Isolation of ribosome particles from meningopneumonitis organisms

In ribonucleic acid (RNA) extracted by phenol and sodium dodecyl sulfate from purified reticulate bodies of meningopneumonitis (MP) organisms, 21S, 16S, and 4S RNA were found by sucrose density gradient sedimentation analysis. When purified reticulate bodies were homogenized by sonic treatment or by treatment with sodium deoxycholate and were fractionated by differential centrifugation, more than 50% of the RNA was recovered in the fraction which was sedimented by centrifugation at 105,000 x g for 2 hr, but not at 13,000 x g for 20 min. From homogenates prepared in this manner, 50S and 30S particles containing RNA were isolated by sucrose density gradient centrifugation. These 50S and 30S particles were also found in lysates of cytoplasmic fractions of infected cells which were labeled by (32)P during 17 to 17.5 hr or 15 to 18 hr after infection. The synthesis of 50S and 30S particles was not inhibited by actinomycin D. When infected cells were homogenized in the presence of 0.01 or 0.02 m MgCl(2), 70S particles were isolated instead of 50S and 30S particles. When dialyzed against low concentrations of MgCl(2), the 70S particles dissociated to 50S and 30S particles. The base ratio of the 70S particles is very similar to that of 16S plus 21S RNA. The characteristics of the 70S, 50S, and 30S particles suggest that these are ribosome particles, similar to bacterial ribosomes.     

Purification and Chemical Composition of Reticulate Bodies of the Meningopneumonitis Organisms

Reticulate bodies of the meningopneumonitis (MP) microorganism were purified from L cells 18 hr after infection by the combination of differential centrifugation in 30% sucrose solution and potassium tartrate density gradient centrifugation. It was ascertained by electron microscopy that purified preparations of reticulate bodies obtained were almost entirely free of host-cell components and of infectious elementary bodies of MP microorganisms. Purified reticulate bodies were easily disrupted by mechanical agitation, and it was observed in shadowed preparation that ribosome-like particles 15 mmu in diameter were scattered from broken reticulate bodies. In shadowed preparations, reticulate bodies were found to range in size from 1.0 to 1.6 mu in diameter, but in cross-section the range was 0.5 to 1.0 mu. In these preparations, the purified reticulate bodies were irregular in shape, round or oval, and were composed of rather homogenous, amorphous, or reticulate material with moderate density. Some particles exhibited a less-dense internal structure, in which a coarse fibrous reticulum was seen. Chemical fractionation of (32)P-labeled purified reticulate bodies showed that they contained three times more ribonucleic acid (RNA) than deoxyribonucleic acid, with the RNA being composed primarily of 21S, 16S, and 4S RNA. No infectivity of purified reticulate bodies could be demonstrated.     

Regulatory role of CD4/L3T4 molecules in IL-2 production by affecting intracellular Ca2+ concentration of T cell clone stimulated with soluble anti-CD3

To elucidate the role of CD4 molecule in T cell activation, the effect of anti-CD4 on T cell IL-2 production was examined by using an alloreactive Th clone. The alloreactive T cell used in the present experiments produced IL-2 in response to soluble anti-CD3 epsilon-chain (anti-CD3) without accessory cell or insoluble antibody carrier. The IL-2 production was suppressed by the addition of anti-CD4 in cultures. An intracellular free Ca2+ concentration ([Ca2+]i) of the T cell clone was elevated by anti-CD3 stimulation, but the elevation was suppressed in the presence of anti-CD4. When the clone was stimulated in Ca2(+)-free medium, the elevation of [Ca2+]i was not observed. When Ca2+ influx was induced by calcium ionophore A23187 or ionomycin, the clone produced IL-2 in response to anti-CD3 in the presence of anti-CD4. When polyclonal T cell line or several other alloreactive T cell clones were examined for their anti-CD3 response, essentially the same results as mentioned above were obtained. Taken together, these results suggest that the slow and sustained elevation of [Ca2+]i is an essential signal for IL-2 production of T cells, and that anti-CD4 suppresses the IL-2 production by interfering the [Ca2+]i elevation. The significance of CD4 molecules in murine T cell activation was discussed.     

Location of a synergistic factor in the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta

A synergistic factor (SyF), which enhanced the infection of nuclear polyhedrosis viruses, was purified from capsules of a Pseudaletia unipuncta granulosis virus (Hawaiian strain) by immune affinity chromatography. The isolated SyF consisted primarily of a protein with molecular mass 98 kDa. The recovery rate depended on the alkali used to dissolve the capsules: the highest rate occurred with 0.05 M Na2CO3-0.05 M NaCl, followed in turn with 0.02-0.05 M NaOH and 0.04 M NaOH-0.05 M glycine. The solubilized components from untreated capsules contained 98- and 100-kDa proteins in addition to the matrix protein (29 kDa) and its decomposed products, while those from heat-treated capsules contained only the 100-kDa protein. Virons liberated from the capsules with the glycine buffer contained three proteins (33, 98, and 100 kDa) serologically related to the SyF. Immunoelectron microscopy of infected tissue and purified virions revealed the localization of the SyF antigens on the viral envelope.