<Emphasis Type="Italic">RNR4</Emphasis> mutant alleles <Emphasis Type="Italic">pso3-1</Emphasis> and <Emphasis Type="Italic">rnr4Δ</Emphasis> block induced mutation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The PSO3 gene of Saccharomyces cerevisiae was molecularly cloned by complementing the cold-sensitivity phenotype of a pso3-1 mutant and was found to be allelic to RNR4, encoding one of the two DNA damage-inducible small subunits of the ribonucleotide reductase (RNR) complex. Compared to a rnr4Δ mutant that allows only very little mutation induction at very low doses of 254_nm ultraviolet light (UVC), the pso3-1 mutant allele confers leakiness in that it permits some DNA damage-induced mutagenesis at low doses of UVC. Similarly, the pso3 mutant is slightly less sensitive to UVC than an rnr4Δ mutant. Cloning and sequencing of the RNR4 locus of the pso3-1 mutant revealed that its intermediate phenotype is attributable to a G → A transition at nucleotide 352, leading to replacement of glycine by arginine [G118R] in the mutant’s protein. Both RNR4 mutant alleles confer significantly less sensitivity to UVC than mutant alleles of non-UVC-mutable REV3, indicating that, apart from nucleotide excision repair, RAD6-dependent error-free DNA repair may still be functional. The phenotype of a strongly reduced UVC-induced mutagenesis for rnr4 mutant alleles has not yet been described; it suggests the importance of this gene for a fully functional RNR providing correct amounts of DNA precursor molecules, thereby, allowing translesion synthesis (error-prone) of UVC-damaged DNA. Stationary phase cells of the rnr4Δ mutant, but not of the original pso3-1 mutant, are swollen with a fourfold to eightfold increase in volume. The central role of RNR in DNA precursor metabolism and its complex regulation allow for several modes of suppression that may influence the phenotypes of RNR4 mutants, especially those containing the leaky pso3-1 mutant allele.     

<Emphasis Type="Italic">Phlebotomus</Emphasis> (<Emphasis Type="Italic">Euphlebotomus</Emphasis>) <Emphasis Type="Italic">mascomai</Emphasis> n. sp. (Diptera–Psychodidae)

A new species of sandfly is described from limestone caves in Thailand. The inclusion of this species in the subgenus Euphlebotomus is justified on the basis of characters of the male genitalia (paramere, basal lobe). The male–female gathering in the same taxon is based on ecological (cavernicolous species), morphological (length of male genital filaments and female spermathecal ducts) and molecular (homology of cytochrome b mt DNA sequences) criteria. A differential diagnosis between Phlebotomus mascomai n. sp. and P. argentipes Annandale & Brunetti, the vector of Leishmania donovani (Laveran & Mesnil) in India, is proposed based on several morphological characters like antennal formula and genitalia.     

The mating type-specific homeodomain genes <Emphasis Type="Italic">SXI1α</Emphasis> and <Emphasis Type="Italic">SXI2a</Emphasis> coordinately control uniparental mitochondrial inheritance in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

In the great majority of sexual eukaryotes, mitochondrial genomes are inherited almost exclusively from a single parent. While many hypotheses have been proposed to explain this phenomenon, very little is known about the genetic elements controlling uniparental mitochondria inheritance. In the bipolar, isogamous basidiomycete yeast Cryptococcus neoformans, progeny from crosses between strains of mating type a (MATa) and mating type α (MATα) typically inherit mitochondrial DNA (mtDNA) from the MATa parent. We recently demonstrated that a mating type α (MATα)-specific gene SXI1α, controls mitochondrial inheritance in C. neoformans. Here, we show that another homeodomain gene SXI2a in the alternative mating type MATa is also required for uniparental mtDNA inheritance in this fungus. Disruption of SXI2a resulted in biparental mtDNA inheritance in the zygote population with significant numbers of progeny inheriting mtDNA from the MATa parent, the MATα parent, and both the MATa and the MATα parents. In addition, progeny from same-sex mating between MATα strains showed a biparental mitochondrial inheritance pattern. Our results suggest that SXI1α and SXI2a coordinately control uniparental mitochondrial inheritance in C. neoformans.     

Cavidine Ameliorates Lipopolysaccharide-Induced Acute Lung Injury <Emphasis Type="Italic">via</Emphasis> NF-κB Signaling Pathway <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Acute lung injury (ALI) is characterized by widespread inflammation in the lungs and alveolar-capillary destruction, causing high morbidity and mortality. Cavidine, isolated from Corydalis impatiens, have been exhibited to have potent anti-inflammatory effects in previous studies. The purpose of this study was to evaluate the protective effect of cavidine on lipopolysaccharide (LPS)-induced ALI and to enunciate the underlying in vivo and in vitro mechanisms. Mice were intraperitoneally administrated with cavidine (1, 3, or 10 mg/kg) at 1 and 12 h, prior to the induction of ALI by intranasal administration of LPS (30 mg/kg). Blood samples, lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested after LPS challenge. Furthermore, we used LPS-induced lung epithelial cells A549 to examine the mechanism of cavidine to lung injury. The results showed that pretreatment with cavidine significantly decreased lung wet-to-dry weight (W/D) ratio, reduced pro-inflammatory cytokine levels including TNF-α and IL-6 in BALF and serum from LPS-stimulated mice, and attenuated lung histopathological changes. In addition, western blot results showed that cavidine inhibited the phosphorylation of nuclear factor-kappaB (NF-κB) p65 and IκBα induced by LPS. In conclusion, our results demonstrate that cavidine protects against LPS-induced acute lung injury in mice via inhibiting of pro-inflammatory cytokine TNF-α and IL-6 production and NF-κB signaling pathway activation. Taken together, cavidine may be useful for the prevention and treatment of pulmonary inflammatory diseases, such as ALI.     

What are the infectious larvae in <Emphasis Type="Italic">Ascaris suum</Emphasis> and <Emphasis Type="Italic">Trichuris muris?</Emphasis>

In the present study, larvae of Ascaris suum and Trichuris muris were investigated by light and electron microscopy after incubation in a hatching medium containing 89% phosphate-buffered saline (pH 7.4), 10% RPMI-1640 and 1% sodiumhypochlorite at 40 and 37 degrees C, respectively. The larvae were obtained from fertilised eggs of the worms during defined phases of development (A. suum, 36th-50th day of development; T. muris, once a week from week 16 to 20). Light and electron micrographs of the larvae gave evidence that the third larval stage of A. suum is probably the infectious stage. The first moult of the larvae had already taken place before the 36th day of incubation starting at day 1. After 36 days of incubation, only the second larval stage was found within eggs. Some of these larvae were coated by a separated sheath so that a second moult of the larvae is reasonable. On the other hand, no sheathed larvae of T. muris were found in the eggs incubated for 20 weeks in distilled water. No signs of moult were seen for 20 weeks neither on light nor on the electron micrographs. Therefore, in T. muris, the first larval stage is the infectious stage, which was proven by means of re-infections of mice 16, 18 or 20 weeks after incubation of the eggs.     

Genetic regulation of arrested development in nematodes: are <Emphasis Type="Italic">age</Emphasis>-1 and <Emphasis Type="Italic">daf</Emphasis>-gene orthologs present in <Emphasis Type="Italic">Dictyocaulus viviparus</Emphasis>?

In opposite to the free-living soil nematode Caenorhabditis elegans, the genetic regulation of hypobiosis or inhibited or arrested development in parasitic nematodes is completely unknown. In C. elegans, the daf-genes or the age-1 gene are of major importance in signaling pathways regulating arrested development. To investigate if orthologs of these genes are present in the bovine lungworm Dictyocaulus viviparus, a PCR analysis with gene-specific primer combinations was performed. No orthologs of the age-1 or daf-genes could be identified in D. viviparus. The possible differences in the role of the daf-genes concerning arrested development in parasitic and free-living nematodes will be discussed.     

The <Emphasis Type="Italic">rpl5</Emphasis>–<Emphasis Type="Italic">rps14</Emphasis> mitochondrial region: a hot spot for DNA rearrangements in <Emphasis Type="Italic">Solanum</Emphasis> spp. somatic hybrids

Southern analysis with rpl5 and rps14 mtDNA gene probes of Solanum tuberosum, S. commersonii and a sample of somatic hybrids detected polymorphisms between parents and the appearance of a novel restriction fragment in various hybrids. In one of them, detailed mtDNA analyses revealed various configurations of the rpl5–rps14 region present at different stoichiometries. Multiple inter-parental recombination events across homologous sequences were assumed to have caused these rearrangements. Sequence similarity searches detected one sequence putatively involved in the recombination upstream of the rpl5 gene. The presence of a second recombinogenic sequence was inferred. We propose two models to explain the mechanism responsible for obtaining the different rpl5–rps14 arrangements shown after somatic hybridization. Variability in the rpl5–rps14 region observed in both the parental species and their somatic hybrids suggests this region is a hot spot for mtDNA rearrangements in Solanum spp.Contribution no. 39 from the Institute of Plant Genetics, Research Division of Portici.Communicated by A. Brennicke     

Prevalence and antibiotic susceptibility of <Emphasis Type="Italic">Ureaplasma urealyticum</Emphasis> and <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> in Xi’an,China

This study analyzed the prevalence and antibiotic susceptibility of urogenital Ureaplasma urealyticum and Mycoplasma hominis isolated in Xi’an, China. A total of 2161 individuals from 2011 to 2015 were included, and antibiotic susceptibility tests were performed by using the Mycoplasma IST kit. Of the individuals studied, 1018 (47.11 %) were identified to be positive for urogenital mycoplasmas. The single U. urealyticum, single M. hominis, and dual U. urealyticum and M. hominis infections accounted for 772 (75.83 %), 66 (6.48 %), and 180 (17.68 %), respectively. The total positive rate was higher in females than in males (58.76 % vs. 28.86 %, p?<?0.001). The highest total positive rate (48.88 %) was observed in individuals aged 25 years to 30 years. In symptomatic and asymptomatic individuals, the positive rates were both higher in females than in males (67.36 % vs. 31.02 %, p?<?0.001 and 42.58 % vs. 7.69 %, p?<?0.001, respectively) and individuals aged 25 years to 30 years, and those aged 30 years to 35 years had the highest positive rates (54.35 and 57.14 %, respectively). The U. urealyticum and M. hominis identified from single or dual infections displayed low resistance rates to josamycin, doxycycline, and minocycline (<10 %) in both the symptomatic and asymptomatic groups. These results suggest that females and individuals with symptoms and younger age had higher mycoplasma infection rates and that josamycin, doxycycline, and minocycline may be recommended for the clinical treatment of patients infected with urogenital mycoplasmas, irrespective of the symptoms.     

First report of <Emphasis Type="Italic">Cryptosporidium</Emphasis><Emphasis Type="Italic">parvum</Emphasis> ‘ferret’ genotype in American mink (<Emphasis Type="Italic">Mustela vison</Emphasis> Shreber 1777)

A total of 51 faecal samples from wild and farmed mink were analysed by a direct immunofluorescence antibody test. Cryptosporidium oocysts were identified in eight, apparently healthy, farmed American mink (Mustela vison). The isolates were identified as Cryptosporidium parvum ‘ferret’ genotype by PCR-RFLP and sequencing analysis of a 341-base-pair fragment of the Cryptosporidium oocyst wall protein (COWP) gene. This is the first report of Cryptosporidium in American mink. Genbank accession numbers of the sequences used in this study C. parvum, ‘bovine’ genotype AF266273; C. hominis, AF266265; C. parvum, ‘mouse’ genotype AF266268, C. wrairi, U35027; C. parvum, ‘ferret’ genotype AF266267; C. meleagridis, AF266266; C. parvum, ‘marsupial’ genotype AF266269; C. parvum, ‘pig’ genotype AF266270; C. canis, AF266274; C. felis, AF266263; C. baileyi, AF266276; C. serpentis, AF266275; C. andersoni, AF266262 and C. muris, AF161579     

Use of a <Emphasis Type="Italic">ura5</Emphasis><Superscript>+</Superscript>–<Emphasis Type="Italic">lys7</Emphasis><Superscript>+</Superscript> cassette to construct unmarked gene knock-ins in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

While the counterselectable Schizosaccharomyces pombe ura4 ⁺ gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4 ⁺ confers 5FOA-resistant (5FOA^R) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA^R mutants. Relative to the same approach using the homologous URA3 ⁺ gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower efficiency of homologous recombination and a relatively high background of spontaneous 5FOA^R colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we first determined that 5FOA^R strains carry mutations in either of two genes; ura4 ⁺ and ura5 ⁺. We then cloned the S. pombe ura5 ⁺ orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5 ⁺ together with the S. pombe lys7 ⁺ gene. Using this doubly marked cassette to disrupt the sck1 ⁺ kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4 ⁻ and ura5 ⁻ mutants by screening 5FOA^R colonies for the loss of the lys7 ⁺ marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that ~95% of 5FOA^R colonies in our studies arose from background ura4 ⁻ and ura5 ⁻ mutations.     

First description of natural <Emphasis Type="Italic">Echinococcus multilocularis</Emphasis> infections in chinchilla (<Emphasis Type="Italic">Chinchilla laniger</Emphasis>) and Prevost’s squirrel (<Emphasis Type="Italic">Callosciurus prevostii borneoensis</Emphasis>)

This report describes for the first time the occurrence of alveolar echinococcosis in two exotic rodent species in Europe. A pet chinchilla (Chinchilla laniger) was euthanized due to a painful enlargement of the abdominal cavity, and a Prevost's squirrel (Callosciurus prevostii borneoensis) was found dead in the enclosure of a zoo. At necropsy, extended liver lesions consisting of small vesicles and cysts were observed in the livers of both animals. Histological examination revealed that these cysts were composed of an outer, homogenous, eosinophilic layer and an inner, cellular germinal layer. The cysts from both animals contained numerous protoscolices. The morphological diagnosis of Echinococcus multilocularis metacestode infections was confirmed by molecular means.     

Expression of the <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis><Emphasis Type="Italic">cefT</Emphasis> gene in <Emphasis Type="Italic">Penicillum chrysogenum</Emphasis> indicates that it encodes an hydrophilic β-lactam transporter

The Acremonium chryrsogenum cefT gene encoding a membrane protein of the major facilitator superfamily implicated in the cephalosporin biosynthesis in A. chrysogenum was introduced into Penicillium chrysogenum Wisconsin 54-1255 (a benzylpenicillin producer), P. chrysogenum npe6 pyrG (-) (a derivative of Wisconsin 54-1255 lacking a functional penDE gene) and P. chrysogenum TA98 (a deacetylcephalosporin producer containing the cefD1, cefD2, cefEF and cefG genes from A. chrysogenum). RT-PCR analysis revealed that the cefT gene was expressed in P. chrysogenum strains. HPLC analysis of the culture broths of the TA98 transformants showed an increase in the secretion of deacetylcephalosporin C and hydrophilic penicillins (isopenicillin N and penicillin N). P. chrysogenum Wisconsin 54-1255 strain transformed with cefT showed increased secretion of the isopenicillin N intermediate and a drastic decrease in the benzylpenicillin production. Southern and northern blot analysis indicated that the untransformed P. chrysogenum strains contain an endogenous gene similar to cefT that may be involved in the well-known secretion of the isopenicillin N intermediate. In summary, the cefT transporter is a hydrophilic beta-lactam transporter that is involved in the secretion of hydrophilic beta-lactams containing alpha-aminoadipic acid side chain (isopenicillin N, penicillin N and deacetylcephalosporin C).     

Suppression of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">rad27</Emphasis> null mutant phenotypes by the 5′ nuclease domain of <Emphasis Type="Italic">Escherichia coli</Emphasis> DNA polymerase I

RNA primer removal from Okazaki fragments during lagging-strand replication and the excision of damaged DNA bases requires the action of structure-specific nucleases, such as the mammalian flap endonuclease 1 (FEN-1). This nuclease contains two conserved motifs enriched with acidic amino acid residues that are important for catalytic function. Similar motifs have been identified in nucleases found in viruses, archebacteria, eubacteria, and in eukaryotes ranging from yeast to humans. Unique among these proteins, the putative FEN-1 homologue in Escherichia coli is contained within the N-terminal region of the DNA polymerase I (PolN). To demonstrate that the cellular functions of FEN-1 reside in PolN, we cloned and expressed the amino terminal domain (323 amino acid residues) of PolI in a Saccharomyces cerevisiae strain lacking the FEN-1 homologue RAD27. Overexpression of PolN suppressed, to varying degrees, phenotypes associated with a rad27 null strain. These include temperature sensitivity, Okazaki fragment processing, a mutator phenotype, a G2/M cell cycle arrest, minichromosome loss, and methyl methane sulfonate sensitivity. We purified Rad27 and PolN proteins in order to determine whether differences in their intrinsic nuclease activities or interaction with proliferating cell nuclear antigen (PCNA) could explain the partial suppression of some phenotypes. We found that the in vitro nuclease activities of Rad27 were more potent than those of PolN and the activity of Rad27, but not PolN, was stimulated by PCNA. We conclude that the N-terminal nuclease domain of E. coli polymerase I encodes a functional homologue of FEN-1.     

Effects of Human Defensin-α<Subscript>1</Subscript> on <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> Trypomastigotes <Emphasis Type="Italic">in Vitro</Emphasis>

Human defensin-α₁ is a biologically active peptide exhibiting a dose-dependent trypanocidal effect in vitro against trypomastigotes and amastigotes of Trypanosoma cruzi line Tulahuen. This effect is determined by fragmentation of parasite DNA reducing the capacity of passaged T. cruzi to invade HeLa cells.     

Impact of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> exposure on <Emphasis Type="Italic">Anopheles dirus</Emphasis>’s fecundity and resistance development

Mosquitoes are important vectors of many infectious diseases. Bacillus sphaericus (Bs) is an ideal larvicide and has attracted more and more attention, recently. However, the fundamental research of its application is very limited, especially on the subsequent impact of Bs exposure on mosquito’s fecundity and resistance emergence. Through bioassay, LC50 and LC95 of Bs in killing Anopheles dirus larvae were determined as 9.793 ± 1.878 IU/L and 62.4 ± 6.438 IU/L at 48 h posttreatment, 7.128 ± 0.913 IU/L and 34.385 ± 12.547 IU/L at 72 h post treatment, respectively. After being treated with a sub-lethal dose of Bs, gravidity, oviposition, hatch, pupation, and eclosion of the surviving mosquitoes were counted and analyzed to elucidate the subsequent effects of Bs exposure on the reproductive capacity of A. dirus. The result interestingly showed that the exposure of Bs significantly reduced the oviposition ability of the surviving A. dirus, without effect on egg formation/gravidity, hatch, pupation, and eclosion. The surviving mosquitoes were also maintained routinely for generations to test the sustained effect of Bs exposure on the fecundity of the offsprings. After conventional breeding for generations, the capacity of egg laying totally recovered. To explore the rules of resistance development, bioassays were performed after treatment twice with a sub-lethal dose of Bs on two continuous generations of A. dirus larvae. The killing efficacies between the Bs treated group and control group were compared. The results showed that LC50 and LC95 increased by 4.35- and 7.37-folds after treatment with the sub-lethal dose of Bs on two consecutive generations, respectively. The results indicated that A. dirus was sensitive to Bs, which could reduce oviposition of the surviving A. dirus. The subsequent effect might help to further decrease the mosquito population. However, a sub-lethal dose of Bs exposure could easily cause resistance development. Our study provides a dose standard and reference for the rational use of Bs, which will be helpful for mosquito control.     

Is <Emphasis Type="Italic">Duhuo Jisheng Tang</Emphasis> containing <Emphasis Type="Italic">Xixin</Emphasis> safe? A four-week safety study

Background  

Though the nephrotoxicity and carcinogenicity of aristolochic acid (AA) are known, its safety in clinical usage is not clear. This study aims to evaluate the safety of Duhuo Jisheng Tang (DJT) in a four-week study to treat osteoarthritis (OA) of the knee.     

<Emphasis Type="Italic">Ex vivo</Emphasis> production of interferon-γ, tumor necrosis factor-α, and interleukin-6 by mouse macrophages during infection with <Emphasis Type="Italic">M. bovis</Emphasis> and <Emphasis Type="Italic">M. tuberculosis</Emphasis> H37Rv

Ex vivo production of IFN-γ, TNF-α, and IL-6 by mouse peritoneal macrophages was studied during successive infection with the vaccine strain M. bovis BCG and virulent strain M. tuberculosis H37Rv. The increase in the concentrations of TNF-α and IL-6 did not depend on the sequence of macrophage infection with the vaccine or virulent strain, but was related to the presence of the vaccine strain M. bovis BCG in the medium. IFN-γ production depended on infection of macrophages with the virulent strain M. tuberculosis H37Rv. The concentration of IFN-γ was maximum during primary infection with the virulent strain and did not increase after successive infection with the virulent and vaccine strain. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 11, pp. 552–555, November, 2007