Human THP-1 leukemia cells differentiate along the monocytic lineage following exposure to phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (VD3). In the monocytic cell line THP-1, PMA treatment resulted in a more differentiated phenotype than VD3, according to adherence, loss of proliferation, phagocytosis of latex beads, and expression of CD11b and CD14. Both differentiating substances induced similar effects in the release of superoxide anions (O2-). VD3-differentiated cells did not release prostaglandin E2 (PGE2), in contrast to PMA-differentiated cells, and in PMA-differentiated cells phospholipase A2 (PLA2) activity and expression was increase. Lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) release was higher in PMA-treated cells. PMA- but not VD3-differentiation resulted in a translocation of protein kinase C (PKC) isoenzymes to membrane fractions. Both differentiating agents up-regulated the expression of PKC isoenzymes. Whereas VD3 elevated mainly the expression of PKC-beta, PMA caused a strong increase in PKC-delta and a weak increase in PKC-alpha, PKC-epsilon, and PKC-zeta expression. These results indicate that phorbol ester and the active metabolite of vitamin D induce different signal pathways, which might result in different achievement of differentiation.

The total content and pattern of gangliosides were determined in the unfractionated sera of 11 healthy human adults and in isolated lipoproteins. The total content of lipid-bound sialic acid was 10.5 3.2 nmol/ml serum. The ganglioside profile consisted of more than ten different components. The major ganglioside was GM3, followed by GD3, GD1,, GM2, GTlb, MG-3 (sialosyllactoneotetraosylceramide), GDlb and GQlb. Traces of four additional gangliosides could not be quantified reliably. Ganglioside patterns did not vary in sera taken from healthy adults of different age and sex. Approximately 98% of human serum gangliosides were transported by serum lipoproteins, predominantly by LDL (66%), followed by HDL (25%) and VLDL (7%). The quantitative distribution of individual gangliosides in VLDL and LDL was almost the same as that in the unfractionated serum; some differences existed with the ganglioside profile in HDL.Gangliosides are sialic-acid-containing glycosphingolipids, ubiquitous in neural and extraneural organs of deuterostomia [l]. They Svennerholm [35, 361, in parenthesis. The nomenclature for MG-3 and MG-4 is according to [17]. GM3, (I13NeuAcLacCer), (N-acetylneuraminosyl)galactosylglucosylceramide; GM2, (I13Neu-AcCgOse3Cer), N-acetylgalactosaminyl-(N-acetylneuraminosy1)galactosylglucosylceramide; G M I , (I13NeuAcGgOse4Cer), galacto-

Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis.

Mammalian ribosome-associated complex (mRAC), consisting of the J-domain protein MPP11 and the atypical Hsp70 homolog (70-homolog) Hsp70L1, can partly complement the function of RAC, which is the homologous complex from Saccharomyces cerevisiae. RAC is the J-domain partner exclusively of the 70-homolog Ssb, which directly and independently of RAC binds to the ribosome. We here show that growth defects due to mRAC depletion in HeLa cells resemble those of yeast strains lacking RAC. Functional conservation, however, did not extend to the 70-homolog partner of mRAC. None of the major human 70-homologs was able to complement the growth defects of yeast strains lacking Ssb or was bound to ribosomes in an Ssb-like manner. Instead, our data suggest that mRAC was a specific partner of human Hsp70 but not of its close homolog Hsc70. On a mechanistic level, ATP binding, but not ATP hydrolysis, by Hsp70L1 affected mRAC's function as a J-domain partner of Hsp70. The combined data indicate that, while functionally conserved, yeast and mammalian cells have evolved distinct solutions to ensure that Hsp70-type chaperones can efficiently assist the biogenesis of newly synthesized polypeptide chains.

NAC acts as a modulator of SRP function. It can bind to signal sequences directly. SRP initially displaces NAC from RNCs; however, when the signal sequence emerges, trimeric NAC·RNC·SRP complexes form. Upon docking NAC·RNC·SRP complexes to the ER, NAC remains bound, allowing NAC to shield cytosolically exposed nascent chain domains.