The Journal of Experimental Medicine

Sellers, Alvin L.; Griggs, Neilyn; Marmorston, Jessie; Goodman, Howard C.

doi: 10.1084/jem.100.1.1pmid: 13163334

Plasma proteins of the rat have been labelled by the in vivo injection of the dye T-1824. From a study of the rate of disappearance of T-1824 from the circulating blood, and the total T-1824 content of the perfused kidney the rate of protein reabsorption from the glomerular fluid by the cells of the renal tubule has been calculated. It is concluded that protein reabsorption by the cells lining the proximal convoluted tubule of the rat kidney proceeds at a rate of at least 5 mg. per hour, equivalent to a daily filtration and reabsorption of 33 per cent of the circulating plasma protein. Footnotes Submitted: 25 February 1954

Dziewiatkowski, Dominic D.

doi: 10.1084/jem.100.1.11pmid: 13163335

The administration of vitamin A to vitamin A-deficient rats resulted in a decreased concentration of inorganic sulfate-sulfur in the serum from a value of 2.5 mg. per cent to 1.8 mg. per cent, the latter being close to the value of 2.0 mg. per cent found in normal rats of the same age. The uptake of sulfate and phosphate by femurs and tibiae of vitamin A-deficient rats was less than that in normal rats of the same age. An increased uptake followed the administration of vitamin A: radioautography indicated that in the case of sulfate, its uptake was particularly increased in the epiphyseal cartilage; an increased uptake of phosphate was particularly evident in the diaphysis immediately adjacent to the epiphyseal cartilage plate. The specific activity of the sulfate-sulfur in the chondroitin sulfate samples isolated from the skeletons of vitamin A-deficient rats fell progressively as the deficiency continued. Following administration of vitamin A, the specific activity approached and exceeded the value given by the sample from the skeletons of normal rats of the same age. A substantial increase was found in the value of the specific activity of the sulfate-sulfur of sulfomucopolysaccharides isolated from skins of vitamin A-deficient rats that had been given vitamin A. Following administration of vitamin A to rats deficient in this vitamin, an increased accumulation of some sulfur-containing material was found in regions of active calcification. Footnotes Submitted: 1 March 1954

Dziewiatkowski, Dominic D.

doi: 10.1084/jem.100.1.25pmid: 13163336

The concentration of inorganic sulfate-sulfur in the serum of vitamin D-deficient rats, 2.6 to 3.5 mg. per cent, was found to be higher than that in the serum of normal rats of the same age, 2.0 mg. per cent. No change was observed following the administration of 25 γ of vitamin D 2 . In accord with the results of others, it was found that a definitely increased deposition of phosphorus in femurs and tibiae had occurred 36 to 48 hours after the administration of vitamin D 2 to vitamin D-deficient rats. An immediate increase in the uptake of sulfate by the skeleton was found using sodium sulfate-S 35 . As measured by the specific activity of sulfate-sulfur in samples of chondroitin sulfate isolated from the skeletons of the vitamin D-deficient animals and from normal controls receiving equal doses of sulfur-35, the rate of synthesis of chondroitin sulfate in rachitic rats is similar to the rate in normal rats of the same age. Likewise, the incorporation of labelled sulfate into the sulfomuco-polysaccharides of the pelts was found to be equal at 12 hours to that in normal rats. Following the administration of vitamin D 2 to deficient animals an increase in the rate of synthesis of the chondroitin sulfate of the skeletons was noted. The radiochemical and radioautographic evidence suggest that there is in vitamin D-deficient rats an impaired utilization of chondroitin sulfate and that vitamin D 2 is able to accelerate this process. Footnotes Submitted: 1 March 1954

Finter, Norman B.; Liu, Oscar C.; Lieberman, Melvin; Henle, Werner

doi: 10.1084/jem.100.1.33pmid: 13163337

The usefulness of the deembryonation technic has been analyzed as a tool in the study of various problems in the growth cycle of influenza virus in the entodermal cells of the allantoic of chick embryos. Various improvements in the deembryonation technic have been described. The method readily permits repeated sampling of the medium at various stages after infection (cumulative growth curves) or frequent exchanges of the medium (differential growth curve). However, the yield of infectious virus or of hemagglutinins is less than that observed in the intact chick embryo. The difference observed is greater than can be accounted for by the reduction in the available host cells and is assumed, therefore, to be due in part to interruption of blood and nutrient supply to the cells. This handicap can be overcome by the combined in ovo -deembryonation technic, in which deembryonation is performed at any desired time after infection of the intact chick embryo, and the medium is collected and analyzed after 1 to 3 hours of further incubation. The value of the technic is demonstrated by the fact that liberation of virus from infected cells can be detected earlier than in the intact egg. Furthermore, it continues at a nearly constant rate for many hours, thus proving to be erroneous previous inference which had been based upon in ovo experiments. The technic also permits readily the addition and subsequent removal of substances that might interfere with viral propagation. As an example a study was made of the effect of the receptor-destroying enzyme of V. cholerae (RDE) when added to the medium of eggs infected prior to deembryonation. By carefully grading the dose of virus and using an appropriate amount of RDE, one-step growth curves were obtained indicating that those cells not directly invaded by the seed virus were subsequently protected against infection by action of the enzyme. The smaller the amount of virus the less RDE was required in order to note a protective effect. With a decrease in the period of exposure to RDE regeneration of cell receptors became increasingly more apparent in that correspondingly greater amounts of virus were produced and liberated late in the incubation periods. These results confirmed and extended those reported by Stone. More extensive applications of these technics will be reported in subsequent papers of this series. Footnotes Submitted: 17 March 1954

Henle, Werner; Liu, Oscar C.; Finter, Norman B.

doi: 10.1084/jem.100.1.53pmid: 13163338

The period and rate of liberation of influenza virus from entodermal cells of the allantois have been studied by deembryonating eggs within a few minutes after infection, exchanging the medium thereafter at hourly intervals and assaying the virus concentration in the harvests thus obtained (differential growth curves). If the inoculum was sufficiently large, presumably all available cells immediately became infected and only 1 infectious cycle was expected to occur. If the inoculum was small, so that only a fraction of the cells adsorbed virus, the infectious process was held to 1 cycle by continuous exposure of the remaining susceptible cells to RDE. In either case, the results obtained indicate that once cells have been infected they produce and liberate virus at nearly constant rates for periods of 30 hours or longer before the yields decrease rapidly. Evidence has been presented which strongly suggests that such prolonged periods of liberation are observed not only in deembryonated eggs but also in the intact chick embryo. Attempts have been made in the discussion to reconcile these findings with previous estimates of the liberation period and to integrate them with histologic observations and electron micrographs of thin sections of infected allantoic membranes having a bearing on the mode of liberation. Footnotes Submitted: 17 March 1954

Wood, Harrison F.; McCarty, Maclyn; Slater, Robert J.

doi: 10.1084/jem.100.1.71pmid: 13163339

A method is described for obtaining crystalline C-reactive protein from serous fluids in which the protein is associated with lipid. Most pathological fluids currently available as a source of this protein appear to fall in this category. Crystalline C-reactive protein has its isoelectric point at pH 4.82 as determined by free electrophoresis in McIlvaine's buffer. Its mobility in the electrophoresis cell, both alone and after addition to normal serum, coincides with that of the ß-globulin fraction of the serum. In contrast to this finding, by the method of zone electrophoresis on a starch supporting medium the protein migrates with the γ 1 -globulin. The significance of this discrepancy is discussed. Studies in the ultracentrifuge indicate an s 20, w of 7.5. Footnotes Submitted: 14 March 1954

Greiff, Donald; Pinkerton, Henry

doi: 10.1084/jem.100.1.81pmid: 13163340

A vacuum sublimation apparatus is described which will permit, ( a ) the removal of water from virus suspensions at temperatures ranging down to –80°C., ( b ) continuous operation with a minimum of attention from the investigator, ( c ) sealing off of samples at operating pressures (10 –5 mm. Hg), ( d ) simultaneous lyophilization of aliquot samples at different temperatures, ( e ) isolation of a portion of the apparatus without disturbing the remainder of the system, and ( f ) determination of the end-point of sublimation without disturbing the samples. The time required for drying 0.1 ml. of influenza virus suspension was shown to increase markedly with decrease of temperature, 8 days being required for dehydration at –80°C. in contrast to 2 days at –30°C. and 1 day at 0°C. Footnotes Submitted: 7 March 1954

Greiff, Donald; Blumenthal, Herman; Chiga, Masahiro; Pinkerton, Henry

doi: 10.1084/jem.100.1.89pmid: 13163341

The infectivity titre of influenza virus-infected allantoic fluid was determined after a variety of procedures involving cyclic slow freezing and thawing, freezing at various rates with subsequent storage at different temperatures freezing at various rates with subsequent dehydration at various temperatures, and different degrees of dehydration. All these factors were found to influence the survival rate of the virus particles. Five freeze-thaw cycles resulted in a fall in titre from 10 –8.6 to 10 –0.8 cycles 2, 3, and 4 causing much greater losses than cycles 1 and 5. Rapid cooling to –40°C. or slow cooling to –80 or 190°C. did not cause significant titre loss, but rapid cooling to temperatures above –40° or slow cooling to temperatures above –80°C. caused definite titre loss. Loss of titre on storage occurred only at temperatures above –40deg;C. The effect of lyophilization depends both on the preliminary treatment and on the dehydration temperature. Better conservation of titre was obtained after preliminary cooling to –190 or –80°C. than after preliminary cooling to higher temperatures. The most effective sublimation temperatures were 0 and –80°.; the least effective was +20°C. Titre losses in suspensions sublimated at –10, –30, and –60°C. were in general intermediate. No loss in titre occurred after preliminary cooling to –80 or –190°C. and subsequent dehydration at –80 or 0°C. The degree of dehydration definitely affects the survival of virus on storage at 0°C., but sublimation for 4 hours at 0°C. gave complete protection against titre loss on storage at this temperature. Possible explanations of the observations made are suggested, based on known physiochemical phenomena such as supercooling, vitrification, variations in size and shape of ice crystals with different freezing speeds, differential enzyme inactivation, changes in salt concentration, and changes in energy levels. Footnotes Submitted: 7 March 1954

Eagle, Harry

doi: 10.1084/jem.100.1.103pmid: 13163342

1. In a previous study, the differing sensitivity of bacterial strains as they occur in nature appeared to be correlated with their correspondingly differing reactivity with penicillin. Presumably, the over-all reactivity of the cell with penicillin paralleled that of the vulnerable cell component(s). However, when penicillin-resistant variants of these strains ( Streptococcus pyogenes, Micrococcus pyogenes, Diplococcus pneumoniae , and Streptococcus faecalis ) were produced by serial passage through increasing concentrations of antibiotic, this correlation between resistance and the ability of the cell to bind penicillin was no longer apparent. Some resistant variants bound more penicillin than the parent, sensitive cell ( Streptococcus faecalis, Micrococcus pyogenes ), some were unchanged in their reactivity ( Diplococcus pneumoniae, Micrococcus pyogenes ), and some bound less ( Streptococcus pyogenes, Micrococcus pyogenes ). One resistant variant of Micrococcus pyogenes at first showed enhanced reactivity with penicillin; on continued passage through antibiotic, there was a further increase in resistance, but now associated with a significantly decreased reactivity. In the case of Diplococcus pneumoniae , a resistant variant at first reacted normally with penicillin; on continued passage in antibiotic, its binding affinity for penicillin gradually decreased, but with no associated further increase in resistance. 2. The reactivity with penicillin of cell-free sonic extracts of the resistant variants paralleled that of the intact organisms. Permeability considerations therefore did not seem involved in the increased resistance produced by serial passage in antibiotic. 3. The penicillin-resistant variants did not have an enhanced capacity to degrade the free intracellular antibiotic. 4. Possible alternative explanations are discussed in the text. Footnotes Submitted: 22 February 1954

Eagle, Harry

doi: 10.1084/jem.100.1.117pmid: 13163343

1. ( a ) Mammalian cells in tissue culture (mouse fibroblasts and malignant human uterine epithelium) did not concentrate penicillin from the culture medium. Even at low concentrations, the cellular accumulation was usually less than that in the surrounding fluid, and most of it was removed by washing. The radioactive material in such eluates was actively bactericidal, and was presumably in large part unchanged penicillin. ( b ) Penicilloic acid, produced by the action of penicillinase, was bound to the same (limited) extent as the active antibiotic. In both these respects mammalian cells behaved like naturally penicillin-resistant bacteria, and unlike such penicillin-sensitive bacteria as Streptococcus pyogenes or Diplococcus pneumoniae . 2. Cell-free sonic extracts of the L strain had the same limited reactivity with penicillin as the intact cells. The relatively minute amounts bound by the cells are therefore not due to their impermeability, but instead reflect the inherently low reactivity of the cellular constituents with penicillin. 3. It is suggested that the relative non-toxicity of penicillin for the mammalian host, and for mammalian cells in tissue culture, may be related to this low order of reactivity with the antibiotic. Footnotes Submitted: 22 February 1954