INTRACELLULAR PHAGE PRECURSOR

1. Staphylococci activated by rapid growth in the presence of excess O₂ and subsequently brought to a resting state by storage in Locke''s solution at 5°C. produce a significant rise in [phage] when added to phage-containing solutions. 2. For satisfactory activation the staphylococci require a period of active growth in the presence of oxygen. Activation proceeds best on the acid side of neutrality although variation in pH from 5 to 9 has relatively little effect. Activated cells retain their phage-augmenting property for from 4 to 24 hours, and this property may be destroyed by heating the cells at temperatures which do not kill them. The critical thermal increment for heat inactivation is 90,000 suggesting that the reaction involves protein denaturation. 3. The reaction between activated cells and phage has the following characteristics: A. It is complete in 1 to 2 minutes after mixing the reactants. B. The increase in phage does not depend upon bacterial growth nor does it involve any untoward effect on the titration system. C. Serum prepared by injecting rabbits with normal live staphylococci or with activated staphylococci when mixed with activated cells before the addition of phage will prevent the customary increase in [phage]. 4. The phage-producing reaction which follows the addition of activated cells to phage can be interpreted in terms of the precursor theory. It is likely that the precursor either is a protein or contains a protein as an essential component. 5. There is no way of deciding at present whether the reaction between phage and precursor represents the hydrolytic cleavage of a protein or whether it is the final step in a synthesis catalyzed by phage.     

THE PHOTODYNAMIC INACTIVATION OF PHAGE PRECURSOR BY METHYLENE BLUE

Methylene blue added to suspensions of activated staphylococci in amounts sufficient to furnish 1 x 10⁵ molecules of dye/bacterium inactivates the phage precursor content of the cells without causing cell death when the mixtures are exposed to strong light of 4000–8000 Å. There is a lag phase of approximately 15 minutes in the photodynamic inactivation of phage precursor by methylene blue. This delay seems to be due to a primary reaction between the cell and methylene blue after the completion of which exposure to light brings about the inactivation of precursor quite promptly.     

THE ACCELERATING EFFECT OF MANGANOUS IONS ON PHAGE ACTION

Dilute solutions of MnCl₂ or MnSO₄ accelerate the lytic effect of phage upon susceptible staphylococci. Under the conditions of our experiments the manganese-containing mixtures lysed regularly 0.5 hour sooner than the controls. The effect is shown to be due to a lowering of the lytic threshold, i.e. the quantity of phage/bacterium requisite for lysis; Mn⁺⁺ reduces the ratio from 54 to about 12. In the presence of Mn⁺⁺ phage distribution is altered and in growing phage-bacteria mixtures the extracellular phage concentration is increased by manganese to approximately 4 times that occurring in the absence of manganese. There appears to be no enhancement of phage formation nor any affect on the rate of bacterial growth. As would be anticipated, for any given initial phage concentration the end titre after completion of lysis is less in the presence of manganese than in its absence. This is due to the reduced lytic threshold produced by Mn⁺⁺, there consequently being less phage needed to bring about lytic destruction of the bacteria.     

FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

1. The reaction between an antistaphlycoccal phage and the homologous bacterium has been studied, applying the following experimental technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining [phage]. (b) Sampling was done at short intervals; i.e., every 0.1 hour. (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of < 95 per cent of free phage. 2. Using these technics, the reaction was followed: (a) with pH maintained at 6.10 and temperature at 28°C. to slow the process; (b) with pH maintained at 7.2 and temperature at 36°C. 3. In addition separate experiments were performed on the sorption of phage by bacteria at 30°, 23°, and 0°C. 4. At pH 6.10 and 28°C. the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant. (b) When 90 per cent of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques; i.e., phage is formed intracellularly, but is retained within cellular confines. (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque). (d) With the concentrations of phage and bacteria used in the experiment carried out at pH 6.1 and 28°C. there are two further increments in [phage]_act. before massive lysis begins. (e) During terminal lysis there are sharp rises in the curves for [total phage]_plaq., [extracellular phage]_act., and [extracellular phage]_plaq.. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being of sufficient size to be removed by the super-cel filters. 5. At pH 7.2 and 36°C. the steps in the phage production curve as determined by activity assay and plaque count are much less prominent than those observed at pH 6.1 and 28°C. However, the plateaus described by Ellis and Delbrück (10) for B. coli and coli phage can be detected also in the present case if frequent samples are taken. 6. The sorption experiments show a significant rise in the rate of phage uptake with increase in temperature, again supporting the view that the reaction involves more than a purely physical adsorption. 7. Delbrück''s objections to: (a) the use of the activity assay for determining [total phage] in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. 8. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in solution or phage bound to living susceptible cells, under the conditions of the experiments reported here and in earlier work. 9. The titration values obtained in the experiments designed to exhibit intracellular phage precursor are not the result of artifacts as Delbrück has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out.     

Mechanism of Inactivation of Bacteriophage J 1 by Dithiothreitol,Cysteine, Mercaptoethanol,or Thioglycollate

The mechanism of inactivation of a double-stranded DNA phage, phage J1 of Lactobacilluscasei, by reducing agents containing thiol group(s) other than glutathione was studied mainly with dithiothreitol (DTT).

Air bubbling, oxidizing agents, and transition metal ions enhanced the rate of phage inactivation by DTT. Partial oxidation of DTT resulted in a more rapid rate of phage inactivation. In contrast, nitrogen bubbling, reducing agents including high concentrations of DTT itself, chelating agents, and radical scavengers prevented phage inactivation. Fully oxidized DTT had no phagocidal effect. These results indicate that the inactivating effect of DTT requires the presence of molecular oxygen and is indirectly caused by free radicals involved in the mechanism of DTT oxidation. The target attacked by DTT in phage particle was not protein but DNA; DTT reacted with DNA to produce single-strand scissions in DNA, which were the cause of inactivation of phage.

This was true also for L-cysteine, 2-mercaptoethanol, and thioglycollate.

Possible mechanisms by which these thiols fail to inactivate phage at high thiol concentrations are also discussed.     

THE RELATIONSHIP BETWEEN BACTERIAL GROWTH AND PHAGE PRODUCTION

1. The effects of temperature and H-ion concentration on the reaction between antistaphylococcus phage and a susceptible staphylococcus have been studied. 2. The temperature optimum for phage production is in the neighborhood of 35°C. and that for bacterial growth is approximately 40°C. 3. With increasing H-ion concentrations there occur: (a) an increase in the lag phase of bacterial growth without any corresponding increase in the lag phase of phage production; (b) a diminution in the total bacterial population accumulating in the medium without any corresponding drop in the total amount of phage formed. 4. With increasing alkalinity there is no pronounced change in the curves of bacterial growth and phage formation. At pH 8.5 the lytic threshold is increased to about 1000 phage units per bacterium instead of 100–140 as is usually the case and the time of lysis is delayed. 5. By adjusting the medium to pH 6 and 28°C. bacterial growth can be completely inhibited while phage production continues at a rapid rate. 6. Apparently, the previously stressed importance of bacterial growth as the prime conditioning factor for phage formation does not hold, for under certain experimental conditions the two mechanisms can be dissociated.     

THE RATE OF BACTERIOPHAGE INACTIVATION BY FILTRATES OF ESCHERICHIA COLI CULTURES

1. The rate of inactivation of an anti-coli phage by filtrates of cultures of the homologous bacteria has been studied. 2. The inactivation rate at 37°C. is proportional to phage concentration and filtrate concentration. 3. At 0°C. the rate of phage inactivation becomes proportional to the square root of the filtrate concentration. 4. A reaction scheme to account for these observations is suggested and discussed. 5. This coli-phage is also inactivated by relatively large concentrations of soluble starch, inulin, gum arabic, and acetylated gum arabic. 6. The inactivation is markedly influenced by salt concentration, being rapid at moderate salt concentrations and slow at high or extremely low salt concentrations. 7. The inactivated phage cannot be regenerated by high salt concentrations, or by soaps.     

Mechanism of Inactivation of Bacteriophage J1 by Glutathione

Mechanism of inactivation of a double-stranded DNA phage, phage Jl of Lactobacillus casei, by reduced form of glutathione (GSH) was studied.

Air (oxygen) bubbling, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by GSH. Partial oxidation of GSH resulted in a more rapid rate of inactivation. In contrast, nitrogen bubbling, reducing agents, chelating agents and radical scavengers prevented the inactivation. Fully oxidized GSH had no phagocidal effect. These results indicate that the inactivating effect of GSH requires the presence of molecular oxygen and is caused by free radical involved in the mechanism of GSH oxidation.

The target of GSH in the phage particle was not the tail protein but DNA. GSH reacted with phage DNA and caused single-strand scissions in the DNA, as exhibited by alkaline sucrose gradient centrifugation; thus inactivating phage.     

THE INACTIVATION OF BACTERIOPHAGE BY MERCURY BICHLORIDE; THE REACTIVATION OF BICHLORIDE-INACTIVATED PHAGE

1. The inactivation of antistaphylococcus bacteriophage suspended in infusion broth at pH 7.6 and 22°C. by HgCl₂ proceeds according to the equation dP/dt = k [HgCl₂] [P_o – P_i] over the range studied. 2. This inactivation can be reversed by precipitation of Hg⁺⁺ with H₂S. In the present experiments the inactivation was carried out until only some 5 per cent of the initial phage remained active. After reactivation the [P] had increased to 100 per cent of the initial [P].     

Experimental variations in sonic fragility of red cells

A magnetostriction oscillator has been used for determining the relative fragility of human red cells to 9000 cycle vibrations under some different environmental conditions. The destruction of the cells is a logarithmic function of time according to the equation See PDF for Equation. Hypertonic saline solution, saponin, preheating, alcohol, and ether in subhemolytic concentrations decrease the fragility of human red cells subjected to sonic oscillation. Hypotonic saline, preheating to 50°C. for 3 minutes, and hemolytic concentrations of ether increase the fragility of red cells to 9 kc. vibrations. Following preheating of the cells, and in the presence of saponin the destruction deviates slightly from a true logarithmic rate of hemolysis.     

The mechanism of the resistance of methicillin-resistant Staphylococcus aureus to phages from the International Collection

The resistance of methicillin-resistant staphylococci to phage 85 is due to the presence of a certain system restriction modification in microbial cells. The loss of the capacity for restricting phage DNA by the cell as the consequence of the loss of the mec determinant is not accompanied by the loss of its capacity for modifying phage DNA.     

EGFR基因重组T7噬菌体疫苗抗Lewis肺癌的实验研究

本研究中制备了表达表皮生长因子受体（EGFR）部分肽段的基因重组T7噬菌体疫苗,并开展了诱导小鼠产生内源性抗EGFR抗体的实验性抗肿瘤作用研究。由T7噬菌体展示系统将7个经筛选的异种属（人源、鸡源）EGFR膜外区片段展示在其壳体次要头蛋白（P10B）上,用所制备的基因重组噬菌体疫苗免疫小鼠,免疫4W后皮下接种Lewis肺癌细胞,10d后分离瘤体并称重,观察各实验组的抗肿瘤效果。WesternBlot检测重组的融合壳蛋白均有EGFR抗原性：高表达EGFR的A431细胞与免疫3W的小鼠抗血清结合并被荧光二抗标记．流式细胞仪检测法确认有抗EGFR抗体产生;各实验组肿瘤均重统计结果显示,P—CL1—670组、P—cp1-130组、P—cp2—136组、P—cp3—145组、P—cp4—142组与空白噬菌体组差异性显著。说明表达EGFR的基因重组噬菌体疫苗诱导产生的内源性抗体,在一定程度上抑制了EGFR阳性肿瘤的生长,为诱导型内源性抗EGFR抗体的肿瘤靶向治疗研究开辟了新的途径。     

EGFR基因重组T7噬菌体疫苗抗Lewis肺癌的实验研究

本研究中制备了表达表皮生长因子受体(EGFR)部分肽段的基因重组T7噬菌体疫苗,并开展了诱导小鼠产生内源性抗EGFR抗体的实验性抗肿瘤作用研究。由T7噬菌体展示系统将7个经筛选的异种属(人源、鸡源)EGFR膜外区片段展示在其壳体次要头蛋白(P10B)上,用所制备的基因重组噬茵体疫苗免疫小鼠,免疫4W后皮下接种Lewis肺癌细胞,10d后分离瘤体并称重,观察各实验组的抗肿瘤效果。Western Blot检测重组的融合壳蛋白均有EGFR抗原性:高表达EGFR的A431 细胞与免疫3W的小鼠抗血清结合并被荧光二抗标记,流式细胞仪检测法确认有抗EGFR抗体产生;各实验组肿瘤均重统计结果显示,P-CL1-670组、P-cp1-130组、P-cp2-136组、P-cp3-145组、 P-cp4-142组与空白噬菌体组差异性显著。说明表达EGFR的基因重组噬菌体疫苗诱导产生的内源性抗体．在一定程度上抑制了EGFR阳性肿瘤的生长．为诱导型内源性抗EGFR抗体的肿瘤靶向治疗研究开辟了新的途径。     

Oxygen protection of bacteriophage T1 against ionizing radiations

Bacteriophage T1 was suspended in distilled water and in phosphate buffer, saturated with oxygen, nitrogen, hydrogen, and carbon monoxide, and irradiated with gamma rays and x-rays. Under the same conditions phage was exposed to hydrogen peroxide. Oxygen acted as a protective agent against both irradiation and hydrogen peroxide inactivation. As a protective agent against irradiation, oxygen was more efficient in distilled water than in buffer. The phage was much more sensitive to irradiation in the presence of hydrogen or nitrogen than in the presence of oxygen. Survivals of phage irradiated in suspensions saturated with hydrogen and with nitrogen did not differ significantly. From this it was concluded that oxygen did not protect T1 by removing atomic hydrogen from the irradiated medium, since the hydrogen-saturated medium increased the yield of atomic hydrogen but did not increase the yield of inactivated phage. It was presumed, therefore, that phage is sensitive to OH radicals and this was confirmed by irradiating phage with UV in the presence of hydrogen peroxide and comparing this survival with the survivals obtained from hydrogen peroxide alone and from UV alone. The combined effect of hydrogen peroxide and UV acting simultaneously was greater than the effect attributable to hydrogen peroxide and UV acting separately. Evidence for sensitivity to HO₂ radicals was considered, and the effect was attributed chiefly to an oxidizing action since phage sensitivity is greater at higher hydrogen ion concentrations, which favor oxidation by HO₂ radicals. Since the OH radical is a more efficient oxidizing agent than O^-, the former being favored in an acid medium, the latter in an alkaline medium, and since the phage is more sensitive in the first situation than in the second, the present tests proved the importance of oxidation as the mechanism of inactivation. Since some inactivation was encountered when phage was exposed to reducing agents, independently of irradiation, it was concluded that phage is somewhat sensitive to reducing agents, but the inactivation attributable to ionizing radiations is due chiefly to oxidation, against which these reducing agents are very efficient protectors. Under no circumstances did hydrogen peroxide protect T1, whether produced by irradiation in the medium or added beforehand to the medium to be irradiated. The first point was investigated by irradiating T1 in the presence of hydrogen and oxygen combined; this produced a higher yield of hydrogen peroxide but a lower survival of T1. In all these tests phage survival under irradiation was directly correlated with oxygen content of the medium rather than with production of hydrogen peroxide. It is proposed that the protective effect of oxygen is due to a reaction between the phage and oxygen, and this complex confers stability upon the phage.     

Role of substrate in reversible activation of proteasomes (multi-protease complexes) by sodium dodecyl sulfate

Previously, we reported that proteasomes (large multi-protease complexes) are present in a latent state in a variety of eukaryotic cells, and can be activated by treatment with various compounds such as sodium dodecyl sulfate (SDS) or poly-lysine (Tanaka et al. (1988) J. Biol. Chem. 263, 16209-16217). In the present study, the mechanism of activation of latent proteasomes by SDS was examined. Latent proteasomes were greatly activated by addition of low concentrations of 0.04 to 0.08% SDS in the presence of substrate. This activation appeared to be reversible, because SDS-activated proteasomes returned to a latent state when the concentration of SDS was reduced by dilution. In contrast, in the absence of substrate, latent proteasomes lost their activity almost completely in an irreversible fashion within a few minutes during treatment with SDS at either 0 or 37 degrees C. Interestingly, SDS-treated proteasomes were markedly protected against this rapid inactivation by either a peptide or protein substrate. Moreover, removal of the substrate after activation of proteasomes caused their rapid irreversible inactivation. These results indicate that the substrate is necessary for reversible activation of latent proteasomes by SDS. This effect of substrate is presumably important in regulation of intracellular protein breakdown by activated proteasomes in eukaryotic cells.     

Growth and phage production of lysogenic B. megatherium

Cell multiplication and phage formation of lysogenic B. megatherium cultures have been determined under various conditions and in various culture media. 1. In general, the more rapid the growth of the culture, the more phage is produced. No conditions or culture media could be found which resulted in phage production without cell growth. 2. Cultures which produce phage grow normally, provided they are shaken. If they are allowed to stand, those which are producing phage undergo lysis. Less phage is produced by these cultures than by the ones which continue to grow. 3. Cells plated from such phage-producing cultures in liquid yeast extract medium grow normally on veal infusion broth agar or tryptose phosphate broth agar, which does not support phage formation, but will not grow on yeast extract agar. 4. Any amino acid except glycine, tyrosine, valine, leucine, and lysine can serve as a nitrogen source. Aspartic acid gives the most rapid cell growth. 5. The ribose nucleic acid content is higher in those cells which produce phage. 6. The organism requires higher concentrations of Mg, Ca, Sr, or Mn to produce phage than for growth. 7. The lysogenic culture can be grown indefinitely in media containing high phosphate concentrations. No phage is produced under these conditions, but the cells produce phage again in a short time after the addition of Mg. The potential ability to produce phage, therefore, is transmitted through cell division. 8. Colonies developed from spores which have been heated to 100°C. for 5 minutes produce phage and hence, infected cells must divide. 9. No phage can be detected after lysis of the cells by lysozyme.     

An extracellular acid stress-sensing protein needed for acid tolerance induction in Escherichia coli

An extracellular induction component (EIC), needed for acid tolerance induction at pH 5.0 in Escherichia coli, arises from an extracellular precursor which senses acid stress and is activated (forming the EIC) by such stress. The precursor, which is a heat-stable protein, was formed by cells which had not been subjected to acid stress, being present in culture media after growth at pH values from 7.0 to 9.0. This stress-sensing molecule was activated to the EIC at pH values from 4.5 to 6.0 but not at pH 6.5 and did not form EIC on incubation at an extremely acidic pH e.g. 2.0. The precursor was not inactivated at pH 2.0. Precursor activation might be reversible, as the EIC lost its ability to induce acid tolerance after incubation at pH 9.0, but regained it if subsequently incubated at pH 5.0. Whereas the sensor formed at pH 7.0 can only be activated at pH 5.0 to 6.0, that synthesized at pH 9.0 can be activated at pH 5.0 to 7.5. Accordingly, this work shows that the acid stress sensor is extracellular, and it is proposed that its presence in the medium rather than in the cells, allows more sensitive and rapid responses to acid stress.     

Control of ornithine decarboxylase in Chinese hamster ovary cells by polyamines. Translational inhibition of synthesis and acceleration of degradation of the enzyme by putrescine, spermidine, and spermine

We have recently isolated, without using any inhibitors, a mutant of Chinese hamster ovary cell line which greatly overproduces ornithine decarboxylase in serum-free culture. Addition of polyamines (putrescine, spermidine, or spermine, 10 microM) or ornithine (1 mM), the precursor of polyamines, to the culture medium of these cells caused a rapid and extensive decay of ornithine decarboxylase activity. At the same time the activity of S-adenosylmethionine decarboxylase showed a less pronounced decrease. Notably, the polyamine concentrations used were optimal for growth of the cells and caused no perturbation of general protein synthesis. Spermidine and spermine appeared to be the principal regulatory amines for both enzymes, but also putrescine, if accumulated at high levels in the cells, was capable of suppressing ornithine decarboxylase activity. The amount of ornithine decarboxylase protein (as measured by radioimmunoassay) declined somewhat more slowly than the enzyme activity, but no more than 10% of the loss of activity could be ascribed to post-translational modifications or inhibitor interaction. Some evidence for inactivation through ornithine decarboxylase-antizyme complex formation was obtained. Gel electrophoretic determinations of the [35S]methionine-labeled ornithine decarboxylase revealed a rapid reduction in the synthesis and acceleration in the degradation of the enzyme after polyamine additions. No decrease in the amounts of the two ornithine decarboxylase-mRNA species, hybridizable to a specific cDNA, was detected, suggesting that polyamines depressed ornithine decarboxylase synthesis by selectively inhibiting translation of the message.     

Inactivation of the T7 coliphage by monofunctional alkylating agents. Action of phage adsorption and injection of its DNA.

Alkylation by ethyl or methyl methanesulfonate to an extent that inactivates more than 99.5% of T7 coliphages has no effect on phage adsorption on Escherichia coli B cells, but decreases the amount of phage DNA injected into the host cells. Depurination interferes with the injection of the phage DNA. Failure to inject the whole phage genome thus appears to be a cause of the immediate as well as of the delayed inactivation of the T7 coliphage treated by monofunctional alkylating agents; the hypothesis that it is the only cause of inactivation, although not very likely, cannot be excluded at the present time.