The Species Problem in a Ciliate with a High Multiple Mating Type System, Euplotes crassus

ABSTRACT. One hundred twenty non-autogamous wild-type strains of Euplotes crassus , collected over seven years, mainly from the Mediterranean coasts, were investigated for their mating interactions. The strains were mixed pair-wise and data from mating reactions were evaluated and organized by means of a specially constructed computer program. The program identified 38 strains with distinctive mating patterns which could be clustered in nine clumps, all of which were connected either directly or indirectly. Thus, all these strains appeared to be components of the same gene pool, even though direct genetic exchange between strains was not possible in every combination. Subsequently, the 38 strains were subjected to cytometric analysis and scored for zymic variations resulting from electrophoretic patterns of five enzyme systems (acid phosphatases, amylases, malic dehydrogenase, malic enzyme, and tetrazolium oxidases) of proved diagnostic value in the identification of Euplotes species. No significant discontinuities correlated with mating patterns was apparent from these analyses. It was concluded that the E. crassus strains analyzed are not properly divided in sibling species and it was consistently suggested to avoid a genetic partitioning of ciliate species endowed with high multiple mating type systems, in which the sets of wild strains brought under investigation with difficulty represent the natural dimensions of the species.     

Two New Species of Plistophora (Microsporidea) from North American Fish with a Synopsis of Microsporidea of Freshwater and Euryhaline Fishes

SYNOPSIS. Two new species of Microsporidea, Plistophora salmonae from steelhead and rainbow trout (Salmo gairdntri) and Plistophora crpedianar from gizzard shad ( Dorosoma cepedianum) are described. Schizonts to spores of P. cepedianae were found at one time within the same cyst, while only sporonts and spores of P. salmonae were found within the cyst.
An illustrated synopsis of the known Microsporidea of freshwater and euryhaline fishes is given.     

Histone H1 interacts preferentially with DNA fragments containing a cisplatin-induced 1,2-intrastrand cross-link

Cisplatin [cis-diamminedichloroplatinum(II) or cis-DDP], but not its stereoisomer transplatin, is suggested to be among the most powerful anticancer agents. It is believed that its therapeutic activity results from its interaction with DNA forming intra- and interstrand crosslinks. During our earlier investigations, we have observed a prominent preference of the linker histone H1 for binding to cis-platinated DNA (containing several different cross-links along the DNA fragment) compared with unmodified or transplatin-modified DNA. This report presents our recent experimental data obtained by band-shift analysis on the binding of H1 to a cisplatin-modified synthetic 34 bp DNA fragment containing a single target d(GG/CC) for 1,2 cis-intra-platination. Results obtained with another nuclear protein with similar DNA-binding properties, HMGB1, are also presented. The experimental data throw light on the precise preference of histone H1 for binding to different types of cisplatin-created cross-links in DNA.     

Concentrations of the Stress Hormone Copeptin Increase upon Hypoglycaemia in Patients with Type 1 Diabetes Dependent of Hypoglycaemia Awareness

Objective

Copeptin, a marker for stress mirroring vasopressin concentrations, has been shown to increase upon insulin-induced hypoglycaemia in patients after transsphenoidal surgery of pituitary adenomas. Patients with type 1 diabetes mellitus are prone to hypoglycaemia, but no data about copeptin levels upon hypoglycaemia are available. Furthermore, the perception of hypoglycaemia can vary from total unawareness to disabling episodes. The aim of this study was to investigate whether copeptin increases upon hypoglycaemia in patients with type 1 diabetes mellitus and is associated with the degree of hypoglycaemia awareness.

Materials and Methods

In this prospective observational study, 17 patients with type 1 diabetes underwent a standardized insulin infusion test. Blood sampling for glucose and copeptin was performed at baseline and after 60 minutes (min). To assess hypoglycaemia associated symptoms the Mood and Symptom Questionnaire (MSQ) was conducted at baseline and after 60 min.

Results

During insulin infusion, blood glucose decreased from 5.1 (SD±0.2) to 3.0 (±0.5) mmol/L at 60 min (p<0.001). Copeptin concentrations increased from 3.2 (±1.7) to 3.8 (±1.9) pmol/L (p = 0.03). Mood and Symptoms Questionnaire scores increased from 14 (±3.0) to 18 (±5.8), (p = 0.006). Patients with good hypoglycaemia awareness had an increase in copeptin from 3.0 (±1.8) to 4.2 (±2.4) pmol/L (p = 0.03) in contrast to patients more unaware of hypoglycaemia who only showed an increase in copeptin from 3.3 (±1.6) to 3.6 (±1.4) pmol/L (p = 0.4). There was a trend to a larger copeptin increase in patients aware of hypoglycemia compared to patients unaware of hypoglycemia (p = 0.074).

Conclusion

Copeptin increases in patients with type 1 diabetes upon insulin induced hypoglycaemia. Interestingly, the copeptin increase seems associated with the degree of hypoglycaemia awareness. This hypothesis warrants further verification.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00515801","term_id":"NCT00515801"}}NCT00515801     

Genetic and Genomic Diversity Studies of Acacia Symbionts in Senegal Reveal New Species of Mesorhizobium with a Putative Geographical Pattern

Acacia senegal (L) Willd. and Acacia seyal Del. are highly nitrogen-fixing and moderately salt tolerant species. In this study we focused on the genetic and genomic diversity of Acacia mesorhizobia symbionts from diverse origins in Senegal and investigated possible correlations between the genetic diversity of the strains, their soil of origin, and their tolerance to salinity. We first performed a multi-locus sequence analysis on five markers gene fragments on a collection of 47 mesorhizobia strains of A. senegal and A. seyal from 8 localities. Most of the strains (60%) clustered with the M. plurifarium type strain ORS 1032^T, while the others form four new clades (MSP1 to MSP4). We sequenced and assembled seven draft genomes: four in the M. plurifarium clade (ORS3356, ORS3365, STM8773 and ORS1032^T), one in MSP1 (STM8789), MSP2 (ORS3359) and MSP3 (ORS3324). The average nucleotide identities between these genomes together with the MLSA analysis reveal three new species of Mesorhizobium. A great variability of salt tolerance was found among the strains with a lack of correlation between the genetic diversity of mesorhizobia, their salt tolerance and the soils samples characteristics. A putative geographical pattern of A. senegal symbionts between the dryland north part and the center of Senegal was found, reflecting adaptations to specific local conditions such as the water regime. However, the presence of salt does not seem to be an important structuring factor of Mesorhizobium species.     

Isolation of Bovine Adenovirus Type 1 from a Calf with Pneumo-Enteritis

During the last decade, an increasing number of bovine adenoviruses have been isolated from calves suffering from more, or less, well-defined syndromes. These have consisted of respiratory disorders of varying severity, enteritis, or a combination of both, which in typical cases has been termed “pneumo-enteritis”. These investigations have been reviewed by Darbyshire (1968). Wilcox (1969) isolated adenoviruses from kerato-conjunctivitis (KC) in cattle. Furthermore, strains have been isolated from apparently healthy animals (Darbyshire 1968), and from tissue cultures prepared from various organs from calves such as kidneys (Scho- pov et al. 1968), and testes (Rondhuis 1968, Bartha & Csontos 1969). At the present time 9 serotypes of bovine adenoviruses exist, as determined by neutralization tests, and these have recently been reviewed by Guenov et al. (1970). However, several strains, some from cases of pneumonia (Cole 1970, Lupini et al. 1970) and others from KC (Wilcox 1969) remain to be typed and compared with the known prototypes, thereby enabling possible new serotypes to be identified. So far, serotypes 1 and 2 (Darbyshire et al. 1969), serotype 3 (Darbyshire et al. 1966) and serotypes 4 and 5 (Aldasy et al. 1965) have been shown to cause pneumo-enteritis, and serotype 6 (Rondhuis 1970) a mild respiratory disease in experimentally infected calves. Similarly, KC has been produced experimentally by Wilcox (1970), while the pathogenicity for experimental animals of the other typed and untyped strains remains to be investigated.     

一个I型神经纤维瘤病家系的基因突变分析

目的：I型神经纤维瘤病是一种常见的常染色体显性遗传病,主要累及皮肤和神经系统。其临床表现多样,主要以”咖啡牛奶斑”、皮肤神经纤维瘤、虹膜Lisch结节、腋窝和腹股沟斑点为特征,I型神经纤维瘤病由NF1基因突变所致,神经纤维瘤蛋白是NFI基因编码蛋白,是一种肿瘤抑制蛋白,可抑制细胞的过度生长。NF1基因突变不仅可导致细胞过度生长,还可增加良性及恶性肿瘤的发生风险。本研究中,我们通过基因突变分析,确定中国东北地区一个伴有先天性白内障的I型神经纤维瘤家系NF1基因的突变位点。方法：通过聚合酶链反应（PCR）和NF1基因直接测序分析对家系中的3名患者及2名健康成员进行基因突变检测,以确定其突变位点。结果：此家系呈常染色体显性遗传。通过基因序列分析发现NF1基因第1140密码子第二个碱基呈杂合子点突变C—G,导致一个无义突变S1140X,家系中健康成员和正常对照未检测到此突变存在。结论：通过NF1基因测序分析,我们发现NF1基因的S1140X突变是引起该家系NF1疾病的致病原因,该突变导致NF1基因终止密码提前,神经纤维瘤素蛋白截短。本研究丰富了我国关于I型神经纤维瘤病在眼科的临床表现。     

青藏高原5种害鼠D型肉毒素抗性相关基因VAMP1的序列分析

突触小泡相关膜蛋白1基因(VAMP1)的变异是导致鼠类对D型肉毒梭毒素灭鼠剂产生抗性的主要原因。本研究利用转录组测序的方法,分析青藏高原地区5种主要害鼠:高原鼠兔(Ochotona curzoniae)、高原鼢鼠(Eospalax baileyi)、长尾仓鼠(Cricetulus longicaudatus)、青海松田鼠(Neodon fuscus)和喜马拉雅旱獭(Marmota himalayana)的VAMP1序列信息。同时,分别采集来自5个地理种群的58只高原鼠兔和59只高原鼢鼠,对VAMP1基因第二外显子区序列进行分析。结果显示,从转录组组装文件中成功获得5种动物的VAMP1基因全序列,长度均为357 bp,共检测到46个核苷酸变异位点和4个氨基酸变异位点,但未发现与D型肉毒素抗性相关的氨基酸位点。对高原鼠兔群体和高原鼢鼠群体的VAMP1基因第二外显子序列的分析显示,高原鼠兔所有个体的序列高度保守,而在高原鼢鼠中则存在一个同义突变位点,但两物种在D型肉毒素抗性相关位点上都未监测出位点变异。该研究结果提示,D型肉毒杀鼠剂在青藏高原地区害鼠防治方面应该可以长期发挥重要作用。     

Diversity of Freshwater <Emphasis Type="Italic">Thioploca</Emphasis> Species and Their Specific Association with Filamentous Bacteria of the Phylum <Emphasis Type="Italic">Chloroflexi</Emphasis>

Phylogenetic diversity among filamentous sulfur-oxidizing bacteria of the genus Thioploca inhabiting freshwater/brackish environments was analyzed in detail. The 16S rRNA gene sequence of Thioploca found in a freshwater lake in Japan, Lake Okotanpe, was identical to that of Thioploca from Lake Ogawara, a brackish lake. The samples of the two lakes could be differentiated by the sequences of their 23S rRNA genes and 16S–23S rRNA internal transcribed spacer (ITS) regions. The 23S rRNA-based phylogenetic relationships between Thioploca samples from four lakes (Lake Okotanpe, Lake Ogawara, Lake Biwa, and Lake Constance) were similar to those based on the 16S rRNA gene sequences. In addition, multiple types of the ITS sequences were obtained from Thioploca inhabiting Lake Okotanpe and Lake Constance. Variations within respective Thioploca populations were also observed in the analysis of the soxB gene, involved in sulfur oxidation. As major members of the sheath-associated microbial community, bacteria of the phylum Chloroflexi were consistently detected in the samples from different lakes. Fluorescence in situ hybridization revealed that they were filamentous and abundantly distributed within the sheaths of Thioploca.     

Morphological and Molecular Redefinition of Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and Aspidisca lynceus (Müller,1773) Ehrenberg, 1859, with Reconsideration of a “Well‐known” Euplotes Ciliate,Euplotes harpa Stein, 1859 (Ciliophora,Euplotida)

We documented the morphology, infraciliature, silverline system, and molecular data of two euplotid species isolated from China, including two populations of the poorly known Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and the previously well described Aspidisca lynceus (Müller, 1773 ) Ehrenberg, 1830. Based on the information available, an improved diagnosis of Euplotes platystoma is given, including: a narrow adoral zone with 44–68 membranelles, 10 frontoventral, 5 transverse, 2 left marginal and 2 caudal cirri, 11–13 dorsal kineties with 17–25 dikinetids in the mid‐dorsal row, and dorsal silverline system of the double‐eurystomus type. The Chinese population of Aspidisca lynceus closely resembles previously described populations. Phylogenetic analyses inferred from SSU rDNA sequences show that E. platystoma is closely related with E. neapolitanus, and the internal position of A. lynceus within this genus is still not robust. A reconsideration of the “well‐known” Euplotes harpa and a comparison of all SSU rDNA sequences of E. harpa in GenBank are provided. We speculate that the sequences available from GenBank under the name of E. harpa are very likely from misidentified materials, that is, the identity of the species currently associated with the SSU rDNA of this “well‐known” form in molecular databases requires further confirmation.     

Polypores new to Japan 1. Species ofPolyporus,with a note onP. hartmanni

Polyporus admirabilis, P. dictyopus, P. guianensis, P. pseudobetulinus, P. tubaeformis andP. udus are reported for Japan for the first time.Polyporus tubaeformis had been confused withP. melanopus, but the Japanese collections were conspecific with Norwegian isolates ofP. tubaeformis. A key to allPolyporus species in Japan is provided.Polyporus hartmanni is reported for the first time outside the Australian continent. As the species has a bipolar heterothallism and produces brown rot, its taxonomic relationship withPolyporus s. str. is discussed.The Japanese Science and Technology Agency (STA) is thanked by the authors for financial support.     

Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism

Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Delta or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Delta mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.     

RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling

The ATR-Chk1 signaling pathway mediates cellular responses to DNA damage and replication stress and is composed of a number of core factors that are conserved throughout eukaryotic organisms. However, humans and other higher eukaryotic species possess additional factors that are implicated in the regulation of this signaling network but that have not been extensively studied. Here we show that RHINO (for Rad9, Rad1, Hus1 interacting nuclear orphan) forms complexes with both the 9-1-1 checkpoint clamp and TopBP1 in human cells even in the absence of treatments with DNA damaging agents via direct interactions with the Rad9 and Rad1 subunits of the 9-1-1 checkpoint clamp and with the ATR kinase activator TopBP1. The interaction of RHINO with 9-1-1 was of sufficient affinity to allow for the purification of a stable heterotetrameric RHINO-Rad9-Hus1-Rad1 complex in vitro. In human cells, a portion of RHINO localizes to chromatin in the absence of DNA damage, and this association is enriched following UV irradiation. Furthermore, we find that the tethering of a Lac Repressor (LacR)-RHINO fusion protein to LacO repeats in chromatin of mammalian cells induces Chk1 phosphorylation in a Rad9- and Claspin-dependent manner. Lastly, the loss of RHINO partially abrogates ATR-Chk1 signaling following UV irradiation without impacting the interaction of the 9-1-1 clamp with TopBP1 or the loading of 9-1-1 onto chromatin. We conclude that RHINO is a bona fide regulator of ATR-Chk1 signaling in mammalian cells.     

RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling

The ATR-Chk1 signaling pathway mediates cellular responses to DNA damage and replication stress and is composed of a number of core factors that are conserved throughout eukaryotic organisms. However, humans and other higher eukaryotic species possess additional factors that are implicated in the regulation of this signaling network but that have not been extensively studied. Here we show that RHINO (for Rad9, Rad1, Hus1 interacting nuclear orphan) forms complexes with both the 9-1-1 checkpoint clamp and TopBP1 in human cells even in the absence of treatments with DNA damaging agents via direct interactions with the Rad9 and Rad1 subunits of the 9-1-1 checkpoint clamp and with the ATR kinase activator TopBP1. The interaction of RHINO with 9-1-1 was of sufficient affinity to allow for the purification of a stable heterotetrameric RHINO-Rad9-Hus1-Rad1 complex in vitro. In human cells, a portion of RHINO localizes to chromatin in the absence of DNA damage, and this association is enriched following UV irradiation. Furthermore, we find that the tethering of a Lac Repressor (LacR)-RHINO fusion protein to LacO repeats in chromatin of mammalian cells induces Chk1 phosphorylation in a Rad9- and Claspin-dependent manner. Lastly, the loss of RHINO partially abrogates ATR-Chk1 signaling following UV irradiation without impacting the interaction of the 9-1-1 clamp with TopBP1 or the loading of 9-1-1 onto chromatin. We conclude that RHINO is a bona fide regulator of ATR-Chk1 signaling in mammalian cells.