Fatty Acid Hydroxamate Formation by Intact Cells of Micrococcus sp.

When azelaic acid was used as a sole carbon source on the growth of Micrococcus sp. which was isolated from soil, intact cells of the organism catalyzed the enzymic condensation of fatty acids with hydroxylamine. Some of the characteristics of fatty acid hydroxamate formation were investigated.

The enzyme activity was tested with azelaic acid compared to other fatty acids. Azelylhydroxamate formation was activated with the addition of reduced glutathione or 2-mercaptoethanol. The reaction was inhibited by p-chloromercuribenzoate (PCMB), ethylene diamine tetraacetate (EDTA), NaF and benzoate.     

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.     

The presence of heat-labile factors interfering with binding analysis of fibrinogen with ferritin in horse plasma

Background

Horse fibrinogen has been identified as a plasma specific ferritin-binding protein. There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated. The aim of this study was to analyze the binding between horse fibrinogen and ferritin.

Findings

Although fibrinogen in horse plasma did not show the binding to ferritin coated on the plate wells, after following heat-treatment (60°C, 30 min) of horse plasma, plasma fibrinogen as well as purified horse fibrinogen bound to plates coated with horse spleen ferritin, but not with its apoferritin which lost heme as well as iron after the treatment of reducing reagent. Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.

Conclusions

Heat-treatment of horse plasma enabled plasma fibrinogen to bind to plate well coated with holo-ferritin. From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

     

Analysis of Factors That Determine Weight Gain during Smoking Cessation Therapy

Cigarette smokers are generally known to gain weight after quitting smoking, and such weight gain is thought to contribute to the worsening of glucose tolerance. While smoking cessation therapy such as nicotine replacement is useful to minimize post-cessation weight gain, substantial gain occurs even during the therapy. The purpose of the present study was to identify factors associated with weight gain during smoking cessation therapy. We evaluated 186 patients(132 males and 54 females)who visited our outpatient clinic for smoking cessation, and successfully achieved smoking abstinence. We performed gender-adjusted regression analysis for the rate of BMI increase from the beginning of cessation to 3 months after initiation. Furthermore, we performed multivariate analysis to investigate factors that determine the BMI increase after smoking cessation. The mean BMI significantly (p<0.0001) increased from 23.5±3.6 kg/m² at the initial consultation to 23.9±3.8 kg/m² at 3 months after the start of therapy. There was no significant difference in the extent of BMI increase between nicotine patch and varenicline therapy groups. Factors significantly correlated with the %BMI increase at 3 months after the start of therapy were triglyceride (p = 0.0006, βa = 0.260), high-density lipoprotein cholesterol (p = 0.0386, βa = −0.168), daily cigarette consumption (p = 0.0385, βa = 0.154), and the Fagerström Test for Nicotine Dependence (FTND) score (p = 0.0060, βa = 0.203). Stepwise multivariate analysis demonstrated that triglyceride and the FTND score were the factors determining the post-cessation BMI increase and that the FTND score was the strongest one. The present study demonstrated that smokers with a high FTND score are more likely to gain weight during smoking cessation therapy. Thus, smokers with a high nicotine dependency may require intervention against weight gain in the cessation clinic.     

An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.     

Seasonal change in xylem growth of <Emphasis Type="Italic">Pinus densiflora</Emphasis> in central Japan

This study investigated the seasonal change in xylem growth of Japanese red pine (Pinus densiflora). Wood cores were sampled at 2-week intervals from April to November in 2012 using the microcoring method. Daily increment rates of tracheid number and tree-ring width were compared with seasonal changes in daily mean temperature and photoperiod. Xylem growth started in early to late May and stopped in late October to early November. The maximum daily increment rates of tracheid number and tree-ring width were in early July. The 95 % confidence intervals of the timing of the maximum daily increment rates included the summer solstice (23 June) with the longest photoperiod, but not the warmest day (30 July). The maximum daily increment rate of xylem growth is thought to be controlled by the photoperiod rather than by temperature. The daily mean temperature exceeded 20 °C after the summer solstice, indicating that temperature is not a limiting factor for xylem growth. This study suggests that the timing of maximum daily increment rates of xylem growth of P. densiflora is controlled by the photoperiod.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.     

Distinctive Effects of Cytochalasin B in Chick Primary Myoblasts and Fibroblasts

Actin-based structures play fundamental roles in cellular functions. However it remains controversial how cells cope with the absence of F-actin structures. This report focuses on short- and long-term effects of cytochalasin B (CB) on actin-complexes in fibroblasts and myoblasts. Thirty min of CB treatment dispersed subplasma actin cortices, lamellipodia, ruffled membranes, stress fibers and adhesion plaques into actin patches in fibroblasts and muscle cells. In contrast, 72 hrs CB treatment showed distinct morphological effects. Fibroblasts became giant multinucleated-finger shaped with 5 to 10 protrusions, 3–8 μm in width, and >200 μm in length. They lacked cortical actin, stress fibers, adhesion plaques and ruffled membranes but contained immense lamelliopodia with abnormal adhesion plaque protein complexes. Muscle cells transformed into multinucleated globular-shaped but contained normal I-Z-I and A-bands, indicating that CB did not interfere with the assembly of myofibrils. Within 30 min after CB removal, finger-shaped fibroblasts returned to their original shape and actin-containing structures rapidly reappeared, whereas muscle cells respond slowly to form elongated myotubes following CB washout. The capacity to grow, complete several nuclear cycles, assemble intermediate filaments and microtubules without a morphologically recognizable actin cytoskeleton raises interesting issues related to the role of the actin compartments in eukaryotic cells.