous and heterologous transforming principles. It is inferred, therefore, that efficiency of transformation does not necessarily indicate closeness of relationship among bacteria. The efficiency of transforming principles (TP's) from three streptococcal strains to induce streptomycin resistance in pneumococcus increased following their reduplication in pneumococcus. At the same time the efficiency of two of these TP's to transform streptococcus was reduced. Resistance to optochin (ethylhydrocupreine), a characteristic of most streptococci, can be transferred to pneumococcus, a naturally susceptible species, by transformation reactions.

Carrier Proteins↗

PubMed1958

Surface phagocytosis; further evidence of its destructive action upon fully encapsulated pneumococci in the absence of type-specific antibody.

Experiments recently reported (18) have been interpreted to indicate that surface phagocytosis plays no significant part in natural antipneumococcal defense. A repetition of these experiments has revealed: (a) that the cellular content of the leucocytic suspensions used in the phagocytic tests was of a different order of magnitude from that of the exudates which usually exist in infected tissues, (b) that the suspensions were too dilute to allow surface phagocytosis of pneumococci to occur, and (c) that the ratio of bacteria to leucocytes. was such that, when a sufficiently concentrated exudate was employed, the pneumococci injured the leucocytes and thus prevented phagocytosis from taking place. When conditions of the tests were suitably controlled, and conventional quantitative methods were employed to measure the end results of the phagocytic reaction, the essential observations relating to surface phagocytosis were fully confirmed. The significance of this non-antibody mechanism of defense in pneumococcal infections was thus further substantiated.

Antibodies↗

PubMed1959

Determination of type in capsulated transformants on pneumococcus by the genome of non-capsulated donor and recipient strains.

Capsulated transformants producing type II polysaccharide have been obtained by reactions between a number of R mutants of type II pneumococcus. All the IIR strains except II-R36 yielded extracts which transformed 2 VIIIR strains to VIIIS. Two of the IIR strains were transformed to VIIIS by an extract of 1 VIIIR strain. The most striking result was the production of both IIS and VIIIS transformants by the action of extracts of both VIIIR strains applied to cells of II-RB. This suggests that the determination of capsular type in pneumococcus does not depend on single allelomorphic genes; it seems more likely that a number of genes may be required to determine type specificity, and that some genes may be common to different types, possibly in connection with common biochemical pathways for some stages of polysaccharide synthesis. This is further indicated by the finding that extracts of capsulated strains of each of the types I, II, III, VII, VIII, XIV, and XVIII are capable of giving IIS transformants with 1 or more of the IIR strains in addition to the expected donor-type transformants. Strain II-R36 was transformed by extracts of all S types except type VII, but the only transformants found were of the donor type. It appears that II-R36 may have multiple genetic deficiencies for type II capsulation, possibly in the form of a deletion. This may be the case also with IIIS strains since only IIIS transformants were found when IIIS extract was applied to any of the IIR strains.

Polysaccharides↗

PubMed1959

Investigations concerning the changes in serum proteins during immunization; the cause of hypoalbuminemia with high gamma globulin values.

On hyperimmunization of rabbits with pneumococcal vaccine, increase in the content of gamma globulin and fall of albumin in the serum are produced. In such hyperimmunized rabbits, investigations of the plasma volume, albumin degradation, and the distribution of the albumin intra- and extravascularly were undertaken by Sterling's method employing rabbit albumin labelled with I(131). The investigations do not suggest any significant alteration in the albumin degradation nor in the distribution between the intra- and extravascular albumin. It was demonstrated that the plasma volume increases simultaneously with increase in the gamma globulin and fall in albumin. It is concluded that the fall in serum albumin observed is due to a regulative mechanism which attempts to maintain the colloid-osmotic pressure by increasing the plasma volume.

Animals↗

PubMed1959

Simultaneous production of two capsular polysaccharides by pneumococcus. I. Properties of a pneumococcus manifesting binary capsulation.

Pneumococci manifesting binary capsulation have been produced through the agency of transformation reactions. Such cells produce two capsular polysaccharides which are closely similar if not identical with those produced by corresponding singly capsulated strains. Binary capsulation in pneumococcus provides a new basis for serologic cross-reactivity in this species.

Cross Reactions↗

PubMed1959

Simultaneous production of two capsular polysaccharides by pneumococcus. II. The genetic and biochemical bases of binary capsulation.

Study of the capsular genome of pneumococcus has shown that it controls a multiplicity of biochemical reactions essential to the synthesis of capsular polysaccharide. Mutation affecting any one of several biochemical reactions concerned with capsular synthesis may result in loss of capsulation without alteration of other biochemical functions similarly concerned. Mutations affecting the synthesis of uronic acids are an important cause of loss of capsulation and of virulence by strains of pneumococcus Type I and Type III. The capsular genome appears to have a specific location in the total genome of the cell, this locus being occupied by the capsular genome of whatever capsular type is expressed by the cell. Transformation of capsulated or of non-capsulated pneumococci to heterologous capsular type results probably from a genetic exchange followed by the development of a new biosynthetic pathway in the transformed cell. The new capsular genome is transferred to the transformed cell as a single particle of DNA. Binary capsulation results from the simultaneous presence within the pneumococcal cell of two capsular genomes, one mutated, the other normal. Interaction between the biochemical pathways controlled by the two capsular genomes leads to augmentation of the phenotypic expression of the product controlled by one and to partial suppression of the product determined by the other. Knowledge of the biochemical basis of binary capsulation can be used to indicate the presence of uronic acid in the capsular polysaccharide of a pneurnococcal type the composition of the capsule of which is unknown.

Mutation↗

PubMed1960

The immunological specificity of type II Pneumococcus and its separation into partial specificities. II.

Quantitative data are given on the cross reactions in Type II antipneumococcal horse sera of plant gums and hemicelluloses containing multiple terminal groupings of glucuronic acid and/or 4-O-methylglucuronic acid. Great variability is shown both in the reactivities of the polysaccharides and in the antibodies in the sera of different animals immunized with the same antigen. The 4-O-methyl substituent on the glucuronic acid residues in a gum often appears to diminish cross-precipitation with antibodies to S II.

Animals↗

PubMed1962

Release of genetic transforming agent from pneumococcal cultures during growth and disintegration.

Genetic transformations of pneumococcus mediated by streptomycin-induced lysates were studied to gain some insight into the nature of freshly released transforming principle, and the influence of the physiologic state of the donor population on the transformation process. It was found that streptomycin could make the DNA of sensitive cells available for transformation of other cells. Living cultures of pneumococcus growing exponentially in ordinary media were also found to discharge significant quantities of genetically active DNA. Such cultures, not treated with any drug, showed no evidence of concomitant cell disintegration or death. Both single markers and small linkage groups could be transferred in transformations mediated by drug-induced lysates and by filtrates of living cultures. The quantity of DNA liberated is small (less than 0.1 microg per ml), but these transformations are at least as efficient as transformations mediated by purified DNA, when compared on the basis of total DNA available. Up to 1 per cent of the cells in an average recipient culture can be transformed by a small quantity of culture fluid. Both in drug-treated and in untreated cultures the amount of transforming activity increased and then decreased during growth of the culture. Although the source of transforming DNA in growing cultures could not be established, the decline in the transforming activity of aging drug-treated or untreated cultures was attributed to the presence of deoxyribonuclease. The release of this nuclease by pneumococcal cultures midway in exponential growth is sufficient to result in a mild degradation of the low concentration of freshly released transforming agent present. Maximal release of active transforming agent by a living culture coincided in time with the development of maximal receptivity to exogenous DNA by that culture. As a result, recombinants could be recovered from appropriately genetically marked strains growing in each other's presence. In view of these results, it seems possible that DNA-mediated transformations might provide, or might have provided, a mechanism of genetic recombination in nature for some bacterial species in which sexual mechanisms may not be available.

DNA, Bacterial↗

PubMed1963

STUDIES ON THE RELATIONSHIP OF ALLOTYPIC SPECIFICITIES TO ANTIBODY SPECIFICITIES IN THE RABBIT.

Purified antibodies against three different antigens and haptens (ovalbumin, dinitrophenol, and Type II pneumococcal polysaccharide) were prepared from specific precipitates from the antisera of several rabbits. The reactions of these preparations with antisera against the allotypic specificities carried by each of the animals forming the antibodies were used to compare the relative concentrations of molecules bearing each specificity in the different solutions of antibodies from each rabbit. These relative concentrations appeared to vary greatly from one preparation to another, although all the allotypic specificities carried by each animal were found in all the preparations of antibodies from that animal. The interpretation of these results is discussed.

Animals↗

PubMed1964

GAMMA GLOBULIN TURNOVER IN RABBITS BEFORE AND DURING HYPERIMMUNIZATION.

The turnover of (131)I-labeled gamma globulin has been determined in rabbits before and during (8 weeks later) hyperimmunization with pneumococcic vaccine, which increased the gamma globulin concentration 5 to 10 times. Before immunization fractional turnover rate was an average of 36 per cent of the plasma pool per day, and the rate of catabolism was 133 mg/kg/day. During hyperimmunization fractional turnover rate was an average of 37 per cent per day, and the rate of catabolism was 1160 mg/kg/day. The observation that the fractional turnover rate is independent of the concentration suggests that the rate of breakdown should have the characteristic of a first order process.

Animals↗

PubMed1964

GAMMA GLOBULIN METABOLISM IN RABBITS DURING THE ANAMNESTIC RESPONSE.

1. Gamma globulin metabolism and distribution were studied employing rabbit gamma globulin (RGG)I(131) 24 times in 13 control rabbits. Similar studies were performed before and during the ananmestic response in 4 rabbits previously sensitized with a polyvalent pneumococcal vaccine. 2. During the ananmestic response, gamma-globulin levels increased from 1.0 to 6.0 gm/100 ml, and the gamma-globulin pool increased from 0.7 to 4.7 gm/kg. There was no change in the intravascular-extravascular partition of gamma globulin. 3. Gamma globulin degradation increased from 0.06 to 0.33 gm/kg/day during the 28 days of the immunization period while gamma globulin synthesis increased even further to average 0.47 gm/kg/day. Following the attainment of elevated gamma globulin levels the fractional rate of RGG-I(131) turnover increased from 8.0 to 12.5 per cent/day. 4. No differences were noted in the metabolism of homologous or autologous gamma globulin regardless of the allotypic specificities.

Animals↗

PubMed1964

STUDIES ON THE PATHOGENESIS OF FEVER. 13. THE EFFECT OF PHAGOCYTOSIS ON THE RELEASE OF ENDOGENOUS PYROGEN BY POLYMORPHONUCLEAR LEUKOCYTES.

1. Phagocytosis promotes the release of endogenous pyrogen from polymorphonuclear leucocytes. 2. The release of pyrogen, though initiated by the phagocytic event, is not synchronous with it. 3. The postphagocytic release mechanism is not inhibited by sodium fluoride and, therefore, appears not to require continued production of energy by the cell. 4. The release process, on the other hand, is inhibited by arsenite, suggesting the participation of one or more sulfhydryl-dependent enzymes in the over-all reaction. 5. Particle for particle, the ingestion of heat-killed rough pneumococci causes the release of approximately 100 times as much pyrogen as the ingestion of polystyrene beads of the same size. 6. The pyrogen release mechanism of polymorphonuclear leucocytes separated directly from blood, unlike that of granulocytes in acute inflammatory exudates, is not readily activated by incubation of the cells in K-free saline. Despite this difference, both blood and exudate leucocytes following phagocytosis release large amounts of pyrogen, even in the presence of K(+). The fact that the postphagocytic reaction is uninhibited by the concentrations of K(+) which are present in plasma and extracellular fluids, suggests that this mechanism of pyrogen release may well operate in vivo. 7. As might be expected from the foregoing observations, the intravenous injection of a sufficiently large number of heat-killed pneumococci causes fever in the intact host. Intravenously injected polystyrene beads, on the other hand, are significantly less pyrogenic. Evidence is presented to support the conclusion that the fever in both instances is caused by pyrogen released from the circulating leucocytes which have phagocyted the injected particles. 8. The possible relationships of these findings to the pathogenesis of fevers caused by acute bacterial infections are discussed.

Animals↗

PubMed1964

CROSS-REACTIONS OF PNEUMOCOCCAL TYPES. QUANTITATIVE STUDIES WITH THE CAPSULAR POLYSACCHARIDES.

Data are given on the amounts of antibody nitrogen precipitated in the crossreactions, often reciprocal, of the specific capsular polysaccharides of the pneumococcal type pairs II and XX, II and XIX, VII and XIV, VII and XVIII, VII and XIX, VIII and XVIII, VIII and XIX, X and XIV, and X and XX. Since little is known of the fine structures of the polysaccharides of Types VII, X, XIX, and XX, rigorous correlations between chemical structure and specificity can not be made, but several tentative conclusions are drawn.

Agglutination↗

PubMed1965

ENHANCEMENTS OF ANTIBIOTIC ACTIVITY OF COLICINE K AND ALTERATION OF SEROLOGICAL PROPERTIES OF COLICINE K AND ENDOTOXINS.

Hemoglobin, its chains, and myoglobin enhance the antibiotic activity of colicine K. These proteins also interact with colicine K and other O antigens to alter their serological activity. The hemoglobin proteins did not alter the serological activities of three Pneumococcus polysaccharides or T4 bacteriophage DNA antigens but did alter the antigenic activity of fetuin. Interaction of hemoglobin and colicine K resulted in a retardation of colicine K antibiotic moiety as measured by gel filtration but did not affect the gel filtration properties of the lipopolysaccharide moiety.

Anti-Bacterial Agents↗

PubMed1965

ANTI-DNA ANTIBODIES IN HYPERIMMUNIZED RABBITS.

Complement-fixing anti-DNA antibodies were detected in a minority of sera of rabbits hyperimmunized with killed Gram-negative bacteria. The C'-fixing property of DNA was lost after DNase treatment. Preferential reactivity with denatured DNA was observed. The antisera reacted with DNA preparations derived from rabbit bone marrow and thymus, calf thymus, pneumococci, salmon sperm, and Escherichia coli. E. coli DNA was less effective than preparations of mammalian and salmon sperm DNA in fixation of C'. Inhibition of DNA C' fixation by nucleotides and nucleosides was observed. The bulk of anti-DNA activity was associated with the low molecular weight antibody fraction.

Animals↗

PubMed1951

Absence of a prosthetic group in a type-specific polysaccharide of pneumococcus.

The specific polysaccharide of Type III pneumococcus, with only 0.05 per cent of N and 0.01 per cent of P, still shows almost maximal precipitation of a Type III antipneumococcus rabbit serum and the expected antigenicity in man. There is therefore no evidence that a prosthetic group is involved in these characteristic activities.

Animals↗

PubMed1953

Synthesis of type III pneumococcal polysaccharide by suspensions of resting cells.

The capsular polysaccharide (SIII) of type III pneumococci was removed enzymatically, and the cells thus deprived of preformed SIII were washed and examined for capacity to synthesize SIII anew. The washed, decapsulated cocci lost their capacity to be agglutinated in type-specific antiserum but again became agglutinable and formed readily measurable amounts of SIII, after suspension in a solution containing only glucose and salts. Maximal SIII synthesis required the presence of glucose, magnesium, potassium and phosphate ions, and oxygen. Other fermentable sugars could be substituted for glucose but then the yield of SIII was reduced. Synthesis of SIII occurred anaerobically but was increased four- to fivefold by oxygenation of the suspension. The effects of pH and of enzyme poisons on the capacity of the cocci to form SIII are described.

Immune Sera↗