EFFECTS OF UV RADIATION ON GROWTH AND PHLOROTANNINS IN FUCUS GARDNERI (PHAEOPHYCEAE) JUVENILES AND EMBRYOS1

Diminishing levels of atmospheric ozone are increasing UV stress on intertidal algae. Early developmental stages tend to be more susceptible to environmental stresses; however, little research has examined how these stages are protected from UV radiation (UVR). Many brown algae contain high levels of phlorotannins, which are thought to function in screening UVR. In this study, we tested the effects of ambient levels of UV‐B and UV‐A on growth and phlorotannin production in 1‐ to 2‐cm juvenile and microscopic postsettlement embryos of the intertidal alga Fucus gardneri Silva. Algae were grown in four light treatments: 1) ambient light; 2) under cellulose acetate, which lowered light quantity but did not affect light quality; 3) under Mylar^TM, which filtered UV‐B; and 4) under Plexiglas^TM, which blocked UV‐A and UV‐B. Over a 3‐week period, UV‐B inhibited and UV‐A enhanced the growth of F. gardneri embryos, whereas the growth of juveniles was not affected. Phlorotannin concentrations of both embryos and juveniles did not differ in any of the light treatments. Our results suggest that embryos of F. gardneri are susceptible to UV light but develop a tolerance to it as they mature. This tolerance may result from increases in phlorotannin concentrations that occur during maturation; however, phlorotannin production in embryonic or juvenile stages is either not induced by UV light or takes more than 3 weeks to occur.     

电针可促进前脑啡肽原mRNA在大鼠脊髓和延髓的表达：原位杂交研究

本实验室以往的研究表明电针可促进脊髓脑啡肽释放,本研究进一步利用原位杂交方法观察电针后前脑啡肽原mRNA在脊髓和延髓的表达。结果表明电针刺激(2Hz,1-2-3mA,30)后24h同侧脊髓背角前脑啡肽原(PPE)-mRNA阳性细胞数高于对侧。电针后对侧延髓腹内侧网状结构[包括延髓网状巨细胞核(Gi)及其α部(GiA)和Gi的外侧旁核(LPGi)]PPE-mRNA阳性细胞数高于同侧。我们推测电针诱发前脑啡肽原mRNA表达增加可弥补因脑啡肽释放增多而导致脑啡肽前体物质的损失,从而参与针刺镇痛的长期效应。     

FISH技术在微生物生态学中的研究及进展

分子生物学技术在微生物生态学研究中具有灵敏、精确和快速的优势,但不能提供微生物的形态学、数量性状、空间分布等信息。荧光原位杂交技术结合了分子生物学的精确性和显微镜的可视性信息,可以在自然生境中监测和鉴定不同的微生物个体,尤其是对难培养和未被培养的微生物进行检测。荧光原位杂交技术被广泛用于微生物群落结构诊断和评价,现已成为微生物分子生态学研究中的热点技术。对荧光原位杂交技术的发展和在微生物分子生态学中的应用进行了综述,探讨了该技术应用中存在的问题和发展前景。     

DETERMINING THE AFFINITIES OF SALT MARSH FUCOIDS USING MICROSATELLITE MARKERS: EVIDENCE OF HYBRIDIZATION AND INTROGRESSION BETWEEN TWO SPECIES OF FUCUS (PHAEOPHYTA) IN A MAINE ESTUARY1

The high degree of morphological plasticity displayed by species of the brown algal genus Fucus L. is well documented. Such variation is especially pronounced for those estuarine taxa lacking holdfasts (termed ecads) that often bear little resemblance to the attached species from which they are derived. To better understand the systematics of salt marsh fucoids, we developed a suite of four microsatellite‐containing loci capable of distinguishing between F. vesiculosus L. and F. spiralis L. The genetic markers were used to determine the relationships of the fucoid ecads F. vesiculosus ecad volubilis (Hudson) Turner and a muscoides‐like Fucus in the Brave Boat Harbor (ME, USA) estuary. Ecad populations had 2‐ to 3‐fold higher levels of heterozygosity than attached populations of F. vesiculosus and F. spiralis. Further, ecads were “intermediate” between F. vesiculosus and F. spiralis in their allele frequencies and genotype composition. Our data indicate that populations of muscoides‐like Fucus in Brave Boat Harbor mainly consist of F₁ hybrids between F. vesiculosus and F. spiralis, whereas F. vesiculosus ecad volubilis may arise through backcrosses between F. vesiculosus and other fertile hybrids. Finally, our data support the hypothesis that introgression has occurred between attached populations of F. vesiculosus and F. spiralis.     

FREEZING STRESS AND OSMOTIC DEHYDRATION IN FUCUS DISTICHUS (PHAEOPHYTA): EVIDENCE FOR PHYSIOLOGICAL SIMILARITY1

The effects of osmotic dehydration and freezing on photosynthesis were studied in the brown alga Fucus distichus L. The data indicated that F. distichus exhibits similar physiological responses to both osmotic dehydration and freezing stress and that these responses resemble those in the literature for the effect of desiccation in air. Both stresses inhibited light-limited (P_subsat) and light-saturated (P_max) photosynthesis measured immediately after plants were reimmersed in seawater. The degree of initial inhibition and subsequent recovery of photosynthesis were proportional to the severity of the dehydration or freezing treatment. P_subsat and P_max recovered completely from osmotic dehydration for 3 h in 200% and 3 hr at – 10°C, but recovery was only partial following 3 h in 300%o or 3 h at – 15°C. In most cases, recovery was complete within 2 h following dehydration, with little further recovery occurring between 2 and 24 h posttreatment. No time-dependent recovery occurred following severe freezing. Observations using the vital stain fluorescein diacetate suggested that the lack of complete recovery might be due to severe damage or death of a proportion of cells in the thallus. There were no clear effects of either osmotic dehydration or freezing on dark respiration (R_d), although R_d was stimulated in all emersed treatments (frozen plants and 5° C controls) immediately following reimmersion. Measurement of chlorophyll fluorescence induction kinetics indicated that both osmotic dehydration and freezing reduced the ratio of variable to maximum florescence (F_v/F_m), indicating a decrease in the quantum efficiency of photosystem I. Based on these data, we suggest that there are common cellular and physiological components involved in the response of fucoid algae to a range of water stresses. This hypothesis was supported by experiments that showed that osmoacclimation in hyperosmotic seawater (51%o)for 2 weeks increased the ability of F. distichus to recover from freezing at – 15° C. During acclimation, mannitol content increased under hyperosmotic conditions and decreased under hypoosmotic conditions. Changes in plasma membrane integrity, determined by fresh weight: dry weight ratio, and amino acid release following freezing indicated an increasing gradient of freezing tolerance from low to high salinity. However, none of these physiological changes fully explained the marked increase in the freezing tolerance of photosynthesis observed in plants acclimated under hyperosmotic conditions.     

直接逆转录原位PCR检测实验性汉坦病毒感染乳鼠脑组织中病毒RNA及其与原位分子杂交的比较

出生后２～３ｄ的昆明种乳鼠,经腹腔接种１００个半数致死量的陈株汉坦病毒,每只０．０５ｍｌ,于接种后１、２、４、６、８、１０、１２、１４ｄ处死动物,每个时间点３～６只不等,取其脑组织固定于４％的多聚甲醛中,石蜡包埋制备５μｍ的连续组织切片,每时间点取１～２例组织切片,用逆转录原位ＰＣＲ（ＲＴ－ＩＳＰＣＲ）方法检测组织中病毒Ｓ片段ＲＮＡ,组织脱蜡后,经ＤＮａｓｅ、蛋白酶Ｋ、消化等予处理,用汉坦病毒ＲＮＡＳ片段特异的一对引物,在组织切片上进行病毒ＲＮＡ的逆转录和ＰＣＲ过程,直接将ｄｉｇｏｘｉｇｅｎｉｎ－１１－ｄＵＴＰ掺入到扩增产物中,经过３０个ＰＣＲ循环后,用碱性磷酸酶标记的抗ｄｉｇｏｘｉｇｅｎｉｎ抗体免疫组化检测扩增产物,连续组织切片用ｄｉｇｏｘｉｇｅｎｉｎ标记的汉坦病毒Ｍ片段Ｇ２编码区ＲＴ－ＰＣＲ扩增产物的和Ｓ片段特异性探针进行原位分子杂交并与ＲＴ－ＩＳＰＣＲ结果进行比较,另外应用免疫组化检测该基因表达产物病毒核抗原（ＮＰ）,结果,ＲＴ－ＩＳＰＣＲ在病毒感染１ｄ的乳鼠脑组织中检测到病毒ＲＮＡ扩增产物,扩增产物定位于神经细胞胞浆内,而原位分子杂交和免疫组化检测阴性。在病毒感染２ｄ及２ｄ以后的乳鼠脑组织中ＲＴ－Ｉ     

应用RNA原位核酸杂交检测乳腺癌石蜡切片中ER mRNA、PR mRNA表达

目的：制备地高辛标记的寡核苷酸探针,检测乳腺癌石蜡切片中雌激素受体（ER）mRNA、孕激素受体（PR）mRNA表达。方法：应用RNA原位核酸杂交（RISH）和免疫组化（IHC）检测技术,结果：（1）278例乳腺癌石蜡切片ERmRNA,PRmRNA阳性率分别为67．3％和61．5％,阴性率分别为32．7％和38．5％,ERmRNA,PRmRNA双阳性,双阴性分别为54．3％和25．6％,ERmRNA阳性PRmRNA阴性或ERmRNA阴性PRmRNA阳性率为20．1％。（2）RISH法的mRNA、PRmRNA阳性率分别为67．3％和61．5％均高于IHC法ER、PR的48．9％和41．2％（P均＜0．01）。（3）ERmRNA与ER、PRmRNA与PR的阳性和阴性符合率分别为7％和75．95。（4）杂产信号在乳腺癌细胞中的分布可分为,柱状上皮的胞质的上下方,胞质内均匀或近胞膜,核周偏位和核内外分布。（5）ERmRNA与ER、PRmRNA阳性率与组织学级别有关,Ⅱ级组中RV的阳性率为83．5％分别高于Ⅰ组中62．4％和Ⅲ组中54．5％（P＜0．01）,Ⅰ、Ⅱ级组中PRV的阳性率为66．7％,67．3％,均高于Ⅲ组中50．6％（P＜0．05）。结论。结论：地高辛标记的ER、PR寡核苷酸探针和RISH是一种较为理想的检测乳腺癌组织中ERmRNA和PRmRNA的分子检测技术。     

食管鳞癌人乳头状瘤病毒感染的原位杂交检测和观察

目的 :检测食管鳞癌中人乳头状瘤病毒 (HPV)的感染情况 ,探讨 HPV与食管癌发病的关系。方法 :用原位杂交技术及免疫组化方法对 33例食管鳞癌手术切除标本进行 HPV检测。结果 :用原位杂交方法检测食管鳞癌中 HPV DNA检出率为 4 5 .5 % (15 / 33)。HPV DNA阳性与食管鳞癌肿瘤细胞的分化程度无关 (P>0 .0 5 )。HPV衣壳蛋白的免疫组化检测均为阴性。结论 :HPV感染可能是引起食管上皮癌变的原因之一 ,与食管鳞癌的发生有关。即使在存在 HPV DNA的病例中 ,HPV衣壳蛋白亦可以不表达     

MORPHOLOGICAL VARIATION WITHIN AND BETWEEN NATURAL POPULATIONS OF NON-TIDE POOL FUCUS DISTICHUS (PHAEOPHYTA) IN NEW ENGLAND1,2

The sources of morphological variation in natural populations of Fucus distichus L. ssp. edentatus (De la Pyiaie) Powell and F. distichus L. ssp, evanescens (C. Agardh) Powell from New England were evaluated. Individuals from different populations were morphologically distinct as judged by population means and analysis of variance. A correlation between the plant's form and its habitat was established by field observation. The broadest material grows in calm estuarine habitats, while the narrowest plants occur in exposed, open coastal areas. Major differences in morphology also appear seasonally. In addition, microhabitat factors such as exposure to wave action and elevation explain some morphological variation. Cultured germlings from distinct populations of “evanescens” and “edentatus” type plants were outplanted to an experimental garden in order to ascertain whether the variation was heritable or environmentally induced. The latter experiments indicate that major differences in morphology are genetically determined.     

AN IN VITRO NITRATE REDUCTASE ASSAY FOR MARINE MACROALGAE: OPTIMIZATION AND CHARACTERIZATION OF THE ENZYME FOR FUCUS GARDNERI (PHAEOPHYTA)1

Measurement of the activity of the enzyme nitrate reductase (NR) may provide a useful index of nitrogen metabolism in marine macroalgae. In several species, including Fucus gardneri P. C. Silva, in vitro assays previously failed to detect NR activity, necessitating the use of in situ (or so-called“in vivo”) assays, which are more loosely controlled and lead to dafficulties in assessing enzyme characteristics such as the half-saturation constant (K_m). In this paper, we describe an in vitro NR assay developed for F. gardneri, in which tissue was homogenized using liquid nitrogen prior to the assay. In contrast to previous studies, enzyme activity was always detectable in F. gardneri collected directly from the field at levels up to 30 nmol nitrate converted to nitrite·min^?1·g^?1 wet weight. The effect of a variety of compounds, commonly added to NR extraction buffers, were tested. Additions of protease inhibitors, bovine serum albumin, and ethylenediamine tetraacetic acid had no consistent effects on NR activity, while polyvinyl pyrrolidone, potassium ferricyanide, and flavin adenine dinucleotide significantly decreased activity. The half-saturation constant (K_m) for NADH was 0.18 (± 0.05) mM and for nitrate, K_m=0.99 (±0.41) mM. Significant NR activity was detected without the addition of nitrate, suggesting that internal pools of nitrate averaging approximately 20 μmol NO₃^?·g^?1 wet weight were present in F. gardneri in February. The distribution of NR activity within the plant was highly variable between individuals, but activities were approximately 5-fold lower in the stipe than in midregions. In plants freshly sampled from the field, NR activity increased 7-fold from February to March, then fell to near-February levels by April. These changes in activity may correspond to seasonal changes in growth rate. The assay, optimized for F. gardneri, was used in several different macroalgal species from different taxa: Porphyra sp., Coralina vancouveriensis Yendo, Ulva sp., Enteromorpha intestinalis (Linnaeus) Nees, Macrocystis integrifolia Bory; and Costaria costatum (C. Agardh) Saunders. For all species tested, NR activity was detectable and, except for one species (Porphya sp.), was equal to or greater than activities measured by other workers using in vivo or in vitro assays for plants under similar conditions.     

鼻咽癌Epstein-Barr病毒的原位分子杂交研究

应用Epstein-Barr病毒编码的小RNA－1（Epstein-Barr virus encoded small RNA-1,EBER-1)分子探针进行原位分子杂交,检测沈阳地区鼻咽癌的Epstein-Barr病毒存在状况及意义。结果发现：EBER－1杂交信号定位于鼻咽癌的癌细胞核,在正常的鼻咽粘膜上皮细胞、间质细胞、粘液腺及淋巴细胞均为阴性。在53例鼻咽癌病例中,37例阳性,阳性率为69.8%。EBER－1的阳性表达与鼻咽癌的分化程度呈负相关,与患的生存期未见明显关系。提示：应用EBER－1分子探针可进行回顾性研究：EB病毒的存在与鼻咽癌的分化程度成负相关。     

地高辛标记探针电镜原位杂交技术探讨

应用ＬｏｗｉｃｒｙｌＫ４Ｍ低温包埋,Ｄｉｇ标记ｃ－ｆｏｓｃＤＮＡ探针对人肝癌组织进行原位杂交,用金标抗Ｄｉｇ抗体进行检测,银增强放大信号处理。电镜观察表明,免疫胶体金颗粒特异性地标记在肝癌细胞胞浆及核内,呈散在分布,金颗粒大小基本一致,背景清晰。     

A COMPARISON OF AIR AND WATER AS ENVIRONMENTS FOR PHOTOSYNTHESIS BY THE INTERTIDAL ALGA FUCUS SPIRALIS (PHAEOPHYTA)1

The response of photosynthesis and respiration of the intertidal brown alga Fucus spiralis L. to light and temperature at ambient and elevated concentrations of inorganic carbon was investigated. The light-saturated rate of photosynthesis was greater in air at 15° C and 20° C, but greater in water at 10° C. Light compensation point and I_k was about 50% lower under submerged relative to emerged conditions, whereas the initial slope of photosynthesis versus irradiance was higher, except at 20° C. Under both submerged and emerged conditions light-saturated photosynthesis was limited to a similar degree (78%, and 65%, respectively) by the availability of inorganic carbon at naturally occuring concentrations. In air, slight desiccation at tissue water contents of about 96% to 92% caused a stimulation in the rate of net photosynthesis to 110–148% of fully hydrated fronds. At lower water contents the rate of net photosynthesis declined linearly with decreasing water content and became zero at a water content of about 15%. Dark respiration declined linearly with tissue water content and remained positive to a water content of 8%. Upon reimmersion the fronds showed a complete recovery within 35 min following desiccation to a water content of 20–30%. Thus F. spiralis seems to be very tolerant to desiccation. Since F. spiralis photosynthesizes effectively in air, even at a higher rate than in water as long as it has not lost a large proportion of its water in desiccation, the alternating exposure to air may be beneficial by increasing the daily carbon gain compared to a fully submerged situation.     

多色荧光原位杂交对昆明山海棠和丙烯酰胺诱发微核染色体组成的研究

采用着丝粒和端粒DNA探针多色荧光原位杂交,分析了昆明山海棠和丙烯酰胺诱导的 NIH3T3细胞微核的染色体组成。结果表明,昆明山海棠和丙烯酰胺诱导的由整条染色体组成的微核分别可达70.7％和65.9％,提示昆明山海棠和丙烯酰胺均有较强的非整倍体毒性。     

HETEROMORPHISM FOR A HIGHLY REPEATED SEQUENCE IN THE NEW ZEALAND FROG LEIOPELMA HOCHSTETTERI

A satellite DNA sequence, Lhl, was cloned from the New Zealand endemic frog Leiopelma hochstetteri. Large tandem arrays of Lh1 were localized by in situ hybridization to the long arm of a small telocentric autosome in some individuals, but these arrays were absent from other individuals. Lh1 is also present in varying amounts on some supernumerary chromosomes in some individuals. Heteromorphism for the presence of Lh1 exists in two populations that have been separated by a sea channel since the end of the Pleistocene, indicating that the heteromorphism either has arisen repeatedly or has persisted for at least 10,000 years. Individuals lacking Lh1 thus appear to be at no significant selective disadvantage. The variation in Lh1 copy number probably results from its interstitial chromosomal location, which exposes it to more frequent unequal crossovers than the pericentromeric or telocentric locations of most satellite DNA. Lh1 may be parasitic or simply inert junk, but in either case it may be deleted or dispersed throughout the rest of the genome through unequal crossing over.     

BDNF，NT-3在猫背根神经元的自分泌和旁分泌作用初探—原位杂交和免疫组织化学研究

探讨脑源性神经营养因子 (BDNF)、神经营养因子 3 (NT- 3)在成年猫背根节 (DRG)神经元是否存在自分泌或旁分泌作用方式。用特异的 trk B、 trk C抗体及 BDNF、 NT- 3的 c RNA探针 ,以免疫组化及原位杂交双标法观察了成年猫 L6DRG神经元 BDNF m RNA与 trk B、 NT- 3 m RNA与 trk C的关系 ,以了解 BDNF、 NT- 3在成年猫 L6 DRG神经元有无自分泌或旁分泌作用。结果表明 ,在成年猫 L6 DRG BDNFm RNA阳性细胞主要是部分中、小神经元 (4 2～ 5 7μm,<42μm) ,而 trk B阳性细胞则主要是部分大神经元 (>5 7μm)。未见双标的 BDNF m RNA和 trk B阳性神经元 ,但见一些双标的卫星细胞。与BDNF者比较 ,大多数大神经元既表达 NT- 3 m RNA,又表达 trk C。这表明在成年猫 L6 DRG神经元 ,NT- 3可能存在自分泌方式。但本文的结果未证明 BDNF在成年猫 L6 DRG神经元存在自分泌。     

高分化及低分化鼻咽癌细胞株中Epstein－Barr病毒潜伏感染膜蛋白（LMP1）基因的原位杂交与克隆及序列分析

应用染色体原位杂交、ＰＣＲ扩增、克隆及核苷酸序列分析等方法,分析了ＣＮＥ１和ＣＮＥ３细胞株中的潜伏感染膜蛋白（ＬＭＰ１）基因。ＣＮＥ１是来自我国东北的高分化鼻咽癌细胞株,ＣＮＥ３是来自广西的低分化鼻咽癌细胞株。染色体原位杂交结果表明,ＣＮＥ１细胞中ＬＭＰ１基因存在于细胞核内,整合在第一号染色体上,ＣＮＥ３中ＬＭＰ１基因则随机存在于细胞核内及多条染色体上。用ＰＣＲ方法分别从ＣＮＥ１及ＣＮＥ３中扩增得到了ＬＭＰ１基因片段（外显子３）,核苷酸序列分析证明,来自ＣＮＥ１的ＬＭＰ１与来自Ｂ９５－８细胞的ＬＭＰ１核苷酸序列同源性极高,达９９．５％,而ＣＮＥ３的ＬＭＰ１基因与Ｂ９５－８的ＬＭＰ１基因同源性为９３％。     

Poly(A)-specific ribonuclease (PARN): An allosterically regulated,processive and mRNA cap-interacting deadenylase

Abstract

Deadenylation of eukaryotic mRNA is a mechanism critical for mRNA function by influencing mRNA turnover and efficiency of protein synthesis. Here, we review poly(A)-specific ribonuclease (PARN), which is one of the biochemically best characterized deadenylases. PARN is unique among the currently known eukaryotic poly(A) degrading nucleases, being the only deadenylase that has the capacity to directly interact during poly(A) hydrolysis with both the m⁷G-cap structure and the poly(A) tail of the mRNA. In short, PARN is a divalent metal-ion dependent poly(A)-specific, processive and cap-interacting 3′–5′ exoribonuclease that efficiently degrades poly(A) tails of eukaryotic mRNAs. We discuss in detail the mechanisms of its substrate recognition, catalysis, allostery and processive mode of action. On the basis of biochemical and structural evidence, we present and discuss a working model for PARN action. Models of regulation of PARN activity by trans-acting factors are discussed as well as the physiological relevance of PARN.     

IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM CATENELLA AND A. TAMARENSE (DINOPHYCEAE) USING DNA PROBES AND WHOLE-CELL HYBRIDIZATION1

Fluorescent DNA probes (cCAT-F1 and cTAM-Fl) complementary to the 3′ end of ribosomal RNA (rRNA) internal transcribed spacer 1 sequences (ITS 1: positions 154–176) of toxic species of Alexandrium catenella (Whedon and Kofoid) Taylor and A. tamarense (Lebour) Taylor were applied to various cultures of the genus Alexandrium and several other phytoplankters using whole-cell fluorescent in situ hybridization. cCAT-F1 and cTAM-F1 reacted with targeted strains of A. catenella (catenella type) and A. tamarense (tamarense type), respectively, and did not react with isolates of A. affine (Inoue et Fukuyo) Balech, A. fraterculus (Balech) Balech, A. insuetum Balech, A. lusitanicum Balech, A. pseudogonyaulux (Biecheler)Horiguchi ex Yuki et Fukuyo comb. nov., nor isolates of Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra (Ehrenberg) Stein, Gymnodinium mikimotoi Miyake et Kominami ex Oda, Skeletonema costatum (Greville) Cleve, Heterosigma akashiwo (Hada) Hada, and Chattonella antiqua (Hada) Ono. DNase I and RNase A treatment showed that probes hybridized to ribosomal DNA, not rRNA. Probes were localized at the bottom of the U-shaped nucleus, a region that corresponds to the nucleolus. The probes are highly specific for particular strains of A. catenella and A. tamarense and are applicable for identifying these species collected from cultured and possibly natural populations.