An oncoprotein from the plant pathogen agrobacterium has histone chaperone-like activity

Protein 6b, encoded by T-DNA from the pathogen Agrobacterium tumefaciens, stimulates the plant hormone-independent division of cells in culture in vitro and induces aberrant cell growth and the ectopic expression of various genes, including genes related to cell division and meristem-related class 1 KNOX homeobox genes, in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b is found in nuclei and binds to several plant nuclear proteins. Here, we report that 6b binds specifically to histone H3 in vitro but not to other core histones. Analysis by bimolecular fluorescence complementation revealed an interaction in vivo between 6b and histone H3. We recovered 6b from a chromatin fraction from 6b-expressing plant cells. A supercoiling assay and digestion with micrococcal nuclease indicated that 6b acts as a histone chaperone with the ability to mediate formation of nucleosomes in vitro. Mutant 6b, lacking the C-terminal region that is required for cell division-stimulating activity and interaction with histone H3, was deficient in histone chaperone activity. Our results suggest a relationship between alterations in nucleosome structure and the expression of growth-regulating genes on the one hand and the induction of aberrant cell proliferation on the other.     

Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.     

Suppression of influenza A virus nuclear antigen production and neuraminidase activity by a nutrient mixture containing ascorbic acid, green tea extract and amino acids

Influenza, one of the oldest and most common infections, poses a serious health problem causing significant morbidity and mortality, and imposing substantial economic costs. The efficacy of current drugs is limited and improved therapies are needed. A unique nutrient mixture (NM), containing ascorbic acid, green tea extract, lysine, proline, N-acetyl cysteine, selenium among other micronutrients, has been shown to exert anti-carcinogenic and anti-atherogenic activity both in vitro and in vivo. Many of the constituents of NM have been shown to have an inhibitory effect on replication of influenza virus and HIV. This prompted us to study the effect of NM on influenza A virus multiplication in infected cells and neuraminidase activity (NA) in virus particles. Addition of NM to Vero or MDCK cells post infection resulted in dose-dependent inhibition of viral nucleoprotein (NP) production in infected cells. NM-mediated inhibition of viral NP was selective and not due to cytotoxicity towards host cells. This antiviral effect was enhanced by pretreatment of virus with the nutrient mixture. Individual components of NM, namely ascorbic acid and green tea extract, also blocked viral NP production, conferring enhanced inhibition when tested in combination. Incubation of cell-free virus with NM resulted in dose-dependent inhibition of associated NA enzyme activity. In conclusion, the nutrient mixture exerts an antiviral effect against influenza A virus by lowering viral protein production in infected cells and diminishing viral enzymatic activity in cell-free particles.     

Buchbesprechungen zu VHL-Erkrankungen

Buchbesprechungen

Buchbesprechungen zu VHL-Erkrankungen     

Endothelial progenitor cells are integrated in newly formed capillaries and alter adjacent fibrovascular tissue after subcutaneous implantation in a fibrin matrix

Vascularization of bioartificial matrices is crucial for successful tissue engineering. Endothelial progenitor cells (EPC) have shown vascularization potential in ischemic conditions and may also support blood vessel formation in tissue-engineered matrices. The aim of our study was to investigate the impact of a well-characterized murine embryonal EPC line (T17b-EPC) on vascularization and fibrovascular granulation tissue formation after suspension in a fibrine matrix followed by subcutaneous implantation in a separation chamber in rats. EPC were fluorescently labelled in vitro prior to implantation. After 3, 7 or 14 days, animals were killed followed by explantation and histological analysis of the constructs. Before the end of the experiment, Bandeirea Simplicifolia lectin was intravenously injected to mark the vascular ingrowth into the implanted constructs. The transplanted cells were histologically detected at all time-points and located almost exclusively within the fibrin matrix at day 3 but the number of cells in the clot continuously decreased over day 7 to day 14. Conversely, cells were detected within the newly formed granulation tissue in increasing numbers from day 3 over day 7 to day 14. Transplanted cells were also found in the intermuscular septa. Cell viability was confirmed by use of an EPC clone expressing β-galactosidase. Fluorescence microscopy demonstrated integration of the transplanted cells in newly formed blood vessels within the fibrovascular granulation tissue adjacent to the fibrin clot. Presence of cells in the fibrin clot lead to thicker granulation tissue and an increased blood vessel diameter compared to cell-free controls. Organ standard controls showed presence of the transplanted cells in spleens at day 14 after transplantation. In summary, EPC exhibited biological activity after subcutaneous implantation in a fibrin matrix by migration from the fibrin clot into the granulation tissue and along intermuscular septae, undergoing differentiation into mature endothelial cells and integration into newly formed blood vessels and altering fibrovascular granulation tissue development. EPC may hold promise to modulate blood vessel formation in bioartificial matrices.     

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration.     

Virulence of Mycobacterium tuberculosis depends on lipoamide dehydrogenase, a member of three multienzyme complexes

Mycobacterium tuberculosis (Mtb) adapts to persist in a nutritionally limited macrophage compartment. Lipoamide dehydrogenase (Lpd), the third enzyme (E3) in Mtb's pyruvate dehydrogenase complex (PDH), also serves as E1 of peroxynitrite reductase/peroxidase (PNR/P), which helps Mtb resist host-reactive nitrogen intermediates. In contrast to Mtb lacking dihydrolipoamide acyltransferase (DlaT), the E2 of PDH and PNR/P, Lpd-deficient Mtb is severely attenuated in wild-type and immunodeficient mice. This suggests that Lpd has a function that DlaT does not share. When DlaT is absent, Mtb upregulates an Lpd-dependent branched-chain keto acid dehydrogenase (BCKADH) encoded by pdhA, pdhB, pdhC, and lpdC. Without Lpd, Mtb cannot metabolize branched-chain amino acids and potentially toxic branched-chain intermediates accumulate. Mtb deficient in both DlaT and PdhC phenocopies Lpd-deficient Mtb. Thus, Mtb critically requires BCKADH along with PDH and PNR/P for pathogenesis. These findings position Lpd as a potential target for anti-infectives against Mtb.     

Prediction of circulatory equilibrium in response to changes in stressed blood volume

Accurate prediction of cardiac output (CO), left atrial pressure (PLA), and right atrial pressure (PRA) is a prerequisite for management of patients with compromised hemodynamics. In our previous study (Uemura et al. Am J Physiol Heart Circ Physiol 286: H2376-H2385, 2004), we demonstrated a circulatory equilibrium framework, which permits the prediction of CO, PLA, and PRA once the venous return surface and integrated CO curve are known. Inasmuch as we also showed that the surface can be estimated from single-point CO, PLA, and PRA measurements, we hypothesized that a similar single-point estimation of the CO curve would enable us to predict hemodynamics. In seven dogs, we measured the PLA-CO and PRA-CO relations and derived a standardized CO curve using the logarithmic function CO = SL[ln(PLA - 2.03) + 0.80] for the left heart and CO = SR[ln(PRA - 2.13) + 1.90] for the right heart, where SL and SR represent the preload sensitivity of CO, i.e., pumping ability, of the left and right heart, respectively. To estimate the integrated CO curve in each animal, we calculated SL and SR from single-point CO, PLA, and PRA measurements. Estimated and measured CO agreed reasonably well. In another eight dogs, we altered stressed blood volume (-8 to +8 ml/kg of reference volume) under normal and heart failure conditions and predicted the hemodynamics by intersecting the surface and the CO curve thus estimated. We could predict CO [y = 0.93x + 6.5, r2 = 0.96, standard error of estimate (SEE) = 7.5 ml.min(-1).kg(-1)], PLA (y = 0.90x + 0.5, r2= 0.93, SEE = 1.4 mmHg), and PRA (y = 0.87x + 0.4, r2= 0.91, SEE = 0.4 mmHg) reasonably well. In conclusion, single-point estimation of the integrated CO curve enables accurate prediction of hemodynamics in response to extensive changes in stressed blood volume.     

The effects of depowered airbags on skin injuries in frontal automobile crashes

The purpose of this study was to determine the effects of depowered frontal airbags on the incidence of skin injuries. The National Automotive Sampling System database files from 1993 to 2000 were examined in a study including 2,246,524 occupants exposed to airbag deployment in the United States. There was no significant difference between full-powered and depowered airbags, with 60.2 percent of those exposed to a full-powered deployment sustaining a skin injury versus 59.5 percent of occupants exposed to a depowered airbag (p = 0.19). Whether occupants were exposed to a full-powered airbag (1,936,485 occupants) or a depowered airbay (310,039 occupants), the majority of skin injuries were to the upper extremity and the face. Regardless of airbag power, the overwhelming majority of the skin injuries were minor (99.8 percent). There was not a significantly greater risk of injury from any source for occupants exposed to a depowered airbag or a full-powered airbag (p = 0.87). The data suggest that the implementation of depowered airbags did not affect the number, seriousness, location, or source of skin injuries.