Long-term incorporation of tritiated adenine into deoxyribonucleic acid and ribonucleic acid by Treponema pallidum (Nichols strain)

1980 ◽  
Vol 29 (3) ◽  
pp. 1040-1049 ◽  
Author(s):  
S J Norris ◽  
J N Miller ◽  
J A Sykes
Keyword(s):  
Ribonucleic Acid ◽  
Trichloroacetic Acid ◽  
Deoxyribonucleic Acid ◽  
High Efficiency ◽  
Treponema Pallidum ◽  
Acid Synthesis ◽  
Penicillin G ◽  
Rabbit Serum ◽  
Partial Purification ◽  
Normal Rabbit Serum

Treponema pallidum (Nichols strain), extracted in medium containing Eagle minimal essential medium 50% fresh, heat-inactivated normal rabbit serum, and 1.0 mM dithiothreitol, was incubated under 3% oxygen in the presence of tritiated nucleic acid precursors. [8-3H]adenine was incorporated with high efficiency into trichloroacetic acid-insoluble material; 2'-deoxyadenosine and uridine were incorporated in lower quantities, and thymine and thymidine were not incorporated. Incorporation of [3H]adenine was inhibited by penicillin G, mitomycin C, actinomycin D, and erythromycin, but was not affected by cycloheximide. Partial purification of nucleic acids from T. pallidum incubated with [8-3H]adenine for 36 to 72 h and subsequent treatment with ribonuclease and deoxyribonuclease revealed that 15 to 20% of the trichloroacetic acid-precipitable counts were resistant to ribonuclease but susceptible to deoxyribonuclease. A simple assay was developed in which NaOH treatment was used to distinguish incorporation into ribonucleic acid and deoxyribonucleic acid. Both ribonucleic acid and deoxyribonucleic acid synthesis continued for 6 days of incubation under 3% O2, whereas incorporation was limited to the first day of incubation in samples incubated under aerobic or anaerobic conditions. T. pallidum thus appears to be capable of significant de novo deoxyribonucleic acid and ribonucleic acid synthesis under microaerobic conditions.

scholarly journals STUDIES ON THE MECHANISM OF THE FORMATION OF THE PENICILLIN ANTIGEN

1961 ◽  
Vol 114 (6) ◽  
pp. 875-940 ◽  
Author(s):  
Bernard B. Levine ◽  
Zoltan Ovary
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Penicillin G ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Human Beings ◽  
Mixed Disulfide ◽  
Diastereomeric Mixture ◽  
Potassium Penicillin

An excess of D-benzylpenicillenic acid (BPE) was reacted with human γ-globulin, human serum albumin, gelatin, and poly-L-lysine in aqueous solution buffered at pH 7.5–8.0. Under these conditions, BPE reacted predominantly with lysine ϵ-amino groups of the proteins to form the mixture of diastereomers of ϵ-N-(D-α-benzylpenicilloyl)-lysine groups (Di-BPO-Lys). BPE reacted also, but to a considerably smaller extent, with cystine disulfide linkages of human γ-globulin and human serum albumin to form D-benzylpenicillenic acid-cysteine mixed disulfide groups (BPE-SS-Cys). Conjugates containing large numbers of BPE or D-penicillamine mixed disulfide groups were prepared by reaction of BPE or D-penicillamine with thiolated human γ-globulin under mild oxidizing conditions. Anti-penicillin antibodies were produced in rabbits by immunization with either potassium penicillin G (PG) or a preincubated mixture of PG with normal rabbit serum (PG-NRS) in complete Freund's adjuvant. Specific precipitation analyses in aqueous and gel media (Ouchterlony), PCA analyses, and specific inhibition of these reactions with haptens were carried out on the rabbit anti-PG and anti-(PG-NRS) sera, using the above conjugates as antigens. The anti-penicillin antibodies were found to be directed against the diastereomeric mixture of N-(D-α-benzylpenicilloyl) groups, predominantly the Di-BPO-Lys groups. By these techniques, no antibodies directed against the BPE-mixed disulfide or the D-penicillamine mixed disulfide groups were detected. Three out of six patients with histories of allergic reactions to PG responded with wheal-and-erythema reactions to the N-(D-α-benzylpenicilloyl) (BPO) groups contained in BPE-human gamma globulin conjugate. Another such patient exhibited serum antibodies specific for the BPO group. One patient being treated with 25 gm per day of PG showed the presence of non-dialyzable antigenic BPO-conjugates in his serum. These results demonstrate that the diastereomeric BPO groups (predominantly Di-BPO-Lys groups) are major antigenic determinant groups responsible for PG hypersensitivity in rabbits and human beings. The possible clinical usefulness of multivalent Di-BPO conjugates and univalent Di-BPO haptens is discussed.

Induction of cellular deoxyribonucleic acid synthesis in butyrate-treated cells by simian virus 40 deoxyribonucleic acid

1981 ◽  
Vol 1 (11) ◽  
pp. 1038-1047
Author(s):  
S Kawasaki ◽  
L Diamond ◽  
R Baserga
Keyword(s):  
Ribonucleic Acid ◽  
Deoxyribonucleic Acid ◽  
Simian Virus 40 ◽  
Acid Synthesis ◽  
Simian Virus ◽  
S Phase ◽  
Temperature Sensitive ◽  
3T3 Cells ◽  
Deoxyribonucleic Acid Synthesis ◽  
G1 Cells

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells, Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in B1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensitive block. These results confirm previous reports that certain virally coded proteins can induce cell deoxyribonucleic acid synthesis in the absence of cellular functions that are required by serum-stimulated cells. Our interpretation of these data is that butyrate inhibited cell growth by inhibiting the expression of genes required for the G0 leads to G1 leads to S transition and that the product of the simian virus 40 A gene overrode this inhibition by providing all of the necessary functions for the entry into the S phase.

scholarly journals DEFICIENCY OF THE SIXTH COMPONENT OF COMPLEMENT IN RABBITS WITH AN INHERITED COMPLEMENT DEFECT

1966 ◽  
Vol 124 (4) ◽  
pp. 773-785 ◽  
Author(s):  
Klaus Rother ◽  
Ursula Rother ◽  
Hans J. Müller-Eberhard ◽  
Ulf R. Nilsson
Keyword(s):  
Complement System ◽  
Hemolytic Activity ◽  
Complement Deficiency ◽  
Rabbit Serum ◽  
Partial Purification ◽  
Normal Rabbit Serum ◽  
Chemically Modified ◽  
System A ◽  
Inactive Form

A strain of rabbits with an inherited complement deficiency was shown to lack the sixth component of the hemolytic complement system. A method was elaborated for the partial purification of this component from normal rabbit serum. Upon injection of partially purified rabbit C'6 into C'6-deficient animals, an antibody was obtained which specifically inhibited the hemolytic activity of C'6. The data suggest that C'6-deficient serum either lacks the C'6 molecule or contains it in a chemically modified and inactive form.

Partial purification of antigens collected during in vitro cultivation of the larval stages of Taenia pisiformis

Parasitology ◽  
1976 ◽  
Vol 72 (3) ◽  
pp. 269-279 ◽  
Author(s):  
M. D. Rickard ◽  
J. C. Katiyar
Keyword(s):  
Protective Immunity ◽  
Culture Medium ◽  
Serum Proteins ◽  
Skin Tests ◽  
Rabbit Serum ◽  
Partial Purification ◽  
In Vitro Cultivation ◽  
Normal Rabbit Serum ◽  
Skin Reactions

SummaryLarvae of Taenia pisiformis were cultured in vitro in medium containing 2·5, 5 or 20% (v/v) of normal rabbit serum (NRS). Greatest development occurred in 20% NRS, and the potency of antigens collected in medium from each culture tested by intradermal (i/d) skin tests in infected rabbits paralleled the in vitro growth rate of larvae. ‘Culture’ antigens from 5% NRS stimulated good immunity in rabbits to a challenge infection with T. pisiformis eggs, although they were poorly reactive in skin tests.T. pisiformis larvae were also cultured in 10% (v/v) of nitrates of serum reduced to one-half of its volume by passage through 300 000 MW cut oif (XM300F) or 100000 MW cut off (XM100F) ultrafiltration membranes. Larvae cultured using XM300F had growth rates comparable with those cultured in 20% NRS, and the antigens released into the culture medium had equal potency in i/d tests and in stimulating protective immunity in rabbits. Larvae did not develop in XM100F orproduce skin-reactive or protective antigens.Crude ‘culture’ antigen from cultures in 20% NRS was separated into 4 fractions by nitration on Sephadex G200. All of these fractions gave i/d skin reactions in infected rabbits. Fraction 3 (F3) was the most active, but was shown by acrylamide gel electrophoresis and immunoelectrophoresis to be highly contaminated with rabbit serum proteins. F3 was separated into fractions on DEAE-Sephadex A50, and the third fraction from this was as active as the original culture medium in i/d skin tests, but had only 5% of the original protein concentration. Electrophoresis demonstrated few serum contaminants, and 2 indistinct protein bands that were not present in a similar fraction of NRS.Neither Sephadex G200 F3 nor DEAE-Sephadex F3 stimulated protective immunity in rabbits, suggesting that antigens stimulating immunity against the establishment of T. pisiformis in rabbits and those provoking cell-mediated immune reactions may be different.

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