Flux balance analysis in the production of clavulanic acid by Streptomyces clavuligerus

In this work, in silico flux balance analysis is used for predicting the metabolic behavior of Streptomyces clavuligerus during clavulanic acid production. To choose the best objective function for use in the analysis, three different optimization problems are evaluated inside the flux balance analysis formulation: (i) maximization of the specific growth rate, (ii) maximization of the ATP yield, and (iii) maximization of clavulanic acid production. Maximization of ATP yield showed the best predictions for the cellular behavior. Therefore, flux balance analysis using ATP as objective function was used for analyzing different scenarios of nutrient limitations toward establishing the effect of limiting the carbon, nitrogen, phosphorous, and oxygen sources on the growth and clavulanic acid production rates. Obtained results showed that ammonia and phosphate limitations are the ones most strongly affecting clavulanic acid biosynthesis. Furthermore, it was possible to identify the ornithine flux from the urea cycle and the α‐ketoglutarate flux from the TCA cycle as the most determinant internal fluxes for promoting clavulanic acid production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1226–1236, 2015     

Caspase-3：治疗神经退行性疾病的新靶点

Caspase-3是caspases家族（一类天冬氨酸特异性酶切半胱氨酸蛋白酶）中的成员,是哺乳动物细胞凋亡的关键蛋白酶.随着研究的深入,发现caspase-3在神经退行性疾病的病理过程中起着很重要的角色.Caspase-3在这些疾病的病理过程中,不仅仅是起着凋亡的效应器作用,还能直接与老年性痴呆症、帕金森氏症、亨廷顿舞蹈病、脊椎小脑失调等疾病的致病蛋白质分子相互作用,参与致病机制.因此,caspase-3是治疗神经退行性疾病的新靶点,寻找caspase-3高效高选择性的抑制剂将为治疗神经退行性疾病提供新的途径.     

Clinanthus microstephium,an Amaryllidaceae Species with Cholinesterase Inhibitor Alkaloids: Structure−Activity Analysis of Haemanthamine Skeleton Derivatives

Plants of the Amaryllidaceae family are well‐known (not only) for their ornamental value but also for the alkaloids that they produce. In this report, the first phytochemical study of Clinanthus genus was carried out. The chemical composition of alkaloid fractions from Clinanthus microstephium was analyzed by GC/MS and NMR. Seven known compounds belonging to three structural types of Amaryllidaceae alkaloids were identified. An epimeric mixture of a haemanthamine‐type compound (6‐hydroxymaritidine) was tested as an inhibitor against acetyl‐ and butyrylcholinesterase enzymes (AChE and BChE, respectively), two enzymes relevant in the treatment of Alzheimer's disease, with good results. Structure–activity relationships through molecular docking studies with this alkaloid and other structurally related compounds were discussed.     

耐镉马克思克鲁维酵母重金属镉吸附特性的研究

菌株YS-K1是从镉污染土壤中筛选获得的一株耐镉马克思克鲁维酵母Kluyveromyces marxianus YS-K1,对其镉吸附特性研究结果表明,该菌对镉具有良好的耐受性和吸附性,即使在镉浓度高达140mg/L的培养基中也能生长,当镉浓度在30mg/L以下时,24h后能吸附溶液中90%以上的镉,且最佳吸附条件为pH6.0,温度30℃。该菌所吸附的镉大部分集中在细胞壁上,占总吸附镉的76%,细胞膜上的占9%,细胞质中的占15%;能量抑制剂2,4-二硝基苯酚(DNP)和二环己基碳二亚胺(DCC),分别能降低菌株15%和22%的镉吸附量。在Kluyveromyces marxianus YS-K1细胞中,锌离子与镉离子有共用的结合位点,锌离子不仅会与镉离子竞争细胞表面的结合位点,减少其对镉的吸附量,而且也会与镉离子竞争进入细胞的通道位点,减少其对镉的积累量,同时能部分恢复镉离子对菌株生长的抑制作用。     

Heterologous expression, purification, and properties of a potato protein inhibitor of serine proteinases

The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.     

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.     

Structure and physiological importance of angiotensin converting enzyme domains

Somatic angiotensin converting enzyme (ACE) consists of two homologous catalytic domains (N- and C-domain), exhibiting different biochemical properties. The catalytically active ACE isoforms consisted of just one domain have been also detected in mammals. Substantial progress in ACE domain research was achieved during the last years, when their crystal structures were determined. The crystal structures of domains in complex with diverse potent ACE inhibitors provided new insights into structure-based differences of the domain active sites. Physiological functions of ACE are not limited by regulation of the cardiovascular system. Recent evidence suggests that the ACE domains may be also involved into control of different physiological functions. The C-terminal catalytic domain plays an important role in the regulation of blood pressure: it catalyzes angiotensin I cleavage in vivo. The N-domain contributes to the processing of other bioactive peptides for which it exhibits high affinity. The role of the N-domain is not ultimately associated with functioning of the rennin-angiotensin system and it contributes processing of other bioactive peptides for which it exhibits high affinity (goralatide, luliberin, enkephalin heptapeptide, beta-amyloid peptide). Domain-selective inhibitors selectively blocking either the N- or C-domain of ACE have been developed.     

Polyamine metabolism during seedling development in rice

The main free amines identified during growth and development of rice seedlings were agmatine, putrescine, spermidine, diaminopropane and tyramine. Amine composition differed according to tissue and stages of development. Conjugated amines were only found in roots. We present evidence that arginine decarboxylase (ADC) regulates putrescine during the development of rice seedlings. When ADC action was blocked by DFMA (-DL-difluoromethylarginine, a specific irreversible inhibitor of ADC), polyamine titers and seedling development were diminished; when agmatine or putrescine was added, normal polyamine titers and growth were restored. The effects of DFMA were concentration dependent. DFMO (-DL-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase or ODC) promoted growth and development at concentrations below 2 mM. This effect was probably related to its unexplained, but consistently observed slight enhancement of rice ADC. When the increase in the concentration of spermidine was prevented by CHA (cyclohexylammonium sulfate), the number of roots increased and the increase in length of leaves and roots was strongly inhibited. The addition of exogenous spermidine at the time of treatment with CHA reversed the inhibition by CHA.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - CHA cyclohexylammonium sulfate     

Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCI (polymerase MI) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg²⁺ in preference to Mn²⁺ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn²⁺ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata β-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCI DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCI, used both Mg²⁺ and Mn²⁺ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is ˜ 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase MI shows similarities to the Crithidia fasciculata β-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.