Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.     

Effects of protease activated receptor (PAR)2 blocking peptide on endothelin-1 levels in kidney tissues in endotoxemic rat mode

Aims

Septic shock, the severe form of sepsis, is associated with development of progressive damage in multiple organs. Kidney can be injured and its functions altered by activation of coagulation, vasoactive-peptide and inflammatory processes in sepsis. Endothelin (ET)-1, a potent vasoconstrictor, is implicated in the pathogenesis of sepsis and its complications. Protease-activated receptors (PARs) are shown to play an important role in the interplay between inflammation and coagulation. We examined the time-dependent alterations of ET-1 and inflammatory cytokine, such as tumor necrosis factor (TNF)-α in kidney tissue in lipopolysaccharide (LPS)-induced septic rat model and the effects of PAR2 blocking peptide on the LPS-induced elevations of renal ET-1 and TNF-α levels.

Main methods

Male Wistar rats at 8 weeks of age were administered with either saline solution or LPS at different time points (1, 3, 6 and 10 h). Additionally, we treated LPS-administered rats with PAR2 blocking peptide for 3 h to assess whether blockade of PAR2 has a regulatory role on the ET-1 level in septic kidney.

Key findings

An increase in ET-1 peptide level was observed in kidney tissue after LPS administration time-dependently. Levels of renal TNF-α peaked (around 12-fold) at 1 h of sepsis. Interestingly, PAR2 blocking peptide normalized the LPS-induced elevations of renal ET-1 and TNF-α levels.

Significance

The present study reveals a distinct chronological expression of ET-1 and TNF-α in LPS-administered renal tissues and that blockade of PAR2 may play a crucial role in treating renal injury, via normalization of inflammation, coagulation and vaso-active peptide.     

Serum Wisteria Floribunda Agglutinin-Positive Mac-2 Binding Protein Values Predict the Development of Hepatocellular Carcinoma among Patients with Chronic Hepatitis C after Sustained Virological Response

Measurement of Wisteria floribunda agglutinin-positive human Mac-2 binding protein (WFA⁺-M2BP) in serum was recently shown to be a noninvasive method to assess liver fibrosis. The aim of this study was to evaluate the utility of serum WFA⁺-M2BP values to predict the development of hepatocellular carcinoma (HCC) in patients who achieved a sustained virological response (SVR) by interferon treatment. For this purpose, we retrospectively analyzed 238 patients with SVR who were treated with interferon in our department. Serum WFA⁺-M2BP values were measured at pre-treatment (pre-Tx), post-treatment (24 weeks after completion of interferon; post-Tx), the time of HCC diagnosis, and the last clinical visit. Of 238 patients with SVR, HCC developed in 16 (6.8%) patients. The average follow-up period was 9.1 years. The cumulative incidence of HCC was 3.4% at 5 years and 7.5% at 10 years. The median pre-Tx and post-Tx WFA⁺-M2BP values were 1.69 (range: 0.28 to 12.04 cutoff index (COI)) and 0.80 (range: 0.17 to 5.29 COI), respectively. The WFA⁺-M2BP values decreased significantly after SVR (P < 0.001). The median post-Tx WFA⁺-M2BP value in patients who developed HCC was significantly higher than that in patients who did not (P < 0.01). Multivariate analysis disclosed that age (> 60 years), sex (male), pre-Tx platelet count (< 15.0×10³/μL), and post-Tx WFA⁺-M2BP (> 2.0 COI) were associated with the development of HCC after SVR.

Conclusion

Post-Tx WFA⁺-M2BP (> 2.0 COI) is associated with the risk for development of HCC among patients with SVR. The WFA⁺-M2BP values could be a new predictor for HCC after SVR.     

Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.Plants have evolved both constitutive and inducible defense mechanisms against herbivores. Constitutive mechanisms include structural defenses (e.g. spines and trichomes) and specific chemical compounds. Constitutive defense mechanisms provide immediate protection against herbivore attacks, although they represent an energy investment by the plant regardless of whether herbivory occurs or not (Mauricio, 1998; Bekaert et al., 2012). By contrast, inducible defense mechanisms do not require an up-front energy cost, although such mechanisms may not be as immediate as constitutive ones when herbivore feeding occurs (Windram et al., 2012). Accordingly, plants exhibit both constitutive and inducible defense mechanisms against herbivory to balance the speed and cost of response. In this regard, it is plausible that the recruitment of abundant primary metabolites for defensive purposes might represent a substantial benefit to plants, providing both a swift and economical defense function.Toxic chemical compounds form an essential part in both constitutive and inducible defense mechanisms. However, these compounds are potentially a double-edged sword for plants, in a sense that they might pose toxic effects for both plants and herbivores. Plants have evolved an intricate binary system that prevents autointoxication by their own chemical compounds. Specifically, a toxic substance is stored in its inactive form and is spatially isolated from specific activating enzymes. These enzymes activate the substance when cells are disrupted by chewing herbivores (Saunders and Conn, 1978; Thayer and Conn, 1981; Morant et al., 2008). One of the most extensively studied binary defense systems is the glucosinolate/myrosinase system, in which the glucosinolate substrate and their hydrolyzing enzyme, a thioglucosidase myrosinase, are compartmentalized. Upon tissue damage, both the substrate and the enzyme come into contact to produce unstable aglycones, and various toxic compounds are then spontaneously produced (Bones and Rossiter, 1996). Another well-known example of the binary system is comprised of cyanogenic glucosides and β-glucosidase (Vetter, 2000; Mithöfer and Boland, 2012). In this system, nontoxic cyanogenic glycoside compounds are stored in the vacuole, whereas, the related glycosidase is localized in the cytoplasm. Upon cell destruction by chewing herbivores, the cyanogenic glycosides are hydrolyzed by glycosidase to yield unstable cyanohydrin that is either spontaneously or enzymatically converted into toxic hydrogen cyanide and a ketone or an aldehyde. Because the binary defense system is efficient and effective, a use of ubiquitous compounds for such systems would provide further benefits for plants.Tetrapyrrole compounds, in particular heme and chlorophyll, are abundant in plant cells. Despite their significant roles in various biological processes including photosynthesis and respiration, many tetrapyrroles are highly toxic to plant and animal cells, if present in excess amounts (Kruse et al., 1995; Meskauskiene et al., 2001). Their photodynamic properties can cause the generation of reactive oxygen species upon illumination, resulting in cell injury or direct cell death. For example, Tapper et al. (1975) showed that a tetrapyrrole compound (pheophorbide a), which is readily converted from dietary chlorophyll through the loss of magnesium and phytol, reduces the growth and survival rates of young albino rats through its photodynamic property. More recently, Jonker et al. (2002) demonstrated that dietary-derived pheophorbide a causes severe damages on the skin of mutant mice that lack a transporter to excrete pheophorbide a from cells. These studies indicate that incorporation of an excessive amount of tetrapyrrole compounds can induce photosensitization in animals. Previous studies also showed that tetrapyrroles have illumination-independent deleterious effects on insects. For example, pheophorbide a affected the assimilation of the plant sterols to synthesize developmental hormones of insects by inhibiting the activity of a key enzyme, cholesterol acyltransferase (Song et al., 2002). Moreover, some tetrapyrroles, including pheophorbide a, have been suggested to induce illumination-independent cell death in plants as well by an unknown mechanism (Hirashima et al., 2009). It is proposed that organisms use the toxicity of tetrapyrroles for their defense systems. The larvae of tortoise beetle (Chelymorpha alternans) even utilize pheophorbide a as a powerful deterrent in the fecal shield to protect themselves from their predators (Vencl et al., 2009). Kariola et al. (2005) suggested that a chlorophyll derivative, chlorophyllide, is involved in the defense against fungi, based on their observations that down-regulation of a chlorophyll-hydrolyzing enzyme, chlorophyllase (CLH), results in increased susceptibility of Arabidopsis (Arabidopsis thaliana) plants to the necrotrophic fungus Alternaria brassicicola.In this study, we examined the possibility that plants use tetrapyrroles for defense against herbivores by analyzing CLH, a well-known hydrolase common in plants. Chlorophyll consists of a tetrapyrrolic macrocycle and a hydrophobic phytol side chain (Fig. 1). Phytol hydrolysis results in the formation of chlorophyllide (Fig. 1), a less hydrophobic chlorophyll derivative, which has photochemical properties similar to chlorophyll. Two different plant enzymes are known to catalyze the cleavage of phytol, pheophytinase (PPH) and CLH. PPH is a chloroplast-located enzyme that specifically catalyzes the removal of phytol from Mg-free chlorophyll catabolites (Schelbert et al., 2009). This enzyme was only recently discovered and has been shown to be responsible for chlorophyll degradation during leaf senescence. By contrast, CLH has a broader substrate specificity and removes the side chain from chlorophyll or other chlorophyll derivatives (McFeeters et al., 1971). CLH activity was first reported in leaf extracts in 1913 (Willstätter and Stoll, 1913), but despite a century of research, in vivo function and intracellular localization of this enzyme remained controversial. Some reports have indicated CLH to localize to chloroplasts (Azoulay Shemer et al., 2008; Azoulay-Shemer et al., 2011), while Schenk et al. (2007), by examining the intracellular localization of transiently expressed CLH-GFP fusions, proposed Arabidopsis CLH to localize outside the chloroplast. Schenk et al. (2007) also reported that the lack of CLH does not affect chlorophyll degradation during leaf senescence. However, it remains possible that CLH is specifically involved in chlorophyll degradation in response to stresses that activate jasmonate signaling, such as wounding or pathogen attack. This hypothesis is based on the observation that the expression of a CLH gene was highest when methyl-jasmonate (MeJA; a derivative of jasmonic acid) was applied to Arabidopsis plants (Tsuchiya et al., 1999).Open in a separate windowFigure 1.Early steps of proposed chlorophyll breakdown pathways. MCS, Magnesium-dechelating substance.Here, we report that CLH is not involved in endogenous chlorophyll breakdown even when leaf senescence was promoted by jasmonate signaling. CLH is shown to localize to the chlorophyll-free endoplasmic reticulum (ER) and the tonoplast of intact plant cells. We found that CLH promotes the conversion of chlorophyll into chlorophyllide when leaf cells are disrupted or when CLH is genetically mislocalized to chloroplasts. To examine the possibility that plants use chlorophyll and CLH to form a binary defense system against herbivores, a generalist herbivore, Spodoptera litura larvae, was employed to investigate the toxicity of Arabidopsis leaves with genetically enhanced CLH activity and purified chlorophyllide. The results support our hypothesis, indicating plants to deploy an abundant photosynthetic pigment for defense against herbivores, which would be economic and provide adaptation benefits to plants. A potential mechanism of chlorophyllide action as part of the plant defense system is discussed based on the examination of chlorophyllide binding to the insect gut.     

Growth factor contents of autologous human sera prepared by different production methods and their biological effects on chondrocytes

To discuss the autologous serum production for cartilage tissue engineering, we compared three kinds of sera: whole blood-derived serum (WBS), platelet-containing plasma-derived serum (PCS), and plasma-derived serum (PDS), on the growth factor contents and their biological effects on human auricular chondrocytes. EGF, VEGF and PDGF levels were highest in WBS, while PCS and PDS followed WBS. The proliferation effects of WBS were the most pronounced, followed by that of PCS, both of which realized a 1000-fold-increase in chondrocyte numbers at the third passage, whereas PDS reached it after passage 4. No significant differences were observed in histology or cartilaginous matrix measurements of tissue-engineered cartilage produced from chondrocytes cultured under different serum conditions. WBS would be clinically useful because of its potent proliferation effects, while PCS, which possibly saves the red cell concentrate, may be an option in cases where there are elevated risks of blood loss.     

Complete Genome Sequence of Finegoldia magna, an Anaerobic Opportunistic Pathogen

Finegoldia magna (formerly Peptostreptococcus magnus), a memberof the Gram-positive anaerobic cocci (GPAC), is a commensalbacterium colonizing human skin and mucous membranes. Moreover,it is also recognized as an opportunistic pathogen responsiblefor various infectious diseases. Here, we report the completegenome sequence of F. magna ATCC 29328. The genome consistsof a 1 797 577 bp circular chromosome and an 189 163bp plasmid (pPEP1). The metabolic maps constructed based onthe genome information confirmed that most F. magna strainscannot ferment most sugars, except fructose, and have variousaminopeptidase activities. Three homologs of albumin-bindingprotein, a known virulence factor useful for antiphagocytosis,are encoded on the chromosome, and one albumin-binding proteinhomolog is encoded on the plasmid. A unique feature of the genomeis that F. magna encodes many sortase genes, of which substratesmay be involved in bacterial pathogenesis, such as antiphagocytosisand adherence to the host cell. The plasmid pPEP1 encodes sevensortase and seven substrate genes, whereas the chromosome encodesfour sortase and 19 substrate genes. These plasmid-encoded sortasesmay play important roles in the pathogenesis of F. magna byenriching the variety of cell wall anchored surface proteins.