Leukocyte activation by malarial pigment

Malarial pigment, a unique hemozoin crystal composed of unit cells of heme dimers, is present in large amounts in circulating monocytes and neutrophils and can persist unchanged in macrophages for several months. In the present study, we investigated the effect of hemozoin not only on macrophages, but also on neutrophils. We used beta-hematin (BH), a chemically synthetic crystal structurally identical to hemozoin, for these studies. In vitro, BH up-regulated the expression of tumor necrosis factor-alpha in whole blood and in isolated peritoneal macrophages, indicating that hemozoin is able to stimulate monocytes. BH stimulated murine peritoneal neutrophils to express macrophage inflammatory protein-2 (MIP-2), a homologue of human interleukin-8 that is used as a marker of neutrophil activation. Injecting BH into the peritoneal cavity resulted in a dose-dependent migration of neutrophils and a high level of myeloperoxidase activity of peritoneal cells. Finally, BH directly induced neutrophil chemotaxis in vitro. Taken together, these results suggest that the malarial pigment hemozoin can activate leukocytes and may participate in the pathology of severe malaria.     

Complex formation of Plk1 and INCENP required for metaphase-anaphase transition

Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Here, we report that Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. We propose that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics.     

Homopiperazine Derivatives as a Novel Class of Proteasome Inhibitors with a Unique Mode of Proteasome Binding

The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (ß 1, ß 2 and ß 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the ß5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a ß5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.     

A Novel Role for Adipose Ephrin-B1 in Inflammatory Response

Aims

Ephrin-B1 (EfnB1) was selected among genes of unknown function in adipocytes or adipose tissue and subjected to thorough analysis to understand its role in the development of obesity.

Methods and Results

EfnB1 mRNA and protein levels were significantly decreased in adipose tissues of obese mice and such reduction was mainly observed in mature adipocytes. Exposure of 3T3-L1 adipocytes to tumor necrosis factor-α (TNF-α) and their culture with RAW264.7 cells reduced EFNB1 levels. Knockdown of adipose EFNB1 increased monocyte chemoattractant protein-1 (Mcp-1) mRNA level and augmented the TNF-α-mediated THP-1 monocyte adhesion to adipocytes. Adenovirus-mediated adipose EFNB1-overexpression significantly reduced the increase in Mcp-1 mRNA level induced by coculture of 3T3-L1 adipocytes with RAW264.7 cells. Monocyte adherent assay showed that adipose EfnB1-overexpression significantly decreased the increase of monocyte adhesion by coculture with RAW264.7 cells. TNF-α-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was reduced by EFNB1-overexpression.

Conclusions

EFNB1 contributes to the suppression of adipose inflammatory response. In obesity, reduction of adipose EFNB1 may accelerate the vicious cycle involved in adipose tissue inflammation.     

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2′:2,1′-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2′:2,1′-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).     

Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties.

Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K(d) approximately 7 microM at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., Wyer, J. R. , Ladbury, J. E., Bell, J. I., Jakobsen, B. K., and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition.     

An outbreak of amebiasis in an institution for the mentally retarded in Japan

The results of an epidemiological survey in a 190-patient institution for mentally retarded were reported. Twenty percent of the patients had either cysts or trophozoites of Entamoeba histolytica in their stools, and 38% were positive serologically. The amebic outbreak revealed a sex-independent but age-dependent distribution; younger patients had more serious symptoms in cases invasive amebiasis. A high prevalence of amebic infection was found in the heavily retarded patients, and the positive cases tended to concentrate in certain training classes. Further demographic analysis suggests that the amebic infection was possibly caused by abnormal behavior of heavily retarded patients.     

Oriental blepharoplasty: single-stitch, nonincision technique

The facial features of Orientals and Caucasians are different in many respects. In Orientals, the concept of beauty differs in each country depending on the cultural background. At present time, blepharoplasty in the Oriental eye is the most common aesthetic procedure performed in Korea and Japan. The objective should not be the caucasianization of the Oriental eye, where desired modifications are usually quite subtle. Generally, patients prefer more defined and invaginated folds, but not necessarily a very high fold, since the latter will often look unnatural or overdone in the Oriental face. We have found several different types of eyelids in the Oriental population. The purpose of this presentation is to present our nonincision technique to create aesthetically pleasing supratarsal folds. Seven-hundred and sixty-two patients were reviewed. We have found that our single-suture method is very reliable and allows the construction of a more even, smooth, and symmetrical fold.     

Increased superoxide production causes coronary endothelial dysfunction and depressed oxygen consumption in the failing heart

This study examined whether increased superoxide (O(2)(-).) production contributes to coronary endothelial dysfunction and decreased coronary blood flow (CBF) in congestive heart failure (CHF). To test this hypothesis, the effects of the low-molecular-weight SOD mimetic M40401 on CBF and myocardial oxygen consumption (MVo(2)) were examined in dogs during normal conditions and after CHF was produced by 4 wk of rapid ventricular pacing. The development of CHF was associated with decreases of left ventricular (LV) systolic pressure, maximum first derivative of LV pressure, MVo(2), and CBF at rest and during treadmill exercise as well as endothelial dysfunction with impaired vasodilation in response to intracoronary acetylcholine. M40401 increased CBF (18 +/- 5%, P < 0.01) and MVo(2) (14 +/- 6%, P < 0.01) in CHF dogs and almost totally reversed the impaired CBF response to acetylcholine. M40401 had no effect on acetylcholine-induced coronary vasodilation, CBF, or MVo(2) in normal dogs. Western blot analysis demonstrated that extracellular SOD (EC-SOD) was significantly decreased in CHF hearts, whereas mitochondrial Mn-containing SOD was increased. Cytosolic Cu/Zn-containing SOD was unchanged. Both increased O(2)(-). production and decreased vascular O(2)(-). scavenging ability by EC-SOD could have contributed to endothelial dysfunction in the failing hearts.