Rosmarinic acid downregulates the oxLDL­induced interaction between monocytes and endothelial cells, in addition to monocyte diapedesis, under high glucose conditions.

Endothelial dysfunction during diabetes has been previously reported to be at least in part attributed to increased oxidized low­density lipoprotein (oxLDL) levels mediated by high glucose (HG) levels. Endothelial inflammation increases the adhesiveness of monocytes to the endothelium in addition to increasing vascular permeability, promoting diabetic atherogenesis. In a previous study, it was reported that oxLDL treatment induced nucleotide­binding domain and leucine­rich repeat containing family, pyrin domain­containing 3 inflammasome activation in endothelial cells (ECs) under HG conditions, in a manner that could be effectively reversed by rosmarinic acid. However, it remains unclear whether oxLDL­mediated inflammasome activation can regulate the interaction between monocytes and ECs. The effects of oxLDL­mediated inflammasome activation on endothelial permeability under HG conditions, in addition to the effects of rosmarinic acid on these oxLDL­mediated processes, also remain poorly understood. Therefore, the present study aimed to elucidate the mechanisms involved in oxLDL­induced endothelial permeability and monocyte diapedesis under HG conditions, in addition to the potential effects of rosmarinic acid. ECs were treated with oxLDL under HG conditions in the presence or absence of ROS scavengers mitoTEMPO and NAC, p38 inhibitor SB203580, FOXO1 inhibitor AS1842856 or transfected with the TXNIP siRNA, before protein expression levels of intercellular adhesion molecule 1 (ICAM­1), vascular cell adhesion molecule­1 (VCAM­1), phosphorylated vascular endothelial­cadherin (VE­cadhedrin), VE­cadherin and zonula occludens­1 (ZO­1) were measured by western blotting. In addition, adhesion assay and Transwell assays were performed. oxLDL was found to significantly increase the expression of ICAM­1 and VCAM­1 in ECs under HG conditions whilst also enhancing the adhesion of monocytes to ECs. This was found to be dependent on the reactive oxygen species (ROS)/p38 MAPK/forkhead box O1 (FOXO1)/thioredoxin interacting protein (TXNIP) signaling pathway. In addition, oxLDL­stimulated ECs under HG conditions exhibited increased phosphorylated VE­cadherin protein levels and decreased ZO­1 protein expression levels compared with those in untreated ECs, suggesting increased endothelial permeability. Furthermore, monocyte transmigration through the endothelial monolayer was significantly increased by oxLDL treatment under HG conditions. These oxLDL­mediated effects under HG conditions were also demonstrated to be dependent on this ROS/p38 MAPK/FOXO1/TXNIP signaling pathway. Subsequently, rosmarinic acid treatment significantly reversed oxLDL­induced overexpression of adhesion molecules and monocyte­EC adhesion, oxLDL­induced endothelial junction hyperpermeability and monocyte transmigration through the endothelial monolayer under HG conditions, in a dose­dependent manner. These results suggest that rosmarinic acid can exert a protective effect against oxLDL­mediated endothelial dysfunction under HG conditions by reducing the interaction between monocytes and ECs in addition to preventing monocyte diapedesis.

Paroxetine Induces Apoptosis of Human Breast Cancer MCF-7 Cells through Ca2+-and p38 MAP Kinase-Dependent ROS Generation.

Depression is more common in women with breast cancer than the general population. Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are widely used for the treatment of patients with depression and a range of anxiety-related disorders. The association between the use of antidepressant medication and breast cancer is controversial. In this study, we investigated whether and how SSRIs induce the death of human breast cancer MCF-7 cells. Of the antidepressants tested in this study (amitriptyline, bupropion, fluoxetine, paroxetine, and tianeptine), paroxetine most reduced the viability of MCF-7 cells in a time-and dose-dependent manner. The exposure of MCF-7 cells to paroxetine resulted in mitochondrion-mediated apoptosis, which is assessed by increase in the number of cells with sub-G1 DNA content, caspase-8/9 activation, poly (ADP-ribose) polymerase cleavage, and Bax/Bcl-2 ratio and a reduction in the mitochondrial membrane potential. Paroxetine increased a generation of reactive oxygen species (ROS), intracellular Ca2+ levels, and p38 MAPK activation. The paroxetine-induced apoptotic events were reduced by ROS scavengers and p38 MAPK inhibitor, and the paroxetine's effect was dependent on extracellular Ca2+ level. Paroxetine also showed a synergistic effect on cell death induced by chemotherapeutic drugs in MCF-7 and MDA-MB-231 cells. Our results showed that paroxetine induced apoptosis of human breast cancer MCF-7 cells through extracellular Ca2+-and p38 MAPK-dependent ROS generation. These results suggest that paroxetine may serve as an anticancer adjuvant to current cancer therapies for breast cancer patients with or without depression.

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3.

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 ß) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.

Decreased expression of heat shock protein 20 in colorectal cancer and its implication in tumorigenesis.

Heat shock protein 20 (HSP20), which is a member of the small heat shock protein family, is known to participate in many pathological processes, such as asthma, intimal hyperplasia, and insulin resistance. However, the function of HSP20 in cancer development is not yet fully understood. In this study, we identified HSP20 as a down-regulated protein in 20 resected colorectal cancer (CRC) specimens compared with their paired normal tissues. Because HSP20 proteins were barely detectable in HCT-116 cells (a human colorectal cancer cell line), recombinant adenovirus encoding HSP20 (Ad-HSP20) was used to induce HSP20 overexpression in HCT-116 cells. Infection of Ad-HSP20, but not control adenovirus (Ad-GFP), reduced viability, and induced massive apoptosis in a time-dependent manner. The forced expression of HSP20 enhanced caspase-3/7 activity and down-regulated the anti-apoptotic Bcl-xL and Bcl-2 mRNA and protein levels. In addition, immunohistochemical analysis of 94 CRC specimens for HSP20 protein showed that reduced HSP20 expression was related to advanced TNM stage, lymph node metastasis, and tumor recurrence. Our study shows, for the first time, that expression of the HSP20 protein has a pro-death role in colorectal cancer cells. Therefore, HSP20 may have value as a prognostic tumor marker and its overexpression might be a novel strategy for CRC therapy.

Spinal cord injury markedly altered protein expression patterns in the affected rat urinary bladder during healing stages.

The influence of spinal cord injury (SCI) on protein expression in the rat urinary bladder was assessed by proteomic analysis at different time intervals post-injury. After contusion SCI between T9 and T10, bladder tissues were processed by 2-DE and MALDI-TOF/MS at 6 hr to 28 days after SCI to identify proteins involved in the healing process of SCI-induced neurogenic bladder. Approximately 1,000 spots from the bladder of SCI and sham groups were visualized and identified. At one day after SCI, the expression levels of three protein were increased, and seven spots were down-regulated, including heat shock protein 27 (Hsp27) and heat shock protein 20 (Hsp20). Fifteen spots such as S100-A11 were differentially expressed seven days post-injury, and seven proteins including transgelin had altered expression patterns 28 days after injury. Of the proteins with altered expression levels, transgelin, S100-A11, Hsp27 and Hsp20 were continuously and variably expressed throughout the entire post-SCI recovery of the bladder. The identified proteins at each time point belong to eight functional categories. The altered expression patterns identified by 2-DE of transgelin and S100-A11 were verified by Western blot. Transgelin and protein S100-A11 may be candidates for protein biomarkers in the bladder healing process after SCI.

Cigarette Smoke Extract-induced Reduction in Migration and Contraction in Normal Human Bronchial Smooth Muscle Cells.

The proliferation, migration, cytokine release, and contraction of airway smooth muscle cells are key events in the airway remodeling process that occur in lung disease such as asthma, chronic obstruction pulmonary disease, and cancer. These events can be modulated by a number of factors, including cigarette smoke extract (CSE). CSE-induced alterations in the viability, migration, and contractile abilities of normal human airway cells remain unclear. This study investigated the effect of CSE on cell viability, migration, tumor necrosis factor (TNF)-α secretion, and contraction in normal human bronchial smooth muscle cells (HBSMCs). Treatment of HBSMCs with 10% CSE induced cell death, and the death was accompanied by the generation of reactive oxygen species (ROS). CSE-induced cell death was reduced by N-acetyl-l-cysteine (NAC), an ROS scavenger. In addition, CSE reduced the migration ability of HBSMCs by 75%. The combination of NAC with CSE blocked the CSE-induced reduction of cell migration. However, CSE had no effect on TNF-α secretion and NF-κB activation. CSE induced an increase in intracellular Ca(2+) concentration in 64% of HBSMCs. CSE reduced the contractile ability of HBSMCs, and the ability was enhanced by NAC treatment. These results demonstrate that CSE treatment induces cell death and reduces migration and contraction by increasing ROS generation in normal HBSMCs. These results suggest that CSE may induce airway change through cell death and reduction in migration and contraction of normal HBSMCs.

Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer.

PURPOSE: The aim of this study is to identify the potential tumor markers that function in carcinogenesis and tumor progression, thus providing important diagnostic and prognostic information. EXPERIMENTAL DESIGN: We performed 2-D gel electrophoresis and MALDI-TOF MS to investigate the differentially expressed proteins in 25 papillary thyroid carcinoma tissues. For validation of candidate proteins and investigation of clinical significance, we performed Western, Northern blot analysis and immunohistochemical staining. RESULTS: Our proteomic analyses revealed significantly decreased annexin A3 expression in papillary thyroid carcinoma at both the protein and mRNA levels, compared with normal thyroid tissue. ANXA3 immunoreactivity was not significantly correlated with lymph node metastasis, multifocality, capsular invasion or perithyroidal extension in thyroid cancer. However, the tumor subgroup with a lymph node metastasis score of >3 displayed significantly lower ANXA3 expression than did subgroups with negative and ≤3 scores (p=0.001). Moreover, ANXA3 expression was markedly lower in large tumors (>1 cm in diameter) than in microcarcinomas (p=0.001). CONCLUSION AND CLINICAL RELEVANCE: Decreased expression of ANXA3 in papillary thyroid cancer supports the idea that ANXA3 may be an effective marker of microcarcinoma, and a negative predictor of papillary thyroid cancer progression.

Proteomic analysis of differentially expressed proteins in bovine endometrium with endometritis.

Endometritis is one of the primary reasons for reproductive failure. In order to investigate endometritis-associated marker proteins, proteomic analysis was performed on bovine endometrium with endometritis. In bovine endometritis, desmin, α-actin-2, heat-shock protein (HSP) 27, peroxiredoxin-6, luteinizing hormone receptor isoform 1, collectin-43 precursor, deoxyribonuclease-I (DNase-I), and MHC class I heavy chain (MHC-Ih) were up-regulated. In contrast, transferrin, interleukin-2 precursor, hemoglobin ß subunit, and potassium channel tetramerisation domain-containing 11 (KCTD11) were down-regulated in comparison to normal endometrium. The proteomic results were validated by semiquantitative-PCR and immunoblot analysis. The mRNA levels of desmin, transferrin, α-actin-2, HSP27, KCTD11, and MHC-Ih were up-regulated by over 1.5-fold, and showed a pattern similar to their proteomic profiles. Desmin and α-actin-2 protein showed positive correlations between proteomic analysis and immunoblot analysis. These results suggest that desmin and α-actin-2 may play important roles in endometritis-related function, and could be useful markers for the diagnosis of bovine endometritis.

Acetylcholine rescues two-cell block through activation of IP3 receptors and Ca2+/calmodulin-dependent kinase II in an ICR mouse strain.

Acetylcholine (ACh) causes early activation events in mouse oocytes, but little is known about its precise role in the early embryonic development of mice. We aimed to determine whether and how ACh is capable of rescuing two-cell block in an in vitro culture system. ACh evoked different transient Ca(2+) patterns showing a higher Ca(2+) peak in the two-cell stage embryos (two-cells) than observed in mature oocytes. In early two-cells subjected to an in vitro two-cell block, xestospongin C (Xes-C), an IP3 receptor antagonist, significantly decreased the level of the ACh-induced Ca(2+) increase. The reduction in the ACh-induced Ca(2+) increase by Xes-C in late two-cells was lower than that in early two-cells. Furthermore, KN62 and KN93, both CaMKII inhibitors, were found to reduce the magnitude of the ACh-induced Ca(2+) increase in early two-cells. The addition of ACh to the culture medium showed an ability to rescue in vitro two-cell block. However, the addition of ACh together with both Xes-C and CaMKII inhibitors or with either inhibitor separately had no effect on the rescue of two-cell block. Long-term exposure of late two-cells to ACh decreased morula and early blastocyst development and ACh had a differential effect on early and late two-cells. These results indicate that ACh likely rescues the in vitro two-cell block through activation of IP3R- and/or CaMKII-dependent signal transduction pathways.

Heat-shock dependent oligomeric status alters the function of a plant-specific thioredoxin-like protein, AtTDX.

We found that Arabidopsis AtTDX, a heat-stable and plant-specific thioredoxin (Trx)-like protein, exhibits multiple functions, acting as a disulfide reductase, foldase chaperone, and holdase chaperone. The activity of AtTDX, which contains 3 tetratricopeptide repeat (TPR) domains and a Trx motif, depends on its oligomeric status. The disulfide reductase and foldase chaperone functions predominate when AtTDX occurs in the low molecular weight (LMW) form, whereas the holdase chaperone function predominates in the high molecular weight (HMW) complexes. Because deletion of the TPR domains results in a significant enhancement of AtTDX disulfide reductase activity and complete loss of the holdase chaperone function, our data suggest that the TPR domains of AtTDX block the active site of Trx and play a critical role in promoting the holdase chaperone function. The oligomerization status of AtTDX is reversibly regulated by heat shock, which causes a transition from LMW to HMW complexes with concomitant functional switching from a disulfide reductase and foldase chaperone to a holdase chaperone. Overexpression of AtTDX in Arabidopsis conferred enhanced heat shock resistance to plants, primarily via its holdase chaperone activity.

Proteomic analysis of rat brains following exposure to electroconvulsive therapy.

Electroconvulsive therapy (ECT) is one of the most effective treatments used in psychiatry to date. The mechanisms of ECT action, however, are the least understood and still unclear. As a tool to elucidate the mechanisms of action of ECT, we employed proteomic analysis based on the identification of differentially expressed proteins after exposure to repeated ECT in rat brains. The expression of proteins was visualized by silver stain after two-dimensional gel electrophoresis. Of 24 differentially expressed protein spots (p<0.05 by Student t-test), six different proteins from 7 spots were identified by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF)/mass spectrometry. Among the identified proteins, there were five dominantly expressed proteins in the ECT-treated rat brain tissues (p<0.05); S100 protein beta chain, 14-3-3 protein zeta/delta, similar to ubiquitin-like 1 (sentrin) activating enzyme subunit 1, suppressor of G2 allele of SKP1 homolog, and phosphatidylinositol transfer protein alpha. The expression of only one protein, ACY1 protein, was repressed (p<0.05). These findings likely serve for a better understanding of mechanisms involved in the therapeutic effects of ECT.

Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A.

Deoxyhypusine hydroxylase (DOHH) is a novel metalloenzyme that catalyzes the final step of the post-translational synthesis of hypusine (Nepsilon-(4-amino-2-hydroxybutyl)lysine) in the eukaryotic translation initiation factor 5A (eIF5A). Hypusine synthesis is unique in that it occurs in only one protein, denoting the strict specificity of the modification enzymes toward the substrate protein. The specificity of the interaction between eIF5A and DOHH was investigated using human eIF5A (eIF5A-1 isoform) and human recombinant DOHH. DOHH displayed a strong preference for binding the deoxyhypusine-containing form of eIF5A, over the eIF5A precursor or the hypusine-containing eIF5A, indicating a role for the deoxyhypusine residue in binding. In addition to the deoxyhypusine residue, a large portion of the eIF5A polypeptide (>20-90 amino acids) is required for effective modification by DOHH. We have identified the amino acid residues of DOHH that are critical for substrate binding by alanine substitution of 36 conserved amino acid residues. Of these, alanine substitution at Glu57, Glu90, Glu208, Glu241, Gly63, or Gly214 caused a severe impairment in eIF5A(Dhp) binding, with a complete loss of binding and activity in the E57A and E208A mutant enzymes. Only aspartate substitution mutants, E57D or E208D, retained partial activity and substrate binding, whereas alanine, glutamine, or asparagine mutants did not. These findings support a proposed model of DOHH-eIF5A binding in which the amino group(s) of the deoxyhypusine side chain of the substrate is primarily anchored by gamma-carboxyl groups of Glu57 and Glu208 at the DOHH active site.

Heme oxygenase-1 protects rat kidney from ureteral obstruction via an antiapoptotic pathway.

This study examined the functional significance of heme oxygenase-1 (HO-1) expression on renal injury induced by ureteral obstruction in the rat kidney. Male Sprague-Dawley rats were divided into three groups, after which unilateral ureteral obstruction (UUO) was performed: untreated (group 1), treated with 30 mg/kg body wt hemin (group 2), and treated with 50 microg/kg body wt zinc (alpha) protoporphyrin eta (ZnPP) and 30 mg/kg hemin (group 3). After 7 and 14 d, histologic changes and the expression of HO-1, Bcl-2, Bad, TGF-beta, and cleaved caspase-3 were examined. Tubular lumens were dilated and epithelial cells were flattened on day 7 after UUO. Interstitial fibrosis and separation of the tubules were markedly increased on day 14. In contrast, the kidneys that were treated with hemin exhibited minimal interstitial fibrosis and flattening of epithelial cells on day 7 and fewer changes on day 14 than in the controls. However, treatment with ZnPP, an inhibitor of HO enzyme activity, eliminated the beneficial effect of hemin on interstitial fibrosis and tubular dilation. Increased HO-1 expression was associated with increased Bcl-2. In the ZnPP-treated rats, Bcl-2 signals were decreased compared with the hemin group. The level of proapoptotic Bad was not changed in any group. The positive cells for cleaved caspase-3 were significantly increased in renal tubular epithelial cells and tubulointerstitial cells in the obstructed rats, and hemin treatment decreased the caspase-3 activation. This study demonstrates that upregulation of HO-1 provides protection against renal injury that follows UUO. This effect is dependent on modulation of the antiapoptotic pathway by HO-1 expression.

Deoxyhypusine hydroxylase is a Fe(II)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected].

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.

The expression of ketohexokinase is diminished in human clear cell type of renal cell carcinoma.

For identification and targeting of tumor-associated marker proteins, the proteome of clear cell type of renal cell carcinoma (RCC) and normal kidney tissues was analyzed by 2-DE. Ketohexokinase (also called fructokinase), which catalyzes the phosphorylation of fructose to fructose 1-phosphate, was identified by MALDI-TOF MS and found to be expressed at low rates in the renal tumor tissues. We found a decreased amount of ketohexokinase mRNA in RCC compared to that observed in the normal kidney tissues by Northern blot. The activity of ketohexokinase in 20 clear cell RCC specimens and the 20 corresponding normal kidneys was investigated, and its activity was shown to be approximately 1.4-fold lower in the RCC specimens than in the normal kidney. Ketohexokinase activity in tumor stage pT3 RCC was 1.5-fold lower than in pT1 RCC. The level of ketohexokinase activity in histological grade 3 RCC was 1.8-fold lower than that in grade 1 cancer. In addition, using in situ hybridization, it was revealed that ketohexokinase in the normal kidney tissue was confined to the proximal tubular epithelial cells, while the expression of ketohexokinase in RCC tissues was extremely low. Our research results show that the expression of human ketohexokinase was diminished in clear cell RCC.

Identification of proteins differentially expressed in the conventional renal cell carcinoma by proteomic analysis.

Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin alpha-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and alpha-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).

Stenotrophomonas maltophilia infection in patients receiving continuous ambulatory peritoneal dialysis.

BACKGROUND: Stenotrophomonas maltophilia is a gram-negative bacillus that has become increasingly recognized as an important nosocomial pathogen, particularly in individuals with severe debilitation or immunosuppression. S. maltophilia is also characterized by its resistance to multiple antibiotics. S maltophilia peritonitis in CAPD (continuous ambulatory peritoneal dialysis) patients is associated with a poor prognosis and loss of CAPD catheter. No report concerning this entity has been presented in Korea. Therefore, we describe and discuss five cases of the S. maltophilia infection associated with CAPD in three patients with peritonitis and two with exit-site infections. METHODS: We performed a retrospective search for episodes of S. maltophilia infections related to CAPD in our renal unit. The baseline levels of hemoglobin, albumin, cholesterol, BUN and creatinine were compared with age, sex and, if possible, the underlying disease-matched controls. RESULTS: All the patients with S. maltophilia peritonitis had diabetes mellitus as the underlying disease. The individual patients also had other significant combined morbidities, such as panhypopituitarism, COPD chronic obstructive pulmonary disease, cerebrovascular accident and myocardial infarction. The level of hemoglobin in these patients was significantly lower than in the controls, and the mean values of serum albumin, creatinine and BUN were also low. CONCLUSION: Immune dysfunction due to uremia, anemia, malnutrition, other comorbidities (e.g. diabetes mellitus), and also, an indwelling peritoneal catheter may be predisposing factors for the S. maltophilia infection in CAPD patients. Once the S. maltophilia infection is diagnosed in CAPD patient, the patient should be treated based on the understanding of this particular organism.

Protein kinase CK2 phosphorylates and interacts with deoxyhypusine synthase in HeLa cells.

Deoxyhypusine is a modified lysine and formed posttranslationally to be the eukaryotic initiation factor eIF5A by deoxyhypusine synthase, employing spermidine as butylamine donor. Subsequent hydroxylation of this deoxyhypusine-containing intermediate completes the maturation of eIF5A. The previous report showed that deoxyhypusine synthase was phosphorylated by PKC in vivo and the association of deoxyhypusine synthase with PKC in CHO cells was PMA-, and Ca(2+)/phospholipid-dependent. We have extended study on the phosphorylation of deoxyhypusine synthase by protein kinase CK2 in order to define its role on the regulation of eIF5A in the cell. The results showed that deoxyhypusine synthase was phosphorylated by CK2 in vivo as well as in vitro. Endogenous CK2 in HeLa cells and the cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced or decreased by the addition of CK2 effectors such as polylysine, heparin, and poly(Glu, Tyr) 4:1. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mainly phosphorylated on threonine residue and less intensely on serine. These results suggest that phosphorylation of deoxyhypusine synthase is CK2-dependent cellular event as well as PKC-mediated effect. However, there were no observable changes in enzyme activity between the phosphorylated and unphosphorylated forms of deoxyhypusine synthase. Taken together, besides its established function in hypusine modification involving eIF5A substrate, deoxyhypusine synthase and its phosphorylation modification may have other independent cellular functions because of versatile roles of deoxyhypusine synthase.

Deoxyhypusine synthase is phosphorylated by protein kinase C in vivo as well as in vitro.

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. We earlier observed that yeast recombinant deoxyhypusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold following treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca(2+), suggesting a possible involvement of PKC. We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in different cell lines in order to define its role on the regulation of eIF5A in the cell. Deoxyhypusine synthase was found to be phosphorylated by endogenous kinases in CHO, NIH3T3, and chicken embryonic cells. The highest degree of phosphorylation was found in CHO cells. Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cells was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly diminished by diacyl ethylene glycol (DAEG), a PKC inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca(2+)/DAG. Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca(2+) plus DAG. Close association of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune coprecipitation and was PMA-, and Ca(2+)/phospholipid dependent. These results suggest that phosphorylation of deoxyhypusine synthase was PKC-dependent cellular event and open a path for possible regulation in the interaction with eIF5A precursor for hypusine synthesis.