Continuous cultivation of the diatom Nitzschia laevis for eicosapentaenoic acid production: physiological study and process optimization

The continuous cultures of the diatom Nitzschia laevis were performed at different dilution rates (D) and feed glucose concentrations (S(0)) to investigate cellular physiological responses and its production potential of eicosapentaenoic acid (EPA). Steady-state cell dry weight, residual glucose concentration, cell growth yield, specific glucose consumption rate, and fatty acid profiles were investigated within the range of D from 0.1 to 1.0 day(-1) (S(0) fixed at 20 g/L) and the range of S(0) from 5 to 35 g/L (D fixed at 0.3 day(-1)), respectively. The highest EPA productivity of 73 mg L(-1) day(-1) was obtained at D = 0.5 day(-1) and S(0) = 20 g/L. However, when the continuous culture achieved high productivities of EPA at certain dilution rates and feed glucose concentrations, glucose in the feed could not be consumed completely. Accordingly, the continuous culture was evaluated in terms of both EPA productivity (P) and glucose assimilation efficiency (E). The parameter eta, defined as the product of P and E, was used as an overall performance index. Since eta is a function of the two independent variables D and S(0), we employed a central composite design to optimize D and S(0) for the highest eta value. Based on the experimental results of the design, a second-order polynomial equation was established to represent the relationship between eta and D and S(0). The optimal values of D and S(0) were subsequently determined as 0.481 day(-1) and 15.56 g/L, respectively by the empirical model. The verification experiment confirmed the validity of the model. Under the optimal conditions, eta value reached 46.5 mg L(-1) day(-1), suggesting a considerably high efficiency of the continuous culture of N. laevis in terms of EPA production and glucose utilization.     

Biochemistry and genetics of interorganelle aminoglycerophospholipid transport

The organelle specific reactions that constitute the biosynthetic pathway for aminoglycerophospholipid synthesis provide an important means for examining the biochemistry and genetics of intracellular lipid transport. Biochemical studies with intact and permeabilized cells, and isolated organelles have defined some of the essential features of lipid transport between the endoplasmic reticulum and mitochondria and Golgi/vacuole. Genetic screens have now also identified mutations and genes that are involved in aminoglycerophospholipid traffic between different membranes in mammalian cells, yeast and bacteria. Increasingly, studies focused upon intermembrane lipid movement are revealing important new information about this essential aspect of membrane biogenesis.     

鲫耳石重量与年龄的关系及其在年龄鉴定中的作用

耳石重量在年龄组间重叠较少,大小相近的个体,年龄大的,即生长慢的耳石重量比年龄小的,即生长快的大,不同龄组之间耳石重量有显著差异（P＜0．05）,按年龄组在耳石重量与相应的体长作图,可初步判断观测年龄的可靠性,分析耳石重量频率分布能分离出体长相近,年龄不同的个体,其结构与耳石年轮观测的基本一致,耳石重量与年龄呈显著线性正相关（P＜0．05）,用耳石重量与年龄关系估算的年龄从耳石上直接读取的年龄无显著差异（P＞0．05）,文中对耳石重量直接用于确定鱼类年龄的可能性作了分析和探讨。     

Three-dimensional structure of manganese superoxide dismutase from Bacillus halodenitrificans,a component of the so-called "green protein"

A so-called "green protein" has been purified from a moderate halophilic eubacterium, Bacillus halodenitrificans (ATCC 49067), under anaerobic conditions. The protein, which might play an important role in denitrification, dissociates mainly into two components after exposure to air: a manganese superoxide dismutase (GP-MnSOD) and a nucleoside diphosphate kinase. As a first step in elucidating the overall structure of the green protein and the role of each component, the 2.8-A resolution crystal structure of GP-MnSOD was determined. Compared with other manganese dismutases, GP-MnSOD shows two significant characteristics. The first is that the entrance to its substrate channel has an additional basic residue-Lys38. The second is that its surface is decorated with an excess of acidic over basic residues. All these structural features may be related to GP-MnSOD's high catalytic activity and its endurance against the special cytoplasm of B. halodenitrificans. The structure of GP-MnSOD provides the basis for recognizing its possible role and assembly state in the green protein.     

Construction of alpha-alpha domains structure in recombinant monkey metallothionein-1

The differences in metal-thiolate coordination and reactivity of mammalian metallothionein (MT) domains are closely related to their distinct, highly conservative cysteine number and position. Monkey metallothionein-1, containing a beta-domain with Cd(3)S(9) cluster and an alpha-domain with Cd(4)S(11) cluster, was used to evaluate the role of cysteine residues in the formation of MT's metal-thiolate clusters. The possible influence of cysteine residues on the binding and stability of MT domains has been examined with the metallothionein mutants: N4C, T27C and N4C/T27C, which possess ten or eleven cysteine residues in the re-constructed beta-domain, respectively. Assisted by study of UV, CD and electrospray ionization mass spectroscopy (ESI-MS) and their reactivity with DTNB (5,5'-dithiobis (2-nitrobenzoic acid)), we found that besides the original alpha-domain, some kinds of new domain containing 4-cadmium-thiolate clusters were formed in the N4C and N4C/T27C mutants of mkMT1. These new domains displayed metal binding and kinetic reactivity with DTNB similar to the alpha-domain. However, the thermal stability of the mutants was less stable than that of WT mkMT1. This might result from the disturbance of the inter-domains hydrogen bonds and of the non-cysteine amino acid residue arrangement.     

Site-specific lipophilic modification of interferon-alpha

Interferon- (IFN),⁴ a cytokine with modulatory activities on many cell types, is useful for treating many types of cancer and infectious diseases. This study investigates whether modification of a protein, using IFN as an example, with a lipophilic group can alter its distribution and kinetic properties in the body. Ser163 of IFN2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFN2a(S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFN2a was purified by gel filtration chromatography. The biological activities of IFN2a(S163C) and lipophilized IFN2a(S163C) were similar to that of IFN2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in Madin-Darby bovine kidney cells. Lipophilized IFN2a(S163C) bound to human serum albumin and cell membranes more readily than did IFN2a. Future experiments will investigate whether lipophilized IFN2a(S163C) has improved pharmacokinetic properties.     

Transforming growth factor-beta increases Escherichia coli K1 adherence,invasion, and transcytosis in human brain microvascular endothelial cells

Escherichia coli K1 traversal of the human brain microvascular endothelial cells (HBMEC) that constitute the blood-brain barrier (BBB) is a complex process involving E. coli adherence to and invasion of HBMEC. In this study, we demonstrated that human transforming growth factor-beta-1 (TGF-beta1) increases E. coli K1 adherence, invasion, and transcytosis in HBMEC. In addition, TGF-beta1 increases RhoA activation and enhances actin condensation in HBMEC. We have previously shown that E. coli K1 invasion of HBMEC requires phosphatidylinositol-3 kinase (PI3K) and RhoA activation. TGF-beta1 increases E. coli K1 invasion in PI3K dominant-negative HBMEC, but not in RhoA dominant-negative HBMEC, indicating that TGF-beta1-mediated increase in E. coli K1 invasion is RhoA-dependent, but not PI3K-dependent. Our findings suggest that TGF-beta1 treatment of HBMEC increases E. coli K1 adherence, invasion, and transcytosis, which are probably dependent on RhoA.     

Route of administration of chimeric BPV1 VLP determines the character of the induced immune responses

To examine the mucosal immune response to papillomavirus virus-like particles (PV-VLP), mice were immunized with VLP intrarectally (i.r.), intravaginally (i.va.) or intramuscularly (i.m.) without adjuvant. PV-VLP were assembled with chimeric BPV-1 L1 proteins incorporating sequence from HIV-1 gp120, either the V3 loop or a shorter peptide incorporating a known CTL epitope (HIVP18I10). Antibody specific for BPV-1 VLP and P18 peptide was detected in serum following i.m., but not i.r. or i.va. immunization. Denatured VLP induced a much reduced immune response when compared with native VLP. Immune responses following mucosal administration of VLP were generally weaker than following systemic administration. VLP specific IgA was higher in intestine washes following i.r. than i.va. immunization, and higher in vaginal washes following i.m. than i.r. or i.va. immunization. No differences in specific antibody responses were seen between animals immunized with BPV-1 P18 VLP or with BPV-1 V3 VLP. Cytotoxic T lymphocyte precursors specific for the P18 CTL epitope were recovered from the spleen following i.m., i.va. or i.r. immunization with P18 VLP, and were similarly detected in Peyer's patches following i.m. or i.r. immunization. Thus, mucosal or systemic immunization with PV VLP induces mucosal CTL responses and this may be important for vaccines for mucosal infection with human papillomaviruses and for other viruses.