Potent anti-R5 human immunodeficiency virus type 1 effects of a CCR5 antagonist, AK602/ONO4128/GW873140, in a novel human peripheral blood mononuclear cell nonobese diabetic-SCID, interleukin-2 receptor gamma-chain-knocked-out AIDS mouse model

We established human peripheral blood mononuclear cell (PBMC)-transplanted R5 human immunodeficiency virus type 1 isolate JR-FL (HIV-1(JR-FL))-infected, nonobese diabetic-SCID, interleukin 2 receptor gamma-chain-knocked-out (NOG) mice, in which massive and systemic HIV-1 infection occurred. The susceptibility of the implanted PBMC to the infectivity and cytopathic effect of R5 HIV-1 appeared to stem from hyperactivation of the PBMC, which rapidly proliferated and expressed high levels of CCR5. When a novel spirodiketopiperazine-containing CCR5 inhibitor, AK602/ONO4128/GW873140 (molecular weight, 614), was administered to the NOG mice 1 day after R5 HIV-1 inoculation, the replication and cytopathic effects of R5 HIV-1 were significantly suppressed. In saline-treated mice (n = 7), the mean human CD4(+)/CD8(+) cell ratio was 0.1 on day 16 after inoculation, while levels in mice (n = 8) administered AK602 had a mean value of 0.92, comparable to levels in uninfected mice (n = 7). The mean number of HIV-RNA copies in plasma in saline-treated mice were approximately 10(6)/ml on day 16, while levels in AK602-treated mice were 1.27 x 10(3)/ml (P = 0.001). AK602 also significantly suppressed the number of proviral DNA copies and serum p24 levels (P = 0.001). These data suggest that the present NOG mouse system should serve as a small-animal AIDS model and warrant that AK602 be further developed as a potential therapeutic for HIV-1 infection.     

A novel phospholipase C, PLC(eta)2, is a neuron-specific isozyme

Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, beta-, gamma-, delta-, epsilon-, zeta-, and eta-type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLC(eta)2. PLC(eta)2 is composed of conserved domains including pleckstrin homology, EF-hand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLC(eta)2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLC(eta)2 was more sensitive to calcium concentration than the PLC activity of the PLCdelta-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLC(eta)2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLC(eta)2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLC(eta)2 in pyramidal cells of the hippocampus. Finally, we found that PLC(eta)2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLC(eta)2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.     

Phospholipase Cdelta4 associates with glutamate receptor interacting protein 1 in testis

We reported previously that phospholipase C (PLC) delta4 is required for calcium mobilization in the zona pellucida-induced acrosome reaction in sperm. Here we focused on the function of the C2 domain of PLCdelta4 and report that glutamate receptor-interacting protein1 (GRIP1) was identified as a binding protein of the PLCdelta4-C2 domain on yeast two-hybrid screening. Physiological interaction of GRIP1 with PLCdelta4 in mouse testis was confirmed by immunoprecipitation with anti-PLCdelta4 antibodies and the association seemed to correlate with the maturation stage of sperm. We also determined that a PDZ-binding motif at the C-terminus of the PLCdelta4-C2 domain is responsible for GRIP1 binding, whereas the sixth or seventh PDZ domain of GRIP1 is essential and sufficient for association with the PLCdelta4-C2 domain. These results indicate that PLCdelta4 binds via its C2 domain to the PDZ6 or PDZ7 domain of GRIP1, and that this association may play a role in spermatogenesis.     

Drastic Ca2+ sensitization of myofilament associated with a small structural change in troponin I in inherited restrictive cardiomyopathy

Six missense mutations in human cardiac troponin I (cTnI) were recently found to cause restrictive cardiomyopathy (RCM). We have bacterially expressed and purified these human cTnI mutants and examined their functional and structural consequences. Inserting the human cTnI into skinned cardiac muscle fibers showed that these mutations had much greater Ca2+-sensitizing effects on force generation than the cTnI mutations in hypertrophic cardiomyopathy (HCM). The mutation K178E in the second actin-tropomyosin (Tm) binding region showed a particularly potent Ca2+-sensitizing effect among the six RCM-causing mutations. Circular dichroism and nuclear magnetic resonance spectroscopy revealed that this mutation does not extensively affect the structure of the whole cTnI molecule, but induces an unexpectedly subtle change in the structure of a region around the mutated residue. The results indicate that the K178E mutation has a localized effect on a structure that is critical to the regulatory function of the second actin-Tm binding region of cTnI. The present study also suggests that both HCM and RCM involving cTnI mutations share a common feature of increased Ca2+ sensitivity of cardiac myofilament, but more severe change in Ca2+ sensitivity is associated with the clinical phenotype of RCM.     

Production of a polymer-forming fusion protein in Escerichia coli strain BL21

In the course of studying [PSI(+)], a yeast prion, we found inadvertently that Escherichia coli strain BL21 overproducing a fusion protein, in which the prion-domain of Sup35 was connected to the C terminus of glutathione S-transferase, grew normally to the stationary phase and rapidly decreased in colony-forming ability thereafter. Evidence indicated that protein polymers consisting mainly of the fusion protein GST-Sup35NM (about 70% of the mass) and its N-terminal fragments were formed in extract prepared from the cells producing GST-Sup35NM. It was further found that cells of strain BL21 accumulated the protein polymers during prolonged cultivation. Based on these results, we contend that the initially observed defect in colony forming ability is the direct or indirect consequence of intracellular formation and accumulation of the protein polymers.     

Distribution of stable DnaA-binding sites on the Bacillus subtilis genome detected using a modified ChIP-chip method.

We developed a modified ChIP-chip method, designated ChAP-chip (Chromatin Affinity Precipitation coupled with tiling chip). The binding sites of Bacillus subtilis Spo0J determined using this technique were consistent with previous findings. A DNA replication initiator protein, DnaA, formed stable complexes at eight intergenic regions on the B. subtilis genome. Characterization of the binding sequences suggested that two factors -- the local density of DnaA boxes and their affinities for DnaA -- are critical for stable binding. We further showed that in addition to autoregulation, DnaA directly modulate the expression of sda in a positive, and ywlC and yydA in a negative manner. Examination of possible stable DnaA-binding sequences in other Bacillus species suggested that DnaA-dependent regulation of those genes is maintained in most bacteria examined, supporting their biological significance. In addition, a possible stable DnaA-binding site downstream of gcp is also suggested to be conserved. Furthermore, potential DnaA-binding sequences specific for each bacterium have been identified, generally in close proximity to oriC. These findings suggest that DnaA plays several additional roles, such as control of the level of effective initiator, ATP-DnaA, and/or stabilization of the domain structure of the genome around oriC for the proper initiation of chromosome replication.