Continuous ethanol production using induced yeast aggregates

Summary The induction of yeast cell aggregates in a column reactor was initiated by packing yeast cell paste of Saccharomyces uvarum into the column, and then YMP broth was fed into the column from the bottom at a linear flow rate of 2.5 cm/h. Thereafter, yeast cells aggregated in the column within 48 h without a supply of oxygen. When this yeast aggregate column reactor was used for continuous ethanol production, a final ethanol concentration of 10.8% (w/v) was obtained from 23% (w/v) of glucose in a YMP broth with a dilution rate of 0.05 h^-1, and 4.9% (w/v) was obtained from 10% (w/v) of glucose with a dilution rate of 0.6 h^-1. The theoretical yield was above 97% in both cases. The ethanol production rates were 13 g¹ h^-1 l^-1 and 90 g¹ h^-1 l^-1 for producing 10.8% (w/v) and 4.9% (w/v) of ethanol respectively. This column reactor was maintained at a steady state for more than one month.     

Early biochemical events after MHC class II-mediated signaling on human B lymphocytes

These studies examined the role of the MHC class II Ag in signal transduction using human B lymphocytes. Early events in signal transduction were considered including the intracellular calcium [Ca2+)i) flux, the activation of phospholipase C, and induction of protein phosphorylation. The (Ca2+)i was enhanced after incubation of B lymphocytes with several mAb anti-HLA class II and cross-linking with rabbit anti-mouse-F(ab')2. We have also demonstrated an enhancement of the (Ca2+)i in response to a suboptimal concentration of a monoclonal anti-IgM either in the presence of or after preincubation with a mAb anti-HLA class II. The activation of phospholipase C was assessed by measuring the generation of inositol phosphates in permeabilized B lymphocytes. mAb anti-HLA-class II of two different epitopes were used to demonstrate both the (Ca2+)i flux and the generation of inositol phosphates. Two-dimensional gel electrophoresis was used to investigate the phosphorylation pattern of resting B lymphocytes and the changes in the pattern after stimulation with soluble mAb anti-HLA-DR, immobilized mAb anti-HLA-DR, and PMA. In addition to the augmentation of phosphorylation observed with regard to phosphoproteins already present in resting B lymphocytes, new phosphorylations were observed after stimulation by any one of the reagents. Furthermore, stimulation by PMA did not result in an identical pattern to that observed after stimulation by mAb anti-HLA class II. An inhibition of the proliferative response to PMA was demonstrated after prestimulation of cells with immobilized mAb anti-HLA-DR, supporting the notion of a shared pathway of activation. In summary, these data demonstrate signal transduction via MHC class II Ag as assessed by three different measures of early events in human B lymphocyte activation and suggest that a protein kinase C pathway is at least partly involved.     

M protein (M1) of influenza virus: antigenic analysis and intracellular localization with monoclonal antibodies.

A panel of 16 monoclonal antibodies recognizing M protein (M1) of influenza virus was generated. Competition analyses resulted in localization of 14 monoclonal antibodies to three antigenic sites. Three monoclonal antibodies localized to site 1B recognized a peptide synthesized to M1 (residues 220 to 236) with enzyme-linked immunosorbent assay titers equivalent to or greater than that seen with purified M1; therefore, site 1B is located near the C terminus of M1. Sites 2 and 3 localize to the N-terminal half of M1. Antigenic variation of M proteins was seen when the monoclonal antibodies were tested against 14 strains of type A influenza viruses. Several monoclonal antibodies showed specific recognition of A/PR/8/34 and A/USSR/90/77 M proteins and little or no reactivity for all other strains tested. Immunofluorescence analysis with the monoclonal antibodies showed migration of M protein to the nucleus during the replicative cycle and demonstrated association of M protein with actin filaments in the cytoplasm. Use of a vaccinia virus recombinant containing the M-protein gene demonstrated migration of M protein to the nucleus in the absence of synthesis of gene products from other influenza virus RNA segments.     

Effect of Protease Inhibitors on Early Events of Apoptosis

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.     

Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β-precursor protein. In our search for proteases that can generate the N-terminus of amyloid β protein (β-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid β protein, and generates amyloidogenic fragments from full-length recombinant amyloid β-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.     

棕色固氮菌缺失nifZ基因的突变种固氮酶MoFe蛋白的纯化和性质

采用 52℃下加热 6 min,后经 DEAE- 52、Sephacryls S- 2 0 0和 Q- Sepharose等柱层析方法 ,分离纯化了棕色固氮菌 (Azotobacter vinelandii)缺失 nif Z基因突变种固氮酶 Mo Fe(Δnif Z Mo Fe)蛋白 ,其纯度达到电泳纯。Δnif Z Mo Fe蛋白的固氮活性为 2 83nmol C2 H2 还原 / (min·mg蛋白 ) ,远低于野生种 Mo Fe蛋白。Δnif Z Mo Fe蛋白对氧更敏感 ;热稳定性略低于野生种。Δnif Z Mo Fe蛋白的可见光吸收光谱与野生种 Mo Fe蛋白极为相似。其圆二色谱和磁圆二色谱在 450～ 550 nm与野生种 Mo Fe蛋白显著不同 ,表明其 P- cluster及其周围环境与野生种 Mo Fe蛋白有所差异。这亦可能是造成缺失 nif Z突变种 Mo Fe蛋白固氮活性低的原因。     

大豆结瘤突变体成分的初步分析及其对硝酸还原酶活性和结瘤的影响

超结瘤大豆（Ｇｌｙｃｉｎｅ ｍ ａｘ （Ｌ．） Ｍｅｒｒ．） ｎｔｓ ３８２ 和不结瘤大豆Ｎｏｄ ４９ 的叶和根组织水提取物经Ｓｅｐｈａｄｅｘ Ｇ２５ 过滤、洗脱,再根据洗脱物对硝酸还原酶（ＮＲ）活性的影响可划分为４ 个组分（ｆｒａｃｔｉｏｎ）样品,即ｎｔｓ ３８２（Ｎｏｄ ４９） Ｆ１、ｎｔｓ ３８２（Ｎｏｄ ４９） Ｆ２、ｎｔｓ ３８２（Ｎｏｄ ４９） Ｆ３ 和ｎｔｓ ３８２（Ｎｏｄ ４９） Ｆ４。其中, ｎｔｓ３８２ Ｆ２ 和Ｆ４ 抑制ＮＲ 活性作用在接种ＵＳＤＡ１１０ 后明显下降, 但接种的ｎｔｓ ３８２ Ｆ２ 却能提高大豆Ｂｒａｇｇ 的结瘤数达一倍, 而接种的ｎｔｓ ３８２ Ｆ３ 和Ｆ４ 的作用不明显。ＮＲ 活性抑制因子不是刺激结瘤的因子, 刺激结瘤的因子主要分布在接种的ｎｔｓ３８２ Ｆ２ 部分中。与这一现象相反, Ｎｏｄ ４９ Ｆ２ 和Ｆ４ 抑制ＮＲ活性的作用在接种后更强, 且也抑制大豆ｎｔｓ ３８２ 的结瘤, 其中Ｎｏｄ ４９ Ｆ４ 抑制结瘤的作用基本不能逆转。抑制结瘤因子主要分布在接过种的Ｎｏｄ ４９ Ｆ４ 部分中     

Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.     

细胞寒害的热力学熵理论(Ⅱ)──超熵产生作为寒害稳定性判据的理论研究

本文从物质和能量交换的角度,运用非平衡态热力学超熵产生理论,分析了寒害定态的稳定性,并建立了超熵产生判据．理论分析所得的结论与实验结果基本相符．     

向日葵幼苗环旋运动的三维轨迹

采用光学显微镜标尺和垂线原理制作的三维空间点测定仪,对向日葵（Ｈｅｌｉａｎｔｈｕｓ ａｎｎｕｕｓＬ．）幼苗的环旋运动进行了连续测量。结果表明：向日葵环旋运动的轨迹有椭圆型、摆动型和不规则型;同一植株在不同生长阶段所表现的环旋运动轨迹不一定相同,同一株龄的不同个体也不一定具有相同的运动轨迹;运动的方向有左旋和右旋,圆周运动光源可以显著地改变运动方向;从三维角度看,在整个下胚轴生长阶段,环旋运动的振幅存在一个由小变大再由大变小的变化规律