REGENERATION AND SEXUAL DIFFERENTIATION OF GRIFFITHSIA JAPONICA (CERAMIACEAE,RHODOPHYTA) THROUGH SOMATIC CELL FUSION1

Hybrid cells were obtained from somatic cell fusion among male, female, and tetrasporangial plants in Griffithsia japonica Okamura by a wound-healing process. Isolated fusion cells regenerated new mature plants with mixed reproductive structures. The plants regenerated from hybrid cells between male and female plants developed into 1) spermatangiate, 2) carpogonial, 3) bisexual with spermatangia and carpogonial branches, 4) mixed-phase with spermatangia and tetrasporangia, or 5) bisexual/mixed-phase plants with spermatangia, carpogonial branches, and tetrasporangia. About 70% of the plants regenerated from hybrid cells between male and female plants produced tetrasporangia that were always formed with spermatangia on a single cell. Some of those tetrasporangia released tetraspores, six of which gave rise to mature plants. The plants regenerated from hybrid cells between male and tetrasporangial plants developed into spermatangiate, tetrasporangiate, or mixed-phase plants with spermatangia and tetrasporangia. The plants regenerated from hybrid cells between female and tetrasporangial plants developed into carpogonial, tetrasporangiate, or mixed-phase plants with carpogonial branches and tetrasporangia. All types of reproductive structures we re functional.     

Effect of nitrogen application on growth and photosynthetic nitrogen use efficiency in two ecotypes of wild strawberry, Fragaria chiloensis

The relationships between increasing nitrogen fertilization and growth, maximum CO₂ assimilation and the initial slope of the CO₂ response curve were studied in 2 ecotypes of wild strawberry, Fragaria chiloensis (L.) Duchn. Nitrogen accumulation of CA11, an ecotype from a low-nutrient dune site, was greater at all nitrogen concentrations than that of RCP37, an ecotype from a higher-nutrient strand site. Maximum CO₂ assimilation, total Rubisco activity, dry weight, and initiation of leaves and crowns were higher in CAI1 than RCP37 as nitrogen treatment was increased from 0 to 200 mg ^l-1, whereas these parameters were lower in CAl1 when fertilized at 300 mg T¹, but not in RCP37. The mean leaf area of CA11 was greater than RCP37 when grown with no supplemental nitrogen, but mean leaf area of the 2 lines was similar under nitrogen fertilization. Maximum CO₂ assimilation and carboxylation efficiency increased with increasing leaf nitrogen in both clones. At equivalent concentrations of leaf nitrogen, RCP37 had higher CO₂ assimilation and carboxylation efficiency than CA11 and the difference between the 2 clones increased as ieaf nitrogen increased. Thus, RCP37 had a higher photosynthetic nitrogen use efficiency than CA11. However, at a given applied nitrogen level, CA11 allocated more nitrogen to a unit of leaf area so that photosynthetic rates were higher than RCP37, except at the highest application of 300 mg ^l-1. The high nitrogen accumulation capacity and resource allocation to fruiting structures (crowns) in CA11 leads us to suggest that this clone may possess genes that could increase fruit yield in cultivated strawberry.     

Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium

Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log-phase and stationary-phase systems. The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and σ^s-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli . The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.     

Enhancement of CO2 tolerance of Chlorella vulgaris by gradual increase of CO2 concentration

Summary Chlorella vulgaris UTEX259 was cultivated using two different methods of gas supply. In one method the CO₂ concentration in bubbled gas was held constant and in the other method it was increased gradually. Algal growth was almost linear after a short period of lag phase in both methods. With the constant CO₂ concentration, the CO₂ fixation rate in the linear growth phase decreased over 10%(v/v) CO₂, while the rate increased up to 6% CO₂. However, the rate was enhanced by using the latter incremental increase method, especially under a higher concentration of CO₂. The maximum rate of CO₂ fixation was 52 mg CO₂/l·h at 20% CO₂ during the gradual increase of CO₂ concentration.     

瑞氏七鳃鳗乳酸脱氢酶（LDH）同工酶凝胶电泳分析

本文用聚丙烯酰胺凝胶电泳方法,对瑞氏七鳃鳗五种不同组织（骨骼肌、心、肾、肠、鳃）中ＬＤＨ同工酶进行了分析研究,结果表明,ＬＤＨ同工酶具有组织特异性,其中骨骼肌中含有五种ＬＤＨ同。酶,即ＬＤＨ１、ＬＤＨ２、ＬＤＨ３、ＬＤＨ４、ＬＤＨ５,鳃含有ＬＤＨ１和ＬＤＨ４而肾和心只含有ＬＤＨ１,肠只含有ＬＤＨ４。     

Use of moving aeration membrane bioreactor for the efficient production of tissue type plasminogen activator in serum free medium

A moving aeration-membrane (MAM) bioreactor was employed for the production of 2 μg/mL of tissue type Plasminogen Activator (tPA) in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating conditions, shear stress was as low as 0.65 dynes/cm² at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2 μg tPA/mL while maintaining a high cell density of 1.0×10⁷ viable cells/mL.     

Establishment of the Dorso-ventral Axis in Xenopus Embryos Is Presaged by Early Asymmetries in β-Catenin That Are Modulated by the Wnt Signaling Pathway

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on β-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that β-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with β-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous β-catenin. Steadystate levels and nuclear accumulation of β-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased β-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of β-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in β-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in β-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.     

HhaI and HpaII DNA methyltransferases bind DNA mismatches, methylate uracil and block DNA repair.

The hydrolytic deamination of 5-methylcytosine (5-mC) to thymine (T) is believed to be responsible for the high mutability of the CpG dinucleotide in DNA. We have shown a possible alternate mechanism for mutagenesis at CpG in which HpaII DNA-(cytosine-5) methyltransferase (M.HpaII) can enzymatically deaminate cytosine (C) to uracil (U) in DNA [Shen, J.-C., Rideout, W.M., III and Jones, P.A., Cell, 71, 1073-1080, (1992)]. Both the hydrolytic deamination of 5-mC and enzymatic deamination of C create premutagenic DNA mismatches (G:U and G:T) with the guanine (G) originally paired to the normal C. Surprisingly, we found that DNA-(cytosine-5) methyltransferases have higher affinities for these DNA mismatches than for their normal G:C targets and are capable of transferring a methyl group to the 5-position of U, creating T at low efficiencies. This binding by methyltransferase to mismatches at the recognition site prevented repair of G:U mismatches by uracil DNA glycosylase in vitro.     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.