Antimicrobial activity of lipoprotein particles containing apolipoprotein Al

Human plasmain vitro inhibits the growth of coagulase negative staphylococci,S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity againstS. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated withS. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation.To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition.These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.     

Flocculation and pentadecane production of a novel filamentous cyanobacterium <Emphasis Type="Italic">Limnothrix</Emphasis> sp. strain SK1-2-1

Objective

A novel filamentous cyanobacterium, a photosynthesizing microorganism, was isolated from a river, and its unique features of flocculation and pentadecane production were characterized.

Results

Microscopic observations and a phylogenetic analysis with 16S rDNA revealed that this strain was a Limnothrix species denoted as the SK1-2-1 strain. Auto cell-flocculation was observed when this strain was exposed to a two-step incubation involving a standing cultivation following a shaking preincubation. Flocculation was enhanced by blue light at a wavelength at 470 nm and irradiation for several hours to 1 day. Moreover, the strain exhibiting exponential cell growth may preferentially accumulate alkanes as pentadecane C₁₅H₃₂ alkane, which may be used as jet fuel, at a range of approximately 1% in the dry cell weight of flocculated cells.

Conclusion

This is the first study on biofuel production using flocculated cells in which the specific manner of production may be regulated by cultivation conditions.

     

Engineering the substrate specificity of Alcaligenes D-aminoacylase useful for the production of D-amino acids by optical resolution

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (AxD-NAase) offers a novel biotechnological application, the production of D-amino acid from the racemic mixture of N-acyl-DL-amino acids. However, its substrate specificity is biased toward certain N-acyl-D-amino acids. To construct mutant AxD-NAases with substrate specificities different from those of wild-type enzyme, the substrate recognition site of the AxD-NAase was rationally manipulated based on computational structural analysis and comparison of its primary structure with other D-aminoacylases with distinct substrate specificities. Mutations of amino acid residues, Phe191, Leu298, Tyr344, and Met346, which interact with the side chain of the substrate, induced marked changes in activities toward each substrate. For example, the catalytic efficiency (k(cat)/K(m)) of mutant F191W toward N-acetyl-D-Trp and N-acetyl-D-Ala was enhanced by 15.6- and 1.5-folds, respectively, compared with that of the wild-type enzyme, and the catalytic efficiency (k(cat)/K(m)) of mutant L298A toward N-acetyl-D-Trp was enhanced by 4.4-folds compared with that of the wild-type enzyme. Other enzymatic properties of both mutants, such as pH and temperature dependence, were the same as those of the wild-type enzyme. The F191W mutant in particular is considered to be useful for the enzymatic production of D-Trp which is an important building block of some therapeutic drugs.     

In vitro HIV-1 selective integration into the target sequence and decoy-effect of the modified sequence

Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5'-(GTCCCTTCCCAGT)(n)(ACTGGGAAGGGAC)(n)-3', and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA.     

Focus Issue on Calcium Signaling: Analyses of a Gravistimulation-Specific Ca2+ Signature in Arabidopsis using Parabolic Flights

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca2+]_c). However, the [Ca2+]_c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca²⁺ response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10⁻⁴g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca2+]_c increase, which corresponds closely to the second sustained [Ca2+]_c increase observed in ground experiments. The [Ca2+]_c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g–2g) into Ca²⁺ signals on a subsecond time scale.Calcium ion (Ca²⁺) functions as an intracellular second messenger in many signaling pathways in plants (White and Broadley, 2003; Hetherington and Brownlee, 2004; McAinsh and Pittman, 2009; Spalding and Harper, 2011). Endogenous and exogenous signals are spatiotemporally encoded by changing the free cytoplasmic concentration of Ca²⁺ ([Ca2+]_c), which in turn triggers [Ca2+]_c-dependent downstream signaling (Sanders et al., 2002; Dodd et al., 2010). A variety of [Ca2+]_c increases induced by diverse environmental and developmental stimuli are reported, such as phytohormones (Allen et al., 2000), temperature (Plieth et al., 1999; Dodd et al., 2006), and touch (Knight et al., 1991; Monshausen et al., 2009). The [Ca2+]_c increase couples each stimulus and appropriate physiological responses. In the Ca²⁺ signaling pathways, the stimulus-specific [Ca2+]_c pattern (e.g. amplitude and oscillation) provide the critical information for cellular signaling (Scrase-Field and Knight, 2003; Dodd et al., 2010). Therefore, identification of the stimulus-specific [Ca2+]_c signature is crucial for an understanding of the intracellular signaling pathways and physiological responses triggered by each stimulus, as shown in the case of cold acclimation (Knight et al., 1996; Knight and Knight, 2000).Plants often exhibit biphasic [Ca2+]_c increases in response to environmental stimuli. Thus, slow cooling causes a fast [Ca2+]_c transient followed by a second, extended [Ca2+]_c increase in Arabidopsis (Arabidopsis thaliana; Plieth et al., 1999; Knight and Knight, 2000). The Ca²⁺ channel blocker lanthanum (La³⁺) attenuated the fast transient but not the following increase (Knight and Knight, 2000), suggesting that these two [Ca2+]_c peaks have different origins. Similarly, hypoosmotic shock caused a biphasic [Ca2+]_c increase in tobacco (Nicotiana tabacum) suspension-culture cells (Takahashi et al., 1997; Cessna et al., 1998). The first [Ca2+]_c peak was inhibited by gadolinium (Gd³⁺), La³⁺, and the Ca²⁺ chelator EGTA (Takahashi et al., 1997; Cessna et al., 1998), whereas the second [Ca2+]_c increase was inhibited by the intracellular Ca²⁺ store-depleting agent caffeine but not by EGTA (Cessna et al., 1998). The amplitude of the first [Ca2+]_c peak affected the amplitude of the second increase and vice versa (Cessna et al., 1998). These results suggest that even though the two [Ca2+]_c peaks originate from different Ca²⁺ fluxes (e.g. Ca²⁺ influx through the plasma membrane and Ca²⁺ release from subcellular stores, respectively), they are closely interrelated, showing the importance of the kinetic and pharmacological analyses of these [Ca2+]_c increases.Changes in the gravity vector (gravistimulation) could work as crucial environmental stimuli in plants and are generally achieved by rotating the specimens (e.g. +180°) in ground experiments. Use of Arabidopsis seedlings expressing apoaequorin, a Ca²⁺-reporting photoprotein (Plieth and Trewavas, 2002; Toyota et al., 2008a), has revealed that gravistimulation induces a biphasic [Ca2+]_c increase that may be involved in the sensory pathway for gravity perception/response (Pickard, 2007; Toyota and Gilroy, 2013) and the intracellular distribution of auxin transporters (Benjamins et al., 2003; Zhang et al., 2011). These two Ca²⁺ changes have different characteristics. The first transient [Ca2+]_c increase depends on the rotational velocity but not angle, whereas the second sustained [Ca2+]_c increase depends on the rotational angle but not velocity. The first [Ca2+]_c transient was inhibited by Gd³⁺, La³⁺, and the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid but not by ruthenium red (RR), whereas the second sustained [Ca2+]_c increase was inhibited by all these chemicals. These results suggest that the first transient and second sustained [Ca2+]_c increases are related to the rotational stimulation and the gravistimulation, respectively, and are mediated by distinct molecular mechanisms (Toyota et al., 2008a). However, it has not been demonstrated directly that the second sustained [Ca2+]_c increase is induced solely by gravistimulation; it could be influenced by other factors, such as an interaction with the first transient [Ca2+]_c increase (Cessna et al., 1998), vibration, and/or deformation of plants during the rotation.To elucidate the genuine Ca²⁺ signature in response to gravistimulation in plants, we separated rotation and gravistimulation under microgravity (μg; less than 10⁻⁴g) conditions provided by parabolic flight (PF). Using this approach, we were able to apply rotation and gravistimulation to plants separately (Fig. 1). When Arabidopsis seedlings were rotated +180° under μg conditions, the [Ca2+]_c response to the rotation was transient and almost totally attenuated in a few seconds. Gravistimulation (transition from μg to 1.5g) was then applied to these prerotated specimens at the terminating phase of the PF. This gravistimulation without simultaneous rotation induced a sustained [Ca2+]_c increase. The kinetic properties of this sustained [Ca2+]_c increase were examined under different gravity intensities (0.5g–2g) and sequences of gravity intensity changes (Fig. 2A). This analysis revealed that gravistimulation-specific Ca²⁺ response has an almost linear dependency on gravitational acceleration (0.5g–2g) and an extremely rapid responsiveness of less than 1 s.Open in a separate windowFigure 1.Diagram of the experimental procedures for applying separately rotation and gravistimulation to Arabidopsis seedlings. Rotatory stimulation (green arrow) was applied by rotating the seedlings 180° under μg conditions, and 1.5g 180° rotation gravistimulation (blue arrow) was applied to the prerotated seedlings after μg.Open in a separate windowFigure 2.Acceleration, temperature, humidity, and pressure in an aircraft during flight experiments. A, Accelerations along x, y, and z axes in the aircraft during PF. The direction of flight (FWD) and coordinates (x, y, and z) are indicated in the bottom graph. The inset shows an enlargement of the acceleration along the z axis (gravitational acceleration) during μg conditions lasting for approximately 20 s. B, Temperature, humidity, and pressure in the aircraft during PF. Shaded areas in graphs denote the μg condition.     

Vasohibin prevents arterial neointimal formation through angiogenesis inhibition

Vasohibin is a VEGF-inducible angiogenesis inhibitor in vascular endothelium. Here we examined the presence of vasohibin in human arterial wall, and found it in endothelium of adventitial microvessels in atherosclerotic lesion. Adventitial angiogenesis is involved in the progression of neointimal formation. Even in the presence of endogenous angiogenesis inhibitors, pathological angiogenesis persists. However, the supplementation of exogenous angiogenesis inhibitors can prevent pathological angiogenesis. We evaluated the potential role of vasohibin in neointimal formation. Adenovirus-mediated human vasohibin gene transfer in mouse liver resulted in the release of vasohibin in plasma and exhibited anti-angiogenic effects at remote sites. This gene transfer inhibited adventitial angiogenesis, macrophage infiltration, and neointimal formation after cuff placement on mouse femoral artery. Vasohibin exhibited no direct effect on migration and proliferation of smooth muscle cells. Thus, vasohibin has an activity to prevent neointimal formation by inhibiting adventitial angiogenesis.     

Direct assay for alpha-amylase using fluorophore-modified cyclodextrins

A simple assay method for alpha-amylase was developed based on fluorophore-modified cyclodextrins (CDs). Four kinds of CD derivatives bearing a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-amine) moiety were prepared as artificial substrates for the assay method. The fluorescence intensity of all the NBD-amine-modified CDs decreased upon addition of Aspergillus oryzae alpha-amylase, indicating a reduction in hydrophobicity near the NBD-amine moiety induced by hydrolysis of the CD ring. NC4gammaCD, having a gamma-CD and an amino-tetramethylene spacer, was the most sensitive substrate for the alpha-amylase assay. The initial rate of hydrolysis of NC4gammaCD displayed a liner correlation to the concentration of the alpha-amylase. NC4gammaCD was sensitive to the alpha-amylase but was not sensitive to guest compounds that were accommodated by the native CDs.     

Molecular Cloning and DNA Analysis of the Orotidine-5′-Phosphate Decarboxylase Gene from the Yeast Saccharomyces exiguus Yp74L-3

The orotidine-5′-phosphate decarboxylase gene of Saccharomyces exiguus Yp74L-3 was cloned as a DNA fragment complementing a ura4 mutation of this yeast. The coding region of the gene is 807 bp in length, and represents 68.7% similarity to the corresponding gene of S. cerevisiae (URA3). The cloned URA4 gene was shown to be located on the 790-kbp Chromosome (chr) VIII of S. exiguus Yp74L-3. The neighbor-joining phylogenetic tree based on the orotidine-5′-phosphate decarboxylase coding sequences indicates that S. exiguus Yp74L-3 is closely related to Kluyveromyces yeasts, as well as to a S. cerevisiae laboratory strain. Received: 4 February 2000 / Accepted: 3 July 2000     

Regulatory mechanisms and function of ERK MAP kinases

Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is a key factor for determining the specificity of cellular responses including cell proliferation, cell differentiation and cell survival. The fidelity of this signaling is regulated by docking interactions as well as scaffolding. Subcellular localization of ERK is controlled by cytoplasmic ERK anchoring proteins that have a nuclear export signal (NES), such as MEK. In quiescent cells, ERK and MEK localize to the cytoplasm. In response to stimulation, dissociation of the MEK-ERK complex is induced and activated ERK translocates to the nucleus. Recently, several negative regulators for Ras/ERK signaling have been identified and their detailed molecular mechanisms have been analyzed. Among them, Sprouty and Sef act as a temporal and a spatial regulator, respectively, for Ras/ERK signaling. Thus, multiple factors are involved in control of Ras/ERK signaling.