Concurrent production of chitin from shrimp shells and fungi

Crustacean shells constitute the traditional and current commercial source of chitin. Conversely, the control of fungal fermentation processes to produce quality chitin makes fungal mycelia an attractive alternative source. Therefore, the exploitation of both of these sources to produce chitin in a concurrent process should be advantageous and is reported here. Three proteolytic Aspergillus niger (strains 0576, 0307 and 0474) were selected from a screening for protease activity from among 34 zygomycete and deuteromycete strains. When fungi and shrimp shell powder were combined in a single reactor, the release of protease by the fungi facilitated the deproteinization of shrimp-shell powder and the release of hydrolyzed proteins. The hydrolyzed proteins in turn were utilized as a nitrogen source for fungal growth, leading to a lowering of the pH of the fermentation medium, thereby further enhancing the demineralization of the shrimp-shell powder. The shrimp-shell powders and fungal mycelia were separated after fermentation and extracted for chitin with 5% LiCl/DMAc solvent. Chitin isolates from the shells were found to have a protein content of less than 5%, while chitin isolates from the three fungal mycelia strains had protein content in the range of 10-15%. The relative molecular weights as estimated by GPC for all chitin samples were in the 10(5) dalton range. All samples displayed characteristic profiles for chitin in their FTIR and solid-state NMR spectra. All chitin samples evaluated with MTT and Neutral Red assays with three commercial cell lines did not display cytotoxic effects.     

Synergistic effects of MAP2 and MAP1B knockout in neuronal migration, dendritic outgrowth, and microtubule organization

MAP1B and MAP2 are major members of neuronal microtubule-associated proteins (MAPs). To gain insights into the function of MAP2 in vivo, we generated MAP2-deficient (map2(-/-)) mice. They developed without any apparent abnormalities, which indicates that MAP2 is dispensable in mouse survival. Because previous reports suggest a functional redundancy among MAPs, we next generated mice lacking both MAP2 and MAP1B to test their possible synergistic functions in vivo. Map2(-/-)map1b(-/-) mice died in their perinatal period. They showed not only fiber tract malformations but also disrupted cortical patterning caused by retarded neuronal migration. In spite of this, their cortical layer maintained an "inside-out" pattern. Detailed observation of primary cultures of hippocampal neurons from map2(-/-)map1b(-/-) mice revealed inhibited microtubule bundling and neurite elongation. In these neurons, synergistic effects caused by the loss of MAP2 and MAP1B were more apparent in dendrites than in axons. The spacing of microtubules was reduced significantly in map2(-/-)map1b(-/-) mice in vitro and in vivo. These results suggest that MAP2 and MAP1B have overlapping functions in neuronal migration and neurite outgrowth by organizing microtubules in developing neurons both for axonal and dendritic morphogenesis but more dominantly for dendritic morphogenesis.     

Cinnamophilin as a novel antiperoxidative cytoprotectant and free radical scavenger

The antioxidant properties of cinnamophilin were evaluated by studying its ability to react with relevant reactive oxygen species, and its protective effect on cultured cells and biomacromolecules under oxidative stress. Cinnamophilin concentration-dependently suppressed non-enzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC50 value of 8.0+/-0.7 microM and iron ion/ADP/ascorbate-initiated rat liver mitochondrial lipid peroxidation with an IC50 value of 17.7+/-0.2 microM. It also exerted an inhibitory activity on NADPH-dependent microsomal lipid peroxidation with an IC50 value of 3.4+/-0.1 microM without affecting microsomal electron transport of NADPH-cytochrome P-450 reductase. Both 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azo-bis(2-amidinopropane) dihydrochloride-derived peroxyl radical tests demonstrated that cinnamophilin possessed marked free radical scavenging capacity. Cinnamophilin significantly protected cultured rat aortic smooth muscle cells (A7r5) against alloxan/iron ion/H2O2-induced damage resulting in cytoplasmic membranous disturbance and mitochondrial potential decay. By the way, cinnamophilin inhibited copper-catalyzed oxidation of human low-density lipoprotein, as measured by fluorescence intensity and thiobarbituric acid-reactive substance formation in a concentration-dependent manner. On the other hand, it was reactive toward superoxide anions generated by the xanthine/xanthine oxidase system and the aortic segment from aged spontaneously hypertensive rat. Furthermore, cinnamophilin exerted a divergent effect on the respiratory burst of human neutrophil by different stimulators. Our results show that cinnamophilin acts as a novel antioxidant and cytoprotectant against oxidative damage.     

Studies on the cardiovascular action of TPY-beta: an antihypertensive agent with antiplatelet activity

1-Pyrrolidinylmethyl-2-naphthol hydrochloride (TPY-beta) has been reported to have hypotensive and bradycardiac effects in anesthetized rats. Whether administration of atropine or bilateral vagotomy affects mean arterial pressure or heart rate was examined. The percentage difference in hypotensive and bradycardiac effect of TPY-beta (0.5 mg/kg) was 63 +/- 5% and 68 +/- 9%, respectively, in unpretreated rats compared to control levels. Atropine pretreatment (0.1 mg/kg, i.v.) significantly reduced the depressant effect of TPY-beta, although heart rate and mean arterial pressure remained 21 +/- 3% and 31 +/- 4%, respectively, as compared to control levels. Vagotomy decreased heart rate and mean arterial pressure response but moderate bradycardiac (13 +/- 2%) and hypotensive (10 +/- 3%) effects still remained as compared to control levels. Unilateral microinjection of 1, 3.3 and 10 nM TPY-beta into the nucleus tractus solitarri elicited a dose-dependent depressor (-10 +/- 2; -20 +/- 3; -25 +/- 3 mmHg) and bradycardiac activities (-20 +/- 4; -26 +/- 5; -55 +/- 10 beats/min). TPY-beta also relaxed the isolated rat aortic rings preconstracted with high extracellular K+ (80 mM) and Ca2+ (1.9 mM). The above findings suggest that the suppressive effects of TPY-beta may involve activation of vagus nerve and a direct inhibition of Ca2+ channel. In addition, TPY-beta inhibited the aggregation of washed rabbit platelets (aggregated by arachidonic acid and collagen) and adhesiveness on fibrinogen-coated surface. The results suggest that TPY-beta possesses antihypertensive and antiplatelet activity.     

猫皮层体感Ⅱ区内脏伤害感受神经元的电生理特性及形态特征

应用胞内记录和标记技术,观察了猫皮质第Ⅱ感觉区内脏大神经代表区的神经元对电刺激内脏大神经反应诱发反应及形态特征。结果表明,在２５１个记录单位中,有１０９个为内脏伤害性感受神经元,其诱发反应分为兴奋性、抑制性及混合性三类。在形式上ＩＳＰＳ及ＥＰＳＰ－ＩＰＳＰ序列反应较多。对其中２１个神经元用神经生物素进行细胞内电泳标记,显示细胞的形态特点是胞体较小,分布于皮质Ⅱ、Ⅲ、Ⅴ层,其中兴奋性和神经元形态多为     

Cloning and tissue distribution of the human B3GALT7 gene, a member of the β1,3-Glycosyltransferase family

We report here the cloning and tissue distribution of the human B3GALT7 gene, a member of the beta1,3-Glycosyltransferase family, structurally related to the beta1,3-Galactosyltransferase family and beta1,3- N -acetylglucosaminyltransferase family, isolated from a human lung cDNA library. B3GALT7 is mapped to chromosome 19q13.2 by browsing the UCSC genomic database. It contains an ORF with length of 1191bp, encoding a protein with a signal peptide sequence and galactosyl-T domain, and its molecular weight and isoelectric point is predicted to be 43.3 kDa and 8.67 respectively. The molecular weight of the protein when expressed in E. coli corresponded to that expected. Northern blotting showed that B3GALT7 was highly expressed in lung, throat and ileum, whereas the expression level was low in tongue, breast, uteri, testis. In addition, it was also demonstrated that B3GALT7 is differentially transcribed in human tumor cell lines.     

一氧化氮对家兔房室结细胞自发活动的电生理效应

应用经典玻璃微电极技术,观察一氧化氮(NO)对家兔房室结细胞自发活动的电生理效应及其作用机制。结果显示：(1)NO供体硝普(SNP,1-1000μmol／L)及SIN-1(100,1000μmol／L)剂量依赖性地抑制房室结细胞的动作电位幅值(APA)、零相最大人士升速度(Vmax)、4期自动除极速度(VDD)及自发放电频率(RSF);(2)应用L型钙通道开放剂Bay K8644(0．25μmol／L),可拮抗SNP对房室结细胞的电生理效应;(3)提高灌流液中钙离子浓度(5mmol／L)也可逆转SNP对起搏细胞的抑制效应;(4)用无钙K-H液灌流房室结,可完全阻断SNP对房事结细胞的抑制效应。(5)应用鸟苷酸环化酶阻断剂甲基美蓝(50μmol／L)对SNP的上述电生理效应无影响。以上结果提示,NO可能是通过cGMP非依赖性途径减弱钙离子内流,进而抑制了家兔房室结细胞的自发电活动。     

Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R₁ progeny. SaPIN2a mRNA was detected in both the R₀ and R₁ transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.Abbreviations CaMV cauliflower mosaic virus - cDNA complementary DNA - NOS nopaline synthase - PAGE polyacrylamide gel electrophoresis - PI proteinase inhibitor - SaPIN2a Solanum americanum proteinase inhibitor IIa - SDS sodium dodecyl sulphate - T-DNA transferred DNA     

Human mesenchymal stem cells tissue development in 3D PET matrices

Human mesenchymal stem cells (hMSCs) are attractive cell sources for engineered tissue constructs with broad therapeutic potential. Three-dimensional (3D) hMSC tissue development in nonwoven poly(ethylene terephthalate) (PET) fibrous matrices was investigated. HMSCs were seeded onto 3D PET scaffolds and were cultured for over 1 month. Their proliferation rates were affected by seeding density but remained much lower than those of 2D controls. Compared to 2D surfaces, hMSCs grown in 3D scaffolds secreted and embedded themselves in an extensive ECM network composed of collagen I, collagen IV, fibronectin, and laminin. HMSCs were influenced by the orientation of adjacent PET fibers to organize the ECM proteins into highly aligned fibrils. We observed the increased expressions of alpha(2)beta(1) integrin but a slight decrease in the expression of alpha(5)beta(1) integrin in 3D compared to 2D culture and found that alpha(V)beta(3) was expressed only in 2D. Paxillin expression was down-regulated in 3D culture with a concomitant change in its localization patterns. We demonstrated the multi-lineage potentials of the 3D tissue constructs by differentiating the cells grown in the scaffolds into osteoblasts and adipocytes. Taken together, these results showed that hMSCs grown in 3D scaffolds display tissue development patterns distinct from their 2D counterparts and provide important clues for designing 3D scaffolds for developing tissue engineered constructs.