Cyano-bridged Fe(II)-Cu(II) bimetallic assemblies: honeycomb-like and pentanuclear structures

A 2D honeycomb-like compound [Fe(CN)₆{Cu(apn)}₃]_n(ClO₄)_2n(H₂O)_4n (1) (apn=N-(3-aminopropyl)-1,3-propanediamine) and a pentanuclear compound [Fe(CN)₆{Cu(dmen)₂}₄](ClO₄)₄ (2) (dmen=N,N-dimethylethylenediamine) have been prepared and characterized. In the synthesis, the use of ferricyanide or ferrocyanide yielded identical products due to reduction of Fe(III) ion to Fe(II) in water. For 1, all cyanide groups of ferrocyanide are bonded to six Cu(II) ions of which two symmetry-related Cu atoms are linked to nitrogen atoms of cyanide ligands bound to the neighboring Fe(II) center, resulting in the honeycomb structure. The variations of the geometries around Cu(II) centers are between ideal trigonal bipyramidal and square pyramidal structures, which may arise from the relative structural arrangements of flexible apn ligands. For 2, all the Cu(II) ions can be seen as square pyramidal geometries composed of basal least-squares planes from four dmen nitrogen atoms and apical nitrogen atoms from cyanide bridge. The Cu-NC angle around Cu centers in 2 is 127.9(7)°, much acuter than that of 1, which is presumably associated with steric interactions between the bulky methyl groups of the dmen ligands on the neighboring Cu ions. Both compounds exhibit very weak antiferromagnetic interactions in the low temperature range.     

Role of glycosylation in the organic anion transporter OAT1

Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential for OAT function. In contrast, a simultaneous replacement of all asparagines in both mOAT1 and hOAT1 impaired the trafficking of the transporters to the plasma membrane. In summary, we provided the evidence that 1) Asp-39 is crucially involved in substrate recognition of OAT1, 2) glycosylation at individual sites is not required for OAT1 function, and 3) glycosylation plays an important role in the targeting of OAT1 onto the plasma membrane. This study is the first molecular identification and characterization of glycosylation of OAT1 and may provide important insights into the structure-function relationships of the organic anion transporter family.     

AHNAK-mediated activation of phospholipase C-gamma1 through protein kinase C

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by the central repeated units (CRUs) of the AHNAK protein in the presence of arachidonic acid. Here we demonstrate that four central repeated units (4 CRUs) of AHNAK act as a scaffolding motif networking PLC-gamma and PKC-alpha. Specifically, 4 CRUs of AHNAK bind and activate PKC-alpha, which in turn stimulates the release of arachidonic acid near where PLC-gamma1 is localized. Moreover, 4 CRUs of AHNAK interacted with PLC-gamma and the concerted action of 4 CRUs with arachidonic acid stimulated PLC-gamma activity. Stimulation of NIH3T3 cells expressing 4 CRUs of AHNAK with phorbol 12-myristate 13-acetate resulted in the increased generation of total inositol phosphates (IP(T)) and mobilization of the intracellular calcium. Phorbol 12-myristate 13-acetate-dependent generation of IP(T) was completely blocked in NIH3T3 cells depleted of PLC-gamma1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-beta isozyme resulting in the generation of a monophasic IP(T) within 30 s in NIH3T3 cells, led to a biphasic pattern for generation of IP(T) in NIH3T3 cells expressing 4 CRUs of AHNAK. The secondary activation of PLC is likely because of the scaffolding activity of AHNAK, which is consistent with the role of 4 CRUs as a molecular linker between PLC-gamma and PKC-alpha.     

A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

We combined the specificity of tumor-specific antibody with the chemokine function of interferon-gamma inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V(H) region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo.     

A simplified procedure for the reduction and alkylation of cysteine residues in proteins prior to proteolytic digestion and mass spectral analysis

A procedure for reduction and alkylation of cysteine residues in proteins was developed using the volatile reagents triethylphosphine and iodoethanol. These reagents may be used to modify proteins in solution, as well as proteins in gel slices, prior to proteolytic digestion and mass spectral analysis. The procedure eliminates several steps with both types of samples. Samples in solution do not need to be desalted following reduction and alkylation, with excess reagent being removed under vacuum. For gel slices, the procedure combines washing, destaining, reduction and alkylation into a single step. The procedure was applied successfully to samples as complex as serum, and we demonstrated alkylation of cysteines to be quantitative in purified proteins. We also were able to reduce and alkylate proteins with these reagents during the gas phase. Elimination of the need for desalting of samples after reaction raised the possibility of automation of the procedure for liquid samples, which is difficult with conventional reduction and alkylation chemistries.     

Metallothionein-III prevents gamma-ray-induced 8-oxoguanine accumulation in normal and hOGG1-depleted cells

Metallothioneins (MT) play an important biological role in preventing oxidative damage to cells. We have previously demonstrated that the efficiency of the protective effect of MT-III against the DNA degradation from oxidative damage was much higher than that of MT-I/II. As an extension of the latter investigation, this study aimed to assess the ability of MT-III to suppress 8-oxoguanine (8-oxoG), which is one of the major base lesions formed after an oxidative attack to DNA and the mutant frequency of the HPRT gene in human fibroblast GM00637 cells upon exposure to gamma-rays. We found that human MT-III expression decreased the level of 8-oxoG and mutation frequency in the gamma-irradiated cells. Using an 8-oxoguanine DNA glycosylase (OGG1)-specific siRNAs, we also found that MT-III expression resulted in the suppression of the gamma-radiation-induced 8-oxoG accumulation and mutation in the OGG1-depleted cells. Moreover, the down-regulation of MT in human neuroblastoma SKNSH cells induced by MT-specific siRNA led to a significant increase in the 8-oxoG level, after exposure to gamma-irradiation. These results suggest that under the conditions of gamma-ray oxidative stress, MT-III prevents the gamma-radiation-induced 8-oxoG accumulation and mutation in normal and hOGG1-depleted cells, and this suppression might, at least in part, contribute to the anticarcinogenic and neuroprotective role of MT-III.     

Association of sustained ERK activity with integrin beta3 induction during receptor activator of nuclear factor kappaB ligand (RANKL)-directed osteoclast differentiation

Osteoclast differentiation is a multi-step process that involves cell proliferation, commitment, and fusion. Some adhesion molecules, including integrin alphavbeta3, have been shown to have roles in osteoclast fusion. In the course of studying with pharmacologic agents known to inhibit protein tyrosine kinases of the Src family, we found that radicicol increased cell fusion during receptor activator of nuclear factor kappaB ligand (RANKL)-driven differentiation of osteoclasts at concentrations far below the ones shown to inhibit its targets in previous studies. Treatments of low doses of radicicol to RAW 264.7 cells that undergo osteoclastic differentiation in the presence of RANKL enhanced the RANKL-induced gene expression of integrin beta3 without any effect on the expression of integrin alphav, which was constitutively high. The cell surface level of integrin alphavbeta3 complexes was consequently augmented by radicicol. In addition, sustained ERK and MEK activation was observed in cells treated with both radicicol and RANKL. More importantly, modulation of ERK activity by the MEK inhibitor U0126 or the gene transduction of a constitutively active form of MEK resulted in a suppression and increment, respectively, of integrin beta3 induction by RANKL. Our data indicate that sustained ERK activity is associated with integrin beta3 induction and subsequent cell surface expression of the alphavbeta3 integrin complex, which may contribute to cell fusion during RANKL-directed osteoclastogenesis.