Epitope Mapping of Human Alpha-Fetoprotein

The epitope structure of human alpha-fetoprotein (AFP) was studied using more than 50 monoclonal antibodies (MAB) to human AFP. These MAB obtained from various world laboratories of the TD-2 AFP Workshops of the International Society for Oncodevelopmental Biology and Medicine (ISOBM-1996-1998-2000) were analyzed by competitive immunoaffinity electrochromatography (IAE) on nitrocellulose membranes (NCM). Five types of interaction of the AFP–MAB complex with the MAB fixed on NCM were found: 1) complete neutralization; 2) partial neutralization; 3) unidirectional neutralization; 4) enhanced binding; 5) lack of interaction. By IAE, 51 MAB were found to recognize 23 different epitopes in the AFP molecule. Based on these findings, an epitope map of AFP was designed which consists of eight epitope clusters and eight individual epitopes. The epitope location is considered with respect to the conformational state of the AFP molecule. Possible causes of the five types of interaction found on neutralization are discussed.     

Significance of rooting depth in mire plants: Evidence from natural <Superscript>15</Superscript>N abundance

Variation in stable nitrogen isotope ratios (¹⁵N) was assessed for plants comprising two wetland communities, a bog-fen system and a flood plain, in central Japan. ¹⁵N of 12 species from the bog-fen system and six species from the flood plain were remarkably variable, ranging from –5.9 to +1.1 and from +3.1 to +8.7, respectively. Phragmites australis exhibited the highest ¹⁵N value at both sites. Rooting depth also differed greatly with plant species, ranging from 5cm to over 200cm in the bog-fen system. There was a tendency for plants having deeper root systems to exhibit higher ¹⁵N values; plant ¹⁵N was positively associated with rooting depth. Moreover, an increasing gradient of peat ¹⁵N was found along with depth. This evidence, together with the fact that inorganic nitrogen was depleted under a deep-rooted Phragmites australis stand, strongly suggests that deep-rooted plants actually absorb nitrogen from the deep peat layer. Thus, we successfully demonstrated the diverse traits of nitrogen nutrition among mire plants using stable isotope analysis. The ecological significance of deep rooting in mire plants is that it enables those plants to monopolize nutrients in deep substratum layers. This advantage should compensate for any consequential structural and/or physiological costs. Good evidence of the benefits of deep rooting is provided by the fact that Phragmites australis dominates as a tall mire grass.     

Xenoestrogenic short ethoxy chain nonylphenol is oxidized by a flavoprotein alcohol dehydrogenase from <Emphasis Type="Italic">Ensifer</Emphasis> sp. strain AS08

The ethoxy chains of short ethoxy chain nonylphenol (NPEO_av2.0, containing average 2.0 ethoxy units) were dehydrogenated by cell-free extracts from Ensifer sp. strain AS08 grown on a basal medium supplemented with NPEO_av2.0. The reaction was coupled with the reduction in 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and phenazine methosulfate. The enzyme (NPEO_av2.0 dehydrogenase; NPEO-DH) was purified to homogeneity with a yield of 20% and a 56-fold increase in specific activity. The molecular mass of the native enzyme was 120 kDa, consisting of two identical monomer units (60 kDa). The gene encoding NPEO-DH was cloned, which consisted of 1,659 bp, corresponding to a protein of 553 amino acid residues. The deduced amino acid sequence agreed with the N-terminal amino acid sequence of the purified NPEO-DH. The presence of a flavin adenine dinucleotide (FAD)-binding motif and glucose–methanol–choline (GMC) oxidoreductase signature motifs strongly suggested that the enzyme belongs to the GMC oxidoreductase family. The protein exhibited homology (40–45% identity) with several polyethylene glycol dehydrogenases (PEG-DHs) of this family, but the identity was lower than those (approximately 58%) among known PEG-DHs. The substrate-binding domain was more hydrophobic compared with those of glucose oxidase and PEG-DHs. The recombinant protein had the same molecular mass as the purified NPEO-DH and dehydrogenated PEG400-2000, NPEO_av2.0 and its components, and NPEOav10, but only slight or no activity was found using diethylene glycol, triethylene glycol, and PEG200. English edition: The paper was edited by a native speaker through American Journal Experts ().     

Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.     

hunchback and Ikaros-like zinc finger genes control reproductive system development in Caenorhabditis elegans

Here we provide evidence for a C2H2 zinc finger gene family with similarity to Ikaros and hunchback. The founding member of this family is Caenorhabditis elegans ehn-3, which has important and poorly understood functions in somatic gonad development. We examined the expression and function of four additional hunchback/Ikaros-like (HIL) genes in C. elegans reproductive system development. Two genes, ehn-3 and R08E3.4, are expressed in somatic gonadal precursors (SGPs) and have overlapping functions in their development. In ehn-3; R08E3.4 double mutants, we find defects in the generation of distal tip cells, anchor cells, and spermatheca; three of the five tissues derived from the SGPs. We provide in vivo evidence that C. elegans HIL proteins have functionally distinct zinc finger domains, with specificity residing in the N-terminal set of four zinc fingers and a likely protein-protein interaction domain provided by the C-terminal pair of zinc fingers. In addition, we find that a chimeric human Ikaros protein containing the N-terminal zinc fingers of EHN-3 functions in C. elegans. Together, these results lend support to the idea that the C. elegans HIL genes and Ikaros have similar functional domains. We propose that hunchback, Ikaros, and the HIL genes arose from a common ancestor that was present prior to the divergence of protostomes and deuterostomes.     

PGR-1〔3-(2-吡啶基)芮醇〕对槟榔植株酯酶同工酶的影响

本试验采用聚丙烯酰胺凝胶电泳法,测定与分析槟榔不同器官、不同品种酯酶同工酶酶谱、酶量、酶带活性和分布状况,结果表明:经PGR-1处理后.槟榔酯酶同工酶带数目、着色程度、酶带活性都比对照的显著增多、加深、提高。 通过检测.清楚地看到槟榔酶带总共有17条,分为三大酶区,Ⅰ区带有酶谱6条,Ⅱ区带有酶谱7条.Ⅲ区带有酶谱4条。各酶区出现新酶带比对照的增多11条,带幅从狭变宽,带色由浅变深,带位迁移从慢变快,酶带活性由弱变强,这是与用药浓度有相关联的。     

Studies of Antarctic yeast for β-glucosidase production

Moss and lichen samples from the region of the Bulgarian base on Livingston Island, Antarctica were examined for the presence of yeasts. Six pure cultures were obtained. They were screened for -glucosidase production and two of them were selected. These were identified as Cryptococcus albidus AL₂ and C. albidus AL₃, according to their morphology, reproductive behaviour, and growth at different temperatures, salt concentrations, nutritional characteristics and various biochemical tests. These strains were examined for biosynthesis of -glucosidase on different carbon sources under aerobic conditions. High exocellular and endocellular activities were obtained when they were grown on cellobiose, methyl--D-glucopyranoside and salicin. The time course of growth and -glucosidase production of the yeast was examined by cultivation in a medium with cellobiose under aerobic conditions at temperatures 18 and 24 °C for 96 h. Cryptococcus albidus AL₂ and C. albidus AL₃ synthesized exocellular enzyme, respectively 58.33 and 55.83 U/ml and endocellular enzyme 137.75 and 205.34 U/ml at 24 °C for 72 h of the cultivation.     

Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects

The expression of the Acidothermus cellulolyticus endoglucanase E1 gene in transgenic tobacco (Nicotiana tabacum) was examined in this study, where E1 coding sequence was transcribed under the control of a leaf specific Rubisco small subunit promoter (tomato RbcS-3C). Targeting the E1 protein to the chloroplast was established using a chloroplast transit peptide of Rubisco small subunit protein (tomato RbcS-2A) and confirmed by immunocytochemistry. The E1 produced in transgenic tobacco plants was found to be biologically active, and to accumulate in leaves at levels of up to 1.35% of total soluble protein. Optimum temperature and pH for E1 enzyme activity in leaf extracts were 81°C and 5.25, respectively. E1 activity remained constant on a gram fresh leaf weight basis, but dramatically increased on a total leaf soluble protein basis as leaves aged, or when leaf discs were dehydrated. E1 protein in old leaves, or after 5h dehydration, was partially degraded although E1 activity remained constant. Transgenic plants exhibited normal growth and developmental characteristics with photosynthetic rates similar to those of untransformed SR1 tobacco plants. Results from these biochemical and physiological analyses suggest that the chloroplast is a suitable cellular compartment for accumulation of the hydrolytic E1 enzyme.     

The testis‐specific gene 1700102P08Rik is essential for male fertility

Male infertility is a rising problem around the world. Often the cause of male infertility is unclear, and this hampers diagnosis and treatment. Spermatogenesis is a complex process under sophisticated regulation by many testis‐specific genes. Here, we report the testis‐specific gene 1700102P08Rik is conserved in both the human and mouse and highly expressed in spermatocytes. To investigate the role of 1700102P08Rik in male fertility, knockout mice were generated by CRISPR‐Cas9. 1700102P08Rik knockout male mice were infertile with smaller testis and epididymis, but female knockout mice retained normal fertility. Spermatogenesis in the 1700102P08Rik knockout male mouse was arrested at the spermatocyte stage, and no sperm were found in the epididymis. The deletion of 1700102P08Rik causes apoptosis in the testis but did not affect the serum concentration of testosterone, luteinizing hormone, and follicle‐stimulating hormone or the synapsis and recombination of homologous chromosomes. We also found that 1700102P08Rik is downregulated in spermatocyte arrest in men. Together, these results indicate that the 1700102P08Rik gene is essential for spermatogenesis and its dysfunction leads to male infertility.     

Aerobic mineralization of 4,4′-dichlorobiphenyl and 4-chlorobenzoic acid by a novel natural bacterial strain that grows poorly on benzoate and biphenyl

Achromobacter xylosoxidans strain IR08 was isolated from soil contaminated with electrical transformer fluid by enrichment culture containing Aroclor 1221 as the sole carbon source. This strain was found to grow on all monochlorobiphenyls, 4,4′-dichlorobiphenyl (4,4′-diCB) and a wide range of other xenobiotic compounds. During growth on 4,4′-diCB, a near-stoichiometric amount of chloride was excreted into the culture fluid in less than 5 days and growth yield was more than twice that achieved on biphenyl. The production of 4-CBA or chlorocatechol as a metabolite was not observed. Quite unusually, coincubation of strain IR08 with 4,4′-diCB and biphenyl at relatively equal concentrations showed preferential utilization of the chlorobiphenyl: 4,4′-diCB was mineralized in less than 5 days concomitant with stoichiometric release of chloride, while biphenyl was poorly degraded. Growth on 2.5 mM CBA also resulted in complete disappearance of the substrate, however, inorganic chloride recovered from the culture broth was less than 65%. The isolation of a dichlorobiphenyl-mineralizing rather than transformation strain such as IR08 is an important advance in an effort to develop effective bioremediation strategy for polychlorinated biphenyl-contaminated soil.