Conjugates between anti-tetanus F(abP P) 2 fragments and the (37^72) fragment of the HIV Tat protein were taken up by chromaffin cells, NG108-15 neurohybridoma cells and Rev-2-T-6 lymphoma cells. The uptake could not be inhibited by competition with (37^72)Tat, but was reduced in the presence of metabolic inhibitors or at low temperature. The disulfide as well as the thioether conjugate were translocated to the cytoplasmic space, but only the disulfide conjugate moderately restored the stimulated transmitter release inhibited by tetanus toxin. Therefore, disulfide conjugates are more promising than thioethers for the neutralization of intracellular antigens. These conjugates provide new tools to study neuroprotection against bacterial neurotoxins.z 1999 Federation of European Biochemical Societies.
Phenylglyoxal (PG) is shown to be a cell surface probe specific for arginine moieties in protein: (1) It does not enter the cell as evidenced by lack of PG in the cytoplasm. (2) It does not cause excessive cell leakage as measured by release of 51Cr. (3) It reacts with positively-charged groups in proteins at the cell surface but not with those of phospholipids at the surface; since pronase removes PG from the surface, but phospholipase C does not. (4) Under the conditions used in these experiments, it reacts virtually exclusively with arginine moieties in protein (Freedman et al., '68; Takahashi, '68; Werber and Sokolovsky, '72). Synchronized cells were exposed to radioactive PG to assess quantity of arginine moieties in protein at the surface. There is a sharp decrease in arginine at the cell surface at entry into G1 phase from M and a 24-fold increase upon entry into S phase. There is a slight drop in exposed arginine in late S phase followed by an increase to 26 times the G1 level immediately prior to mitosis. Lactoperoxidase-catalyzed iodination of tyrosine moieties in protein at the surface of synchronized cells shows a very gradual increase in protein as the cells move through the cycle and increase in size. Since the increase in arginine moieties in protein at the surface does not reflect a similar increase in total protein at the surface, an arginine-rich protein appears to be exposed at the cell surface during the division-related phases of the cell cycle.
Herpes simplex virions produced by infected human carcinoma No. 2 (HEp-2) cells appear in two general forms, unenveloped (naked) and enveloped. These two forms were separated by zone centrifugation at 18 000 g for 55 min through a 10 to 30% (w/v) sucrose density gradient. The three bands containing virus particles were found by electron microscopy to contain empty naked particles, full naked particles, and enveloped particles. The number of virus particles and the number of plaque-forming units (p.f.u.) were determined for each of the three types of particles. In the band containing enveloped virus particles the infectivity was 1 p.f.u./120 particles; the filled naked particle band yielded 1 p.f.u./60 000 particles; and the empty naked particle band yielded 1 p.f.u./960 000 particles. All of the infectivity of the starting material was recovered from the gradient after centrifugation. It appears that the plaque-forming efficiency of naked virus particles is extremely low, but is not negligible. Particle counts of enveloped full capsids indicate that empty and filled naked particles are enveloped indiscriminately.The infectivity of naked HSV particles as well as the number of naked particles that retain a core is increased when virus is harvested in pH 8.3 medium containing 0.01 M Na citrate, conditions that are unfavorable for DNase activity. The infectivity and morphology of enveloped particles are not affected by this harvesting method.
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