Metal ion binding properties and conformational states of calcium- and integrin-binding protein

Calcium- and integrin-binding protein (CIB) is a novel member of the helix-loop-helix family of regulatory calcium-binding proteins which likely has a specific function in hemostasis through its interaction with platelet integrin alphaIIbbeta(3). The significant amino acid sequence homology between CIB and other regulatory calcium-binding proteins such as calmodulin, calcineurin B, and recoverin suggests that CIB may undergo a calcium-induced conformational change; however, the mechanism of calcium binding and the details of a structural change have not yet been investigated. Consequently, we have performed a variety of spectroscopic and microcalorimetric studies of CIB to determine its calcium binding characteristics, and the subsequent conformational changes that occur. Furthermore, we provide the first evidence for magnesium binding to CIB and determine the structural consequences of this interaction. Our results indicate that in the absence of any bound metal ions, apo-CIB adopts a folded yet highly flexible molten globule-like structure. Both calcium and magnesium binding induce conformational changes which stabilize both the secondary and tertiary structure of CIB, resulting in considerable increases in the thermal stability of the proteins. CIB was found to bind two Ca(2+) ions in a sequential manner with dissociation constants (K(d)) near 0.54 and 1.9 microM for sites EF-4 and EF-3, respectively. In contrast, CIB bound only one Mg(2+) ion to EF-3 with a K(d) near 120 microM. Together, our results suggest that CIB may exist in multiple structural and metal ion-bound states in vivo which may play a role in its regulation of target proteins such as platelet integrin.     

Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.     

ACE1 polymorphism and progression of SARS

We have hypothesized that genetic predisposition influences the progression of SARS. Angiotensin converting enzyme (ACE1) insertion/deletion (I/D) polymorphism was previously reported to show association with the adult respiratory distress syndrome, which is also thought to play a key role in damaging the lung tissues in SARS cases. This time, the polymorphism was genotyped in 44 Vietnamese SARS cases, with 103 healthy controls who had had a contact with the SARS patients and 50 controls without any contact history. SARS cases were divided into either non-hypoxemic or hypoxemic groups. Despite the small sample size, the frequency of the D allele was significantly higher in the hypoxemic group than in the non-hypoxemic group (p=0.013), whereas there was no significant difference between the SARS cases and controls, irrespective of a contact history. ACE1 might be one of the candidate genes that influence the progression of pneumonia in SARS.     

Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne

An investigation was made of the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant Ananas comosus (pineapple) in malate metabolism during CAM phase III. Pineapple mitochondria showed very high malate dehydrogenase (MDH), and low malic enzyme (ME) and glutamate-oxaloacetate transaminase (GOT) activities. The mitochondria readily oxidized succinate and NADH with high rates and coupling, while they only oxidized NADPH in the presence of Ca(2+). Pineapple mitochondria oxidized malate with low rates under most assay conditions, despite increasing malate concentrations, optimizing pH, providing cofactors such as coenzyme A, thiamine pyrophosphate, and NAD(+), and supplying individually external glutamate or GOT. However, providing glutamate and GOT simultaneously strongly increased the rates of malate oxidation. The OAA easily permeated the mitochondrial membranes to import into or export out of pineapple mitochondria during malate oxidation, but the mitochondria did not consume external Asp or alpha-KG. These results suggest that OAA played a significant role in the mitochondrial malate metabolism of pineapple, in which malate was mainly oxidized by active mMDH to produce OAA which could be exported outside the mitochondria via a malate-OAA shuttle. Cytosolic GOT then consumed OAA by transamination in the presence of glutamate, leading to a large increase in respiration rates. The malate-OAA shuttle might operate as a supporting system for decarboxylation in phase III of PCK-CAM pineapple. This shuttle system may be important in pineapple to provide a source of energy and substrate OAA for cytosolic PCK activity during the day when cytosolic OAA and ATP was limited for the overall decarboxylation process.     

Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity

Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.     

Stress relaxation of human ankles is only minimally affected by knee and ankle angle

Comprehensive characterization of stress relaxation in musculotendinous structures is needed to create robust models of viscoelastic behavior. The commonly used quasi-linear viscoelastic (QLV) theory requires that the relaxation response be independent of tissue strain (length). This study aims to characterize stress relaxation in the musculotendinous and ligamentous structures crossing the human ankle (ankle-only structures and the gastrocnemius muscle–tendon unit, which crosses the ankle and knee), and to determine whether stress relaxation is independent of the length of these structures. Two experiments were conducted on 8 healthy subjects. The first experiment compared stress relaxation over 10 min at different gastrocnemius muscle–tendon unit lengths keeping the length of ankle-joint only structures fixed. The second experiment compared stress relaxation at different lengths of ankle-joint only structures keeping gastrocnemius muscle–tendon unit length fixed. Stress relaxation data were fitted with a two-term exponential function (T=G₀+G₁e^?λ₁t+G₂e^?λ₂t). The first experiment demonstrated a significant effect of gastrocnemius muscle–tendon unit length on G₁, and the second experiment demonstrated an effect of the length of ankle-joint only structures on G₂, λ₁ and λ₂ (p<0.05). Nonetheless, the size of effects on stress relaxation was small (ΔG/G<10%), similar to experimental variability. We conclude that stress relaxation in the relaxed human ankle is minimally affected by changing gastrocnemius muscle–tendon unit length or by changing the lengths of ankle-joint only structures. Consequently quasi-linear viscoelastic models of the relaxed human ankle can use a common stress relaxation modulus at different knee and ankle angles with minimal error.     

Beta7 integrin deficiency suppresses B cell homing and attenuates chronic ileitis in SAMP1/YitFc mice

Lymphocyte recruitment to intestinal tissues depends on β(7) integrins. In this study, we studied disease severity and lymphocyte recruitment into the small intestine in SAMP1/YitFc mice, which develop chronic ileitis with similarity to human Crohn's disease. To assess the role of β(7) integrins in chronic ileitis, we generated SAMP1/YitFc lacking β(7) integrins (SAMP1/YitFc Itgb7(-/-)) using a congenic strain developed via marker-assisted selection. We analyzed ileal inflammation in SAMP1/YitFc and SAMP1/YitFc Itgb7(-/-) mice by histopathology and the distribution of T and B lymphocytes in the mesenteric lymph nodes (MLNs) by flow cytometry. Short-term (18 h) adoptive transfer experiments were used to study the in vivo homing capacity of T and B lymphocytes. In both young (<20 wk) and old (20-50 wk) SAMP1/YitFc Itgb7(-/-) mice, ileitis was reduced by 30-50% compared with SAMP1/YitFc mice. SAMP1/YitFc Itgb7(-/-) mice showed a dramatic 67% reduction in the size of their MLNs, which was caused by a 85% reduction in lymphocyte numbers and reduced short-term B cell homing. Flow cytometric analysis revealed a highly significant decrease in the percentage of B cells in MLNs of SAMP1/YitFc Itgb7(-/-) mice. Cotransfer of SAMP1/YitFc MLN B cells but not SAMP1/YitFc Itgb7(-/-) MLN B cells along with CD4(+) T cells resulted in exacerbated ileitis severity in SCID mice. Our findings suggest that β(7) integrins play an essential role in spontaneous chronic ileitis in vivo by promoting homing of disease-exacerbating B cells to MLNs and other intestinal tissues.     

A homologue of the breast cancer-associated gene BARD1 is involved in DNA repair in plants

hBRCA1 and hBARD1 are tumor suppressor proteins that are involved as heterodimer via ubiquitinylation in many cellular processes, such as DNA repair. Loss of BRCA1 or BARD1 results in early embryonic lethality and chromosomal instability. The Arabidopsis genome carries a BRCA1 homologue, and we were able to identify a BARD1 homologue. AtBRCA1 and the putative AtBARD1 protein are able to interact with each other as indicated by in vitro and in planta experiments. We have identified T-DNA insertion mutants for both genes, which show no visible phenotype under standard growth conditions and are fully fertile. Thus, in contrast to animals, both genes have no indispensable role during development and meiosis in plants. The two single as well as the double mutant are to a similar extent sensitive to mitomycin C, indicating an epistatic interaction in DNA crosslink repair. We could further demonstrate that in Arabidopsis BARD1 plays a prominent role in the regulation of homologous DNA repair in somatic cells.     

Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia

Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.     

Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines

Gold immunolabeling combined with negative staining (GINS) provides a valuable immunocytochemical approach that allows a direct ultrastructural definition of all viral vaccine constituents that share common antigenic features with pathogenic viral particles. These results have implications for the development of viral vaccines since it has been demonstrated that incomplete viral particles such as natural empty capsides and Rotavirus-like particles lacking the infective genome are potential candidates for the production of neutralizing antibodies. Furthermore comparative results of the application of GINS to either inactivated vaccines or unfixed samples provide direct evidence that even after inactivation specific antigenic sites are still available for gold immunolabeling.