Summary. CD27 is a marker of memory B cells and its interaction with its ligand, CD70, is very important for differentiation into plasma cells. Although CD27 is detected on normal plasma cells, its expression is significantly reduced with the progression of multiple myeloma (MM), including monoclonal gammopathy of undetermined significance (MGUS). CD27+ myeloma cells are thought to represent an early phase of myeloma, as CD27+ plasma cells from MM patients were found to be composed of normal plasma cells (CD19+/CD38++) and myeloma cells (CD19–/CD38++), and monoclonality was detected in the CD27+/CD38++ fraction. Given that the lack of CD27 on plasma cells is related to myelomagenesis and that the pro‐apoptotic protein Siva is thought to bind to the cytoplasmic tail of CD27, we analysed alterations of cell growth and genes caused by co‐culturing CD27‐transfected myeloma cell lines (U266, KMS‐5) with CD70‐transfected NIH3T3 cells. CD27–CD70 interaction could not induce apoptosis in either type of myeloma transfectant, and binding between Siva and CD27 was not detected. cDNA microarray (human apoptosis CHIP) analysis showed a significant upregulation of expression of the ectodermal neural cortex 1 (ENC1) gene by CD27–CD70 interaction compared with CD27 transfection alone. These findings show that the relationship between the loss of CD27 and oncogenesis of plasma cells is not simple. It remains unclear whether the lack of CD27 leads to evasion of apoptosis.

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/ hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.Chaperonins, a ubiquitous class of molecular chaperones, are double-ring assemblies of about 60-kDa subunits. Each ring has a large central cavity in which a non-native protein can undergo productive folding in an ATP-dependent manner (1, 2). Chaperonin complexes change their conformations to close their chamber and encapsulate the bound substrate, providing a protected environment for protein folding. Opening and closing of the folding chamber is controlled by a conformational cycle driven by ATP binding and hydrolysis. Chaperonins are divided in two groups as follows: group I and group II chaperonins. Group I chaperonins are found in bacteria and endosymbiotic organelles (mitochondria and chloroplasts), and subsets of bacterially related homologs in methanogens (3). On the other hand, group II chaperonins are found in archaea (known as thermosome) (4) and the eukaryotic cytosol (chaperonin-containing t-complex polypeptide-1 or TCP-1 ring complex) (1, 5). Whereas group I chaperonins are homo-oligomeric, eukaryotic and archaeal group II chaperonins are generally hetero-oligomeric (6). All chaperonins share a similar subunit architecture *

Induction of cytochrome P-450s by 3-methylcholanthrene (MC) and phenobarbital (PB) and distribution of P-450s in the rat liver nuclear envelope were investigated by biochemical analyses and ferritin immunoelectron microscopy using specific antibodies against the major molecular species of MC-and P13-induced cytochrome P-450. It was found, in agreement with Kasper (J . Biol. Chem., 1971, 246 : 577-581), that the total amount of cytochrome P-450s determined by biochemical analysis was markedly increased by MC, but not by PB, treatment. Immunoelectron microscopic analysis, however, showed marked and slight increases in ferritin labeling by MC and PB treatment, respectively . The latter finding was interpreted as resulting from the induction of a particular molecular species of PB-induced cytochrome P-450s . Ferritin immunoelectron microscopic analysis of intact isolated nuclei, naked nuclei from which the outer membrane of the nuclear envelope was partially detached (mechanically), and isolated nuclear envelopes have shown that the ferritin particles are found exclusively on the cytoplasmic face of the outer nuclear envelopes . Neither the nucleoplasmic face of the inner membrane of the nuclear envelope nor the cisternal face of both membranes of the nuclear envelope showed any labeling with ferritin . This indicates that cytochrome P-450 is located only on the outer membrane of the nuclear envelope and does not diffuse laterally into the domain of the inner membrane of the nuclear envelope across the nuclear pores . Our results suggest that a marked heterogeneity exists in the enzyme distribution between the outer and inner membrane of the nuclear envelope and that microsomal marker enzymes such as cytochrome P-450 exist exclusively in the outer membrane . In addition, it appears that cytochrome P-450 is probably not a transmembrane protein but an intrinsic protein located on the cytoplasmic face of the outer membrane of the nuclear envelope .The nuclear envelope, which is composed of an inner and an outer membrane, forms the limiting barrier between the nucleoplasm and cytoplasm in all eucaryotic cells and occupies a strategic position in the control and regulation of cellular metabolism. The cytoplasmic surface of the outer leaflet of the nuclear envelope is studded with ribosomes and is sometimes continuous with the endoplasmic reticulum (29,30). In addition to these structural similarities, the presence of a number of enzymes common in the nuclear envelope and endoplasmic reticulum has been reported (5, 6, 9, 31) . For example, hepatic microsomal marker enzymes such as NADPH-cytochrome c reductase and cytochrome P-450 are also present in the nuclear envelope (4,(13)(14)(15)25) . Thus it has been claimed that these 212 two membrane structures cannot be distinguished from one another and that they are practically identical (5) .It is possible, however, to point out several differences between the two membrane structures. In general, the specific enzymatic activities of the nuclear envelope are lower tha...

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Amphotericin B (AmB, 1) is known to assemble together and form an ion channel across biomembranes, by which the drug presumably exerts its antimicrobial activity. To access the whole architecture of this channel assemblage, the understanding of binary interaction between AmB molecules is of prime importance because the dimeric interaction is the basis of the assemblage. In this context, we have recently reported covalently conjugated AmB dimers such as 2 and 3 with a long linker, which show prominent hemolytic potency and ion-channel activity. To evaluate the effect of the length and hydrophilicity of linker parts on the activity, we prepared new dimers bearing tartarate linkages (4 and 5). Especially, 5 exhibited potent hemolytic activity (EC50, 0.03 microM) surpassing those of AmB, 2, and 3. Measurements of UV and CD spectra of 5 in liposomes indicated that AmB portions of 5 could adopt appropriate arrangements in molecular assemblage in spite of the short linkage, and also indicated that the assemblage formed by 5 appeared more stable than AmB. These short-tethered dimers are expected to be a promising tool to reveal the mechanism of dimeric interaction in the ion channel formed by AmB.