Production and Purification of a New Chillproofing Enzyme and Its Properties

Chillproofing enzyme was obtained from broth cultures of Serratia marcescens B–103. This extracellular enzyme, tentatively, named the S-enzyme was highly purified from the culture supernatant by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G–200 and column chromatography on DEAE-Sephadex A–50.

The purified preparation appeared homogeneous on a ultracentrifugation with a sedimentation coefficient of 3.14 S and a molecular weight of 38,000~45,000 determined by the method of Whitaker.

The S-enzyme hydrolyzed various proteins at pH 4~6 and at low temperature hydrolyzed nitrogenous substances which may cause chill haze in beer. So the chillproofing activity of the S-enzyme may be due to its proteolytic activity.

The S-enzyme was stable at 4°C at pH 5~10.5. It was completely inactivated by heating at 60°C for 10 min, and was inactivated by Hg²⁺ and Pb²⁺ and activated by Mn²⁺, Ca²⁺. Mg²⁺ and Zn²⁺     

Some Physicochemical Properties and Substrate Specificity of Acid Protease of Rhodotorula glutinis K-24

Some physicochemical properties, amino acid composition, and substrate specificity of the acid protease of Rhodotorula glutinis K-24 were determined. The molecular weight was estimated to be about 30,000 by the sedimentation equilibrium method. This value also coincided with the data obtained from Andrews’s method.

The isoelectric point was pH 4.5, and the amino terminal amino acid was identified to be alanine. The enzyme contained 14.5% of nitrogen and was composed of 285 residues of amino acid. Substrate specificity toward synthetic peptides was similar to that of pepsin, but its activity was considerably weak.

The enzyme was inactivated by diazoacetyl glycine ethylester, p-bromophenacyl bromide, et al., which attacked the active center of pepsin.     

Selective inhibition of heme oxygenase-2 activity by analogs of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole): Exploration of the effects of substituents at the N-1 position

Several analogs based on the lead structure of 1-(4-chlorobenzyl)-2-(pyrrolidin-1-ylmethyl)-1H-benzimidazole (clemizole) were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). Many of the compounds were found to be potent and highly selective for the HO-2 isozyme (constitutive), and had substantially less inhibitory activity on the HO-1 isozyme (inducible). The compounds represent the first report of highly potent and selective inhibitors of HO-2 activity, and complement our suite of selective HO-1 inhibitors. The study has revealed many candidates based on the inhibition of heme oxygenases for potentially useful pharmacological and therapeutic applications.     

Alkaloid constituents from flower buds and leaves of sacred lotus (Nelumbo nucifera,Nymphaeaceae) with melanogenesis inhibitory activity in B16 melanoma cells

Methanolic extracts from the flower buds and leaves of sacred lotus (Nelumbo nucifera, Nymphaeaceae) were found to show inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the methanolic extracts, a new alkaloid, N-methylasimilobine N-oxide, was isolated together with eleven benzylisoquinoline alkaloids. The absolute stereostructure of the new alkaloid was determined from chemical and physicochemical evidence. Among the constituents isolated, nuciferine, N-methylasimilobine, (?)-lirinidine, and 2-hydroxy-1-methoxy-6a,7-dehydroaporphine showed potent inhibition of melanogenesis. Comparison of the inhibitory activities of synthetic related alkaloids facilitated characterization of the structure-activity relationships of aporphine- and benzylisoquinoline-type alkaloids. In addition, 3–30 μM nuciferine and N-methylasimilobine inhibited the expression of tyrosinase mRNA, 3–30 μM N-methylasimilobine inhibited the expression of TRP-1 mRNA, and 10–30 μM nuciferine inhibited the expression of TRP-2 mRNA.     

Rho of Plant GTPase Signaling Regulates the Behavior of Arabidopsis Kinesin-13A to Establish Secondary Cell Wall Patterns

Plant cortical microtubule arrays determine the cell wall deposition pattern and proper cell shape and function. Although various microtubule-associated proteins regulate the cortical microtubule array, the mechanisms underlying marked rearrangement of cortical microtubules during xylem differentiation are not fully understood. Here, we show that local Rho of Plant (ROP) GTPase signaling targets an Arabidopsis thaliana kinesin-13 protein, Kinesin-13A, to cortical microtubules to establish distinct patterns of secondary cell wall formation in xylem cells. Kinesin-13A was preferentially localized with cortical microtubules in secondary cell wall pits, areas where cortical microtubules are depolymerized to prevent cell wall deposition. This localization of Kinesin-13A required the presence of the activated ROP GTPase, MICROTUBULE DEPLETION DOMAIN1 (MIDD1) protein, and cortical microtubules. Knockdown of Kinesin-13A resulted in the formation of smaller secondary wall pits, while overexpression of Kinesin-13A enlarged their surface area. Kinesin-13A alone could depolymerize microtubules in vitro; however, both MIDD1 and Kinesin-13A were required for the depolymerization of cortical microtubules in vivo. These results indicate that Kinesin-13A regulates the formation of secondary wall pits by promoting cortical microtubule depolymerization via the ROP-MIDD1 pathway.     

Caveolin-1 is a competitive inhibitor of heme oxygenase-1 (HO-1) with heme: identification of a minimum sequence in caveolin-1 for binding to HO-1

Heme oxygenase (HO) catalyzes the O(2)-dependent degradation of heme to biliverdin IXα, carbon monoxide (CO), and free ferrous iron through a multistep mechanism. Electrons required for HO catalysis in mammals are provided by NADPH-cytochrome P450 reductase. Recently, Kim et al. reported for the first time that HO, especially inducible HO-1, appears in caveolae and showed that caveolin-1, a principal isoform of the caveolin family, physically interacts with HO-1 [ Jung , N. H. et al. ( 2003 ) IUBMB Life 55 , 525 - 532 ; Kim , H. P. et al. ( 2004 ) FASEB J. 18 , 1080 - 1089 ]. In the present study, we confirmed by immunoprecipitation experiments that rat HO-1 and rat caveolin-1 (residues 1-101) directly interact with each other and that the HO-1 activity is inhibited by caveolin-1 (1-101). The 82-101 residues of caveolin-1 (CAV(82-101)), called the caveolin scaffolding domain, play essential roles in caveolin-related protein-protein interactions. The HO-1 activity is also inhibited by CAV(82-101) in a competitive manner with hemin, and a hemin titration experiment showed that CAV(82-101) interferes with hemin binding to HO-1. The enzyme kinetics and surface plasmon resonance experiments gave comparable K(i) and K(D) values of 5.2 and 1.0 μM for CAV(82-101), respectively, with respect to the interaction with HO-1. These observations indicated that CAV(82-101) and hemin share a common binding site within the HO-1 protein. The identified caveolin binding motif (FLLNIELF) of rat HO-1 is incomplete compared to the proposed consensus sequence. The affinity between HO-1 and CAV(82-101), however, was almost completely or remarkably eliminated by replacement of Phe(207) and/or Phe(214) with Ala, indicating that HO-1 binds to caveolin-1 via this motif. Among the peptide fragments derived from CAV(82-101), i.e., CAV(82-91), CAV(87-96), CAV(92-101), and CAV(97-101), CAV(92-101) and CAV(97-101) are able to inhibit the HO-1 activity to a similar extent; thus, the five-amino acid sequence (residues 97-101) is considered to be a minimum sequence for binding to HO-1.     

Regulation of cancer stem cell properties by CD9 in human B-acute lymphoblastic leukemia

Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some of the cases still exhibit therapy-resistant. We have previously reported that CD9 was expressed heterogeneously in B-ALL cell lines and CD9⁺ cells exhibited an asymmetric cell division with greater tumorigenic potential than CD9⁻ cells. CD9⁺ cells were also serially transplantable in immunodeficient mice, indicating that CD9⁺ cell possess self-renewal capacity. In the current study, we performed more detailed analysis of CD9 function for the cancer stem cell (CSC) properties. In patient sample, CD9 was expressed in the most cases of B-ALL cells with significant correlation of CD34-expression. Gene expression analysis revealed that leukemogenic fusion proteins and Src family proteins were significantly regulated in the CD9⁺ population. Moreover, CD9⁺ cells exhibited drug-resistance, but proliferation of bulk cells was inhibited by anti-CD9 monoclonal antibody. Knockdown of CD9 remarkably reduced the leukemogenic potential. Furthermore, gene ablation of CD9 affected the expression and tyrosine-phosphorylation of Src family proteins and reduced the expression of histone-deubiquitinase USP22. Taken together, our results suggest that CD9 links to several signaling pathways and epigenetic modification for regulating the CSC properties of B-ALL.     

Reproductive failure,possible maternal infanticide,and cannibalism in wild moustached tamarins, <Emphasis Type="Italic">Saguinus mystax</Emphasis>

Maternal infanticide in wild non-human primates has only been reported twice. In this paper, we report a possible new case of infanticide and cannibalism within a series of four successive reproductive failures in wild moustached tamarins, Saguinus mystax. Necropsy and genetic analyses of the corpses enabled us to rule out any pathology, and to determine paternity. The mother was seen biting and then eating the head of its own infant during a period when another female was pregnant and gave birth just 1 month later. Before that, the perpetrator had given birth to twins three times successfully when four to five adult and subadult males were present in the group. Although we do not know for certain that the infant was alive when the mother started biting it, our field observations preceding the event suggest it probably was. The possible infanticide case and the two cases of births and early death of the infants occurred while only two to three adult males were present in the group. This could be the second case of maternal infanticide reported in the genus Saguinus and the similar circumstances suggest a common pattern. We discuss these events in the light of the different functional explanations of infanticide and conclude that parental manipulation was the most likely: the mother could have terminated the investment in offspring that had low chances of survival in a group with low availability of helpers.     

Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain

Planarians have robust regenerative ability dependent on X-ray-sensitive pluripotent stem cells, called neoblasts. Here, we report that planarians can regenerate dopaminergic neurons after selective degeneration of these neurons caused by treatment with a dopaminergic neurotoxin (6-hydroxydopamine; 6-OHDA). This suggests that planarians have a system to sense the degeneration of dopaminergic neurons and to recruit stem cells to produce dopaminergic neurons to recover brain morphology and function. We confirmed that X-ray-irradiated planarians do not regenerate brain dopaminergic neurons after 6-OHDA-induced lesioning, suggesting that newly generated dopaminergic neurons are indeed derived from pluripotent stem cells. However, we found that the majority of regenerated dopaminergic neurons were 5-bromo-2'-deoxyuridine-negative cells. Therefore, we carefully analyzed when proliferating stem cells became committed to become dopaminergic neurons during regeneration by a combination of 5-bromo-2'-deoxyuridine pulse-chase experiments, immunostaining/in situ hybridization, and 5-fluorouracil treatment. The results strongly suggested that G(2) -phase stem cells become committed to dopaminergic neurons in the mesenchymal space around the brain, after migration from the trunk region following S-phase. These new findings obtained from planarian regeneration provide hints about how to conduct cell-transplantation therapy for future regenerative medicine.