Dopamine Metabolites in Rat Cisternal Cerebrospinal Fluid: Major Contribution from Extrastriatal Dopamine Neurones

The interpretation of central 3,4-dihydroxyphenylethylamine (dopamine, DA) metabolism, as indicated by determinations in rat cisternal CSF, was investigated using intrastriatal injection of the DA neurotoxin 6-hydroxydopamine (6-OHDA) and intraperitoneal injection of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). DA turnover was subsequently determined by measurement of the rate of accumulation of total 3,4-dihydroxyphenylacetic acid and homovanillic acid (DOPAC + HVA) in the CSF after probenecid was given. Two days later the rats were killed, and metabolism of DA and 5-hydroxytryptamine (5-HT) was investigated by determining levels of the amines and their metabolites in brain regions. Although 6-OHDA greatly decreased striatal DA metabolism, this was not paralleled by DA turnover as indicated by CSF, as this fell only moderately and approximately in parallel with results for the brain as a whole. 5-HT metabolism was essentially unaltered. DSP4 considerably depleted noradrenaline and caused smaller decreases of 5-HT metabolism in some regions. However, DA metabolism was not significantly affected, either in brain or CSF, which suggests that noradrenaline neurones make only a small contribution to central DA metabolism. Results as a whole suggest that DOPAC and HVA concentrations in rat cisternal CSF reflect whole brain DA metabolism and derive predominantly from DA neurones in extrastriatal regions of the brain.     

Clarification of animal cell cultures on a large scale by continuous centrifugation

Summary Animal cells from 80-L and 2000-L fed batch fermentations were removed by a prototype disc stack centrifuge in order to achieve a fast and reliable separation of solids from large quantities of cell culture fluids. The clarification capacity was excellent for animal cells but particles remained in the liquid phase and affected further downstream processing of the cell-free harvest fluid. No significant loss of product was observed. A number of parameters were monitored to optimize process conditions for use with animal cells.     

A scale‐down mimic for mapping the process performance of centrifugation,depth and sterile filtration

In the production of biopharmaceuticals disk‐stack centrifugation is widely used as a harvest step for the removal of cells and cellular debris. Depth filters followed by sterile filters are often then employed to remove residual solids remaining in the centrate. Process development of centrifugation is usually conducted at pilot‐scale so as to mimic the commercial scale equipment but this method requires large quantities of cell culture and significant levels of effort for successful characterization. A scale‐down approach based upon the use of a shear device and a bench‐top centrifuge has been extended in this work towards a preparative methodology that successfully predicts the performance of the continuous centrifuge and polishing filters. The use of this methodology allows the effects of cell culture conditions and large‐scale centrifugal process parameters on subsequent filtration performance to be assessed at an early stage of process development where material availability is limited. Biotechnol. Bioeng. 2016;113: 1934–1941. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Reversibility of Cisternal Stack Formation During Hypoxic Hypoxia and Subsequent Reoxygenation in Cerebellar Purkinje Cells

Cisternal stacks are induced during hypoxia, which may be associated with intracellular Ca²⁺ regulation. Although neurons are divided internally in different compartments, little is known about regional differences in cisternal stack formation. We investigated the effects of hypoxic hypoxia and later reoxygenation on cisternal stack formation and other ultrastructual changes in the proximal dendrite, dendritic spine, and cell body of cerebellar Purkinje cells in rats. After brief hypoxic events, cisternal stacks appeared predominantly in the proximal dendrites and after longer hypoxic events in dendritic spines and cell body. Following reoxygenation, cisternal stacks disappeared first in the cell body, followed by the dendritic spines, then the proximal dendrites. These results showed that stack formation occurred at different degrees and time courses among the three regions, and the effect was reversible, which suggests that these compartments are differentially sensitive to hypoxia.     

Catalytic amidolysis of amino acid p-nitroanilides using transition state analogue imprinted artificial enzymes: Cooperative effect of pyridine moiety

Enzyme-like polymer catalysts with the imprints of phosphonate transition state analogue (TSA) lined along with imidazole and pyridine moieties were synthesized using methacryloyl-l-histidine and 4-vinylpyridine as the functional monomers and phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate – the TSA of hydrolytic reaction as the template for the amidolysis of N-benzyloxycarbonyl-l-phenylalanine p-nitroanilide (Z-l-Phe-PNA). Polymers containing different functional groups can act together to provide catalytic activity and selectivity superior to what can be obtained from monofunctional analogues. The higher rate acceleration exhibited by the bifunctional polymer over the monofunctional polymers indicates cooperative catalysis of imidazole and pyridine moieties. The optimum catalytic competence is shown by the bifunctional polymer containing imidazole and pyridine moieties in 2:1 M ratio which may be due to alignment of the functional groups in proper H-bond distance. In addition to the non-covalent interactions like hydrogen bonding or π-stacking interactions between the functional groups of the polymer and the template, 3D-microcavities complementary to the geometry of the template are necessary for effective shape selective binding. Michaelis-Menten kinetics implies that only the catalysts with imidazole moieties act as enzyme-like catalysts and imidazole is the key catalytic function of the enzyme mimics.     

Maximizing power production in a stack of microbial fuel cells using multiunit optimization method

This study demonstrates real‐time maximization of power production in a stack of two continuous flow microbial fuel cells (MFCs). To maximize power output, external resistances of two air–cathode membraneless MFCs were controlled by a multiunit optimization algorithm. Multiunit optimization is a recently proposed method that uses multiple similar units to optimize process performance. The experiment demonstrated fast convergence toward optimal external resistance and algorithm stability during external perturbations (e.g., temperature variations). Rate of the algorithm convergence was much faster than in traditional maximum power point tracking algorithms (MPPT), which are based on temporal perturbations. A power output of 81–84 mW/L_A (A = anode volume) was achieved in each MFC. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Evaluation of a single-use disk stack centrifuge for improved efficiency and sustainability at 1000 L GMP manufacturing scale

Direct depth filtration is an established technology for single-use harvest operation. Advantages of direct depth filtration include familiarity with depth filtration in downstream processes and simplicity of the operation. Drawbacks include low capacity, large footprint, labor-intensive set-up, high water use, and high waste in the form of discarded filters. Single-use centrifugation is emerging as an alternative to depth filtration for the single-use harvest step. Within the single-use centrifugation space, disc stack centrifugation represents the newest entrant. In this study, we evaluated the performance of the GEA kytero single-use disc stack centrifuge to clarify two monoclonal antibody-producing cell culture fluids. The separation performance of the GEA kytero centrifuge varied between the two cell culture fluids, with differences in centrate turbidity and centrate filterability measured. A comparison was then performed to determine resource savings, compared to direct two-stage depth filtration, when using a GEA kytero centrifuge to harvest a 1000 L bioreactor. The analysis concluded that replacement of the first stage of depth filters with a GEA kytero centrifuge has the potential to decrease the required second stage depth filtration area by up to 80%. The decrease in depth filter area resulting from the use of the GEA kytero would result in a decrease in the harvest step footprint, a decrease in buffer volume required to prime and rinse depth filters, and a decrease in the volume of plastic waste. An economic comparison of the GEA kytero single-use centrifuge against a direct depth filtration step found that for a 1000 L harvest step, the GEA kytero centrifuge may reduce costs by up to 20% compared with two-stage direct depth filtration.     

Solution structures of a 30-residue amino-terminal domain of the carp granulin-1 protein and its amino-terminally truncated 3-30 subfragment: implications for the conformational stability of the stack of two beta-hairpins

Carp granulins are members of an emerging class of proteins with a sequence motif encoding a parallel stack of two to four beta-hairpins. The carp granulin-1 protein forms a stack of four beta-hairpins, whereas its amino-terminal fragment appears to adopt a very stable stack of two beta-hairpins in solution. Here we determined a refined three-dimensional structure of this peptide fragment to examine potential conformational changes compared with the full-length protein. The structures were calculated with both a traditional method and a fast semiautomated method using ambiguous NMR distance restraints. The resulting sets of structures are very similar and show that a well-defined stack of two beta-hairpins is retained in the peptide. Conformational rearrangements compensating the loss of the carboxy-terminal subdomain of the native protein are restricted to the carboxy-terminal end of the peptide, the turn connecting the two beta-hairpins, and the Tyr(21) and Tyr(25) aromatic side chains. Further removal of the Val(1) and Ile(2) residues, which are part of the first beta-hairpin and components of two major hydrophobic clusters in the two beta-hairpin structure, results in the loss of the first beta-hairpin. The second beta-hairpin, which is closely associated with the first, retains a similar but somewhat less stable conformation. The invariable presence of the second beta-hairpin and the dependence of its stability on the first beta-hairpin suggest that the stack of two beta-hairpins may be an evolutionary conserved and autonomous folding unit. In addition, the high conformational stability makes the stack of two beta-hairpins an attractive scaffold for the development of peptide-based drug candidates.     

Distribution and morphological changes of the Golgi apparatus during Drosophila spermatogenesis

In spermatogenesis, the Golgi apparatus is important for the formation of the acrosome, which is a sperm‐specific organelle essential for fertilization. Comprehensive examinations of the spatiotemporal distribution and morphological characterizations of the Golgi in various cells during spermatogenesis are necessary for functional analyses and mutant screenings in the model eukaryote Drosophila. Here, we examined the distribution and morphology of the Golgi during Drosophila spermatogenesis with immunofluorescence and electron microscopy. In pre‐meiotic germ cells, the Golgi apparatuses were distributed evenly in the cytoplasm. In contrast, they were located exclusively in two regions near the poles during the meiotic metaphase, where they were segregated prior to the chromosomes. In cells in anaphase to telophase, the Golgi were predominantly left behind in the equatorial region between the separating daughter nuclei. After completion of meiosis, the dispersed Golgi were assembled at the apical side of the spermatid nucleus to form the acrosome. Further investigation of the Golgi distribution in β2‐tubulin mutants showed aberrant and uneven distributions of the Golgi among sister cells in the meiotic spermatocytes and in the post‐meiotic spermatids. At the ultrastructural level, the Golgi apparatus in pre‐meiotic spermatocytes comprised a pair of stacks. The two stacks were situated adjacent to each other, as if they had duplicated before entering into meiotic division. These results highlight the dynamic nature of the Golgi during spermatogenesis and provide a framework for analyzing the correlations between the dynamics of the Golgi and its function in sperm development.     

三叶虫萤幼虫叠背行为

为了探究三叶虫萤幼虫的行为学特征,促进三叶虫萤的资源利用与人工养殖,本文采用控制变量法以三叶虫萤末龄幼虫为研究对象分析了幼虫在不同时间和空间密度的叠背现象,采取LY-WN超清显微系统拍照分析了三叶虫萤幼虫的背腹部结构与足部结构。结果表明,三叶虫萤各龄幼虫均表现出独特的叠背行为,且按照叠背的重合度区分,可分为相对叠背和集群叠背。叠背率随种群密度增加而增加,叠背率与种群密度的相关系数为0.8358。三叶虫萤幼虫的足含有较多的刚毛,且跗节延伸有倒勾;背部较宽,且具有起伏和分节结构;腹部含有大量小而细的刚毛和起伏结构。因此,三叶虫萤幼虫的叠背行为可能与足部结构、背腹部结构、接触时间、种群密度具有较大关系,且幼虫的叠背行为可能对其栖息、防卫、取食、迁移等行为活动具有重大意义。