ILPIP,a novel anti-apoptotic protein that enhances XIAP-mediated activation of JNK1 and protection against apoptosis

We have previously described a new aspect of the Inhibitor of Apoptosis (IAP) family of proteins anti-apoptotic activity that involves the TAK1/JNK1 signal transduction pathway (1,2). Our findings suggest the existence of a novel mechanism that regulates the anti-apoptotic activity of IAPs that is separate from caspase inhibition but instead involves TAK1-mediated activation of JNK1. In a search for proteins involved in the XIAP/TAK1/JNK1 signaling pathway we isolated by yeast two-hybrid screening a novel X chromosome-linked IAP (XIAP)-interacting protein that we called ILPIP (hILP-Interacting Protein). Whereas ILPIP moderately activates JNK family members when expressed alone, it strongly enhances XIAP-mediated activation of JNK1, JNK2, and JNK3. The expression of a catalytically inactive mutant of TAK1 blocked XIAP/ILPIP synergistic activation of JNK1 thereby implicating TAK1 in this signaling pathway. ILPIP moderately protects against interleukin-1beta converting enzyme- or Fas-induced apoptosis and significantly potentiates the anti-apoptotic activity of XIAP. In vivo co-precipitation experiments show that both ILPIP and XIAP interact with TAK1 and tumor necrosis factor receptor-associated factor 6. Finally, expression of ILPIP did not affect the ability of XIAP to inhibit caspase activation, further supporting the idea that XIAP protection against apoptosis is achieved by two separate mechanisms: one requiring JNK1 activation and a second involving caspase inhibition.     

Interactions of opioid and chemokine receptors: oligomerization of mu,kappa, and delta with CCR5 on immune cells

In this study, we examined the signaling pathways for extracellular signal-related protein kinase (ERK) activation by three structurally different peroxisome proliferator activated receptor-gamma (PPARgamma) agonists. In murine C2C12 myoblasts, treatment with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and GW1929 leads to ERK1/2 phosphorylation in a time- and concentration-dependent manner. Consistent with ERK phosphorylation, mitogen activated protein/ERK kinase (MEK) phosphorylation as well as Raf-1 kinase activity are also accordingly stimulated, while the constitutive Ser259 phosphorylation of Raf-1 is decreased. The ERK phosphorylation induced by PPARgamma agonists is not blocked by the PKC inhibitors GF109203X and Ro31-8220, the PI3K inhibitor wortmannin, the Ras inhibitor FPTI, the negative mutant of Ras, or the PPARgamma antagonist bisphenol A diglycidil ether. Expression of PPARgamma2 without DNA binding domain or with a nonphosphorylatable mutant (S112A) fails to change ERK phosphorylation by 15d-PGJ(2). On the contrary, the ERK phosphorylation by PPARgamma agonists is inhibited by the MEK inhibitor PD98059, GSH, and permeable SOD mimetic MnTBAP. Chemiluminescence study reveals that these three PPARgamma agonists are able to induce superoxide anion production, with an efficacy similar to their action on ERK phosphorylation. Consistent with this notion, we also show that superoxide anion donor 2,3-dimethoxy-1,4-naphoquinone elicits ERK phosphorylation. In this study, we for the first time demonstrate a novel mechanism, independent of Ras activation but initiated by superoxide anion production, for PPARgamma agonists to trigger the Raf-MEK-ERK1/2 signaling pathway.     

The N-terminal domain of SV40 large T antigen represses the HER2/neu-mediated transformation and metastatic potential in breast cancers

HER2/neu is known to be overexpressed in approximately 40% of human breast and ovarian cancers and it is associated with increased metastasis and poor prognosis. We have shown previously that the N-terminal domain of simian virus 40 large T antigen (LT425) can act as a transforming suppressor of the HER2/neu oncogene in human ovarian cancer. In the present study, we demonstrate that LT425 can also repress the transforming properties of HER2/neu-overexpressing human breast cancer cells. In addition, the results of a chemotaxis assay and an in vitro chemoinvasion assay further suggest that LT425 can also suppress the metastatic potential of the HER2/neu-transformed breast cancer cells. Taken together, these data clearly suggest that the inhibition of the expression of p185 HER2/neu tyrosine kinase by LT425 is capable of suppressing the HER2/neu-mediated transformation and metastatic potential in breast cancers.     

The fatty acid composition of the serum phospholipids of children with sickle cell disease in Nigeria

The purpose of this study was to determine the fatty acid composition of the serum phospholipids of children with sickle cell disease (SCD) in Nigeria and to compare the relative fluidity of the acyl chains of the serum phospholipids of controls versus the subjects with SCD. It is widely accepted that the fatty acid composition of an individual's serum phospholipids reflects that of their tissue phospholipids. An alteration in the fatty acid composition of membrane phospholipids could affect critical membrane-dependent enzymes and processes (e.g., ion and solute transport, hormone-receptor interactions, signal transduction pathways). We found a significant reduction in the content of polyunsaturated n-3 fatty acids in the phospholipids of subjects with SCD which could result in a reduction of the fluidity of their tissue membranes. Specifically, there was a 40-50% reduction in the proportion of total n-3 fatty acids in subjects with SCD. On the basis of calculated melting points and double bond indices of the acyl chains of the serum phospholipids, the phospholipids of the children with SCD are less fluid relative to those of their healthy counterparts. In addition, we determined that linoleic acid, arachidonic acid, and stearic acid were the major determinants of the fluidity of the acyl chains of the serum phospholipids of the healthy controls and children with SCD.     

Stool antigen assay to screen H. pylori infection and to assess the success of 3-Day and 7-Day eradication therapy in the patients with partial gastrectomy

Background. Even after partial gastrectomy, Helicobacter pylori may persist in the residual stomach but be less abundant in the bacterial load. H. pylori stool antigen is a reliable noninvasive tool to detect H. pylori infection in patients without gastrectomy. We thus test whether [ 1 ] the course of H. pylori eradication therapy could be diminished [ 2 ]; stool antigen can effectively detect H. pylori infection for the patients with gastrectomy. Methods. One hundred and eight patients who had undergone partial gastrectomy were enrolled to receive panendoscopy and provided stool samples for H. pylori stool antigen within 3 days after endoscopy. The H. pylori‐infected patients were then randomized to receive either a 3‐ or 7‐day triple therapy for H. pylori eradication. Six weeks later, to evaluate the success of H. pylori eradication, patients received a follow‐up endoscopy and again provided stool samples for H. pylori stool antigen. Results. Seventy out of 108 patients, proven to have H. pylori infection, were evenly randomized into 3‐day and 7‐day therapy groups. The H. pylori eradication rates were similar between the 3‐day and 7‐day triple therapy (90.9 vs. 93.8%, p > .05). Before therapy, the H. pylori stool antigen was 93% sensitive and 100% specific to detect H. pylori. After therapy, H. pylori stool antigen remain 100% sensitive and 88.3% specific to detect the failure of eradication therapy. Conclusion. H. pylori stool antigen is a highly reliable tool to screen H. pylori infection before therapy and to assess the success of eradication therapy in partial gastrectomy patients. To eradicate H. pylori infection for patients with partial gastrectomy, the duration of triple therapy can be shortened.     

Solution structure of the DNA-binding domain of interleukin enhancer binding factor 1 (FOXK1a)

Interleukin enhancer binding factor (ILF) binds to the interleukin-2 (IL-2) promoter and regulates IL-2 gene expression. In this study, the 3D structure of the DNA-binding domain of ILF was determined by multidimensional NMR spectroscopy. NMR structure analysis revealed that the DNA-binding domain of ILF is a new member of the winged helix/forkhead family, and that its wing 2 contains an extra alpha-helix. This is the first study to report the presence of a C-terminal alpha-helix in place of a typical wing 2 in a member of this family. This structural difference may be responsible for the different DNA-binding specificity of ILF compared to other winged helix/forkhead proteins. Our deletion studies of the fragments of ILF also suggest that the C-terminal region plays a regulatory role in DNA binding.     

Penoscrotal extramammary Paget's disease: a review of 33 cases in a 20-year experience

Extramammary Paget's disease in men most frequently involves the penoscrotal area. The uncertainty of the outcome and of the relationship to the underlying adnexal carcinoma and associated internal malignancy still exists. From 1982 to 2001, 33 patients with penoscrotal extramammary Paget's disease were treated and followed up. Therapeutic modalities included carbon dioxide laser ablation (two patients) and local wide excision (31 patients). Split-thickness skin graft (22 patients), local scrotal flap (six patients), and primary closure (three patients) were utilized to reconstruct the penoscrotal defects after local wide excision. An underlying adnexal carcinoma occurred in seven of 33 patients (21.2 percent). The incidence of associated internal malignancy was 9.1 percent (three of 33 patients), including one concurrently and two nonconcurrently associated malignancies. Eight of 33 patients had local recurrence, representing an incidence of 24.2 percent. Three patients (9.1 percent) had distant metastasis and ultimately died of metastatic carcinoma. Of these patients, 31 were grouped according to the degrees of involvement: limited to the epidermis (group 1, n = 14), involvement of the adnexal gland and/or hair follicle (group 2, n = 10), and the presence of an underlying adnexal carcinoma (group 3, n = 7). Local wide excision with subsequent reconstruction by split-thickness skin graft was favored in this series. Patients with an underlying adnexal carcinoma or pathological invasion of the dermis (group 2 or 3) had a worse prognosis than patients without. From this study, it is difficult to address the particular relationship between the outcome and the associated internal malignancy.     

Patterns of initial recurrence and prognosis after sentinel lymph node biopsy and selective lymphadenectomy for melanoma

The histologic status of the sentinel lymph node is a highly significant prognostic factor for patients with clinically localized cutaneous melanoma. The patterns of initial treatment failure of patients with positive sentinel lymph node biopsy versus those with negative results have not been well described. The purpose of this study was to determine the relative prognostic importance of sentinel lymph node status and to compare patterns of initial treatment failure and prognosis of node-positive versus node-negative cutaneous melanoma patients staged by sentinel lymph node biopsy and selective lymphadenectomy. The authors reviewed the pertinent demographic and surgical data in a consecutive series of patients with cutaneous melanoma who underwent sentinel lymph node staging of nonpalpable regional nodes. Sentinel lymph node biopsy was performed using a combination of blue dye and radiolocalization. Patients with positive biopsy results underwent selective lymphadenectomy, whereas those with negative results were observed. Site(s) and date(s) of initial recurrence and death were determined, and disease-free and overall survival probabilities were compared between positive and negative groups using the log-rank test and multivariable Cox regression analysis. Between February of 1994 and August of 2000, 408 patients with melanoma underwent sentinel lymph node biopsy to stage 518 regional lymph node basins. Mean Breslow tumor thickness was 2.27 mm (range, 0.2 to 14.0 mm). Eighty-five patients (20.8 percent) had at least one histologically positive sentinel lymph node, and selective lymphadenectomy yielded additional positive lymph nodes in 18 of 84 patients (21.4 percent). Recurrences were noted in 70 patients (17 percent) at a median follow-up period of 31.4 months. Recurrences were more frequent in patients with positive biopsy results (36.5 percent) than in those with negative results (12.1 percent, p < 0.0001). Distant sites of initial recurrence were more likely in the positive group than in the negative group (71 percent versus 49 percent of recurrences, respectively; p = 0.06). The false-negative rate for sentinel lymph node staging was 4.5 percent and overall accuracy was 99 percent compared with clinical follow-up. Disease-free and overall survival correlated significantly with tumor thickness, ulceration, sentinel lymph node status, and the number of tumor-positive lymph nodes (two-sided p < 0.0001 for all comparisons). Multivariable analysis revealed that sentinel lymph node status (p = 0.003), tumor thickness (p = 0.016), ulceration (p = 0.006), and age (p = 0.003) were significant independent predictors of survival for the entire group. Tumor thickness and ulceration were significant predictors of recurrence and survival in sentinel node-negative patients but not in sentinel node-positive patients. Sentinel lymph node histology is possibly the most important negative predictor of early recurrence and survival in patients with American Joint Committee on Cancer stage I and II melanoma. The number of positive lymph nodes provides additional prognostic information. Although sentinel node-negative patients are a prognostically favorable group, various combinations of local and regional recurrences comprise the most common pattern of initial relapse after a negative sentinel lymph node biopsy result.     

Effects of prenyl pyrophosphates on the binding of PKCgamma with RACK1

Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus.     

Angeli's salt induces neurotoxicity in dopaminergic neurons in vivo and in vitro

In this study, we investigated the hypothesis that the pro-oxidative properties of Angeli's salt (AS), a nitroxyl anion (HNO/NO -) releasing compound, cause neurotoxicity in dopaminergic neurons. The pro-oxidative properties were demonstrated in vitro by measuring hydroxylation products of salicylate and peroxidation of lipids under various redox conditions. AS (0-1000 μM) released high amounts of hydroxylating species in a concentration dependent manner. AS also increased lipid peroxidation in brain homogenates at concentrations below 100 μM, while inhibiting it at 1000 μM concentration. The AS induced pro-oxidative effects were completely suppressed by copper (II), which converts nitroxyl anion to nitric oxide, as well as by a potent nitroxyl anion scavenger glutathione. Neurotoxicity towards dopaminergic neurons was tested in rat nigrostriatal dopaminergic system in vivo and by using primary mesencephalic dopaminergic neuronal cultures in vitro . Intranigral infusion of AS (0-400 nmol) caused neurotoxicity reflected as a dose dependent decrease of striatal dopamine seven days after treatment. The effect of the 100 nmol dose was more pronounced when measured 50 days after the infusion. Neurotoxicity was also confirmed as a decrease of tyrosine hydroxylase positive neurons in the substantia nigra. Neither sulphononoate, a close structural analog of AS, nor sodiumnitrite caused changes in striatal dopamine, thus reflecting lack of neurotoxicity. In primary dopaminergic neuronal cultures AS reduced [ 3 H] dopamine uptake with concentrations over 200 μM confirming neurotoxicity. In line with the quite low efficacy to increase lipid peroxidation in vitro , infusion of AS into substantia nigra did not cause increased formation of fluorescent products of lipid peroxidation. These results support the hypothesis that AS derived species oxidize critical thiol groups, rather than membrane lipids, potentially leading to protein oxidation/dysfunction and demonstrated neurotoxicity. These findings may have pathophysiological relevance in case of excess formation of nitroxyl anion.