Coevolution of Cryptosporidium tyzzeri and the house mouse (Mus musculus)

Two house mouse subspecies occur in Europe, eastern and northern Mus musculus musculus (Mmm) and western and southern Mus musculus domesticus (Mmd). A secondary hybrid zone occurs where their ranges meet, running from Scandinavia to the Black Sea. In this paper, we tested a hypothesis that the apicomplexan protozoan species Cryptosporidium tyzzeri has coevolved with the house mouse. More specifically, we assessed to what extent the evolution of this parasite mirrors divergence of the two subspecies. In order to test this hypothesis, we analysed sequence variation at five genes (ssrRNA, Cryptosporidium oocyst wall protein (COWP), thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1), actin and gp60) in C. tyzzeri isolates from Mmd and Mmm sampled along a transect across the hybrid zone from the Czech Republic to Germany. Mmd samples were supplemented with mice from New Zealand. We found two distinct isolates of C. tyzzeri, each occurring exclusively in one of the mouse subspecies (C. tyzzeri-Mmm and C. tyzzeri-Mmd). In addition to genetic differentiation, oocysts of the C. tyzzeri-Mmd subtype (mean: 4.24 × 3.69 μm) were significantly smaller than oocysts of C. tyzzeri-Mmm (mean: 4.49 × 3.90 μm). Mmm and Mmd were susceptible to experimental infection with both C. tyzzeri subtypes; however, the subtypes were not infective for the rodent species Meriones unguiculatus, Mastomys coucha, Apodemus flavicollis or Cavia porcellus. Overall, our results support the hypothesis that C. tyzzeri is coevolving with Mmm and Mmd.     

复合不育剂EP-1对小鼠空间记忆与焦虑行为的影响

EP-1是由炔雌醚和左炔诺孕酮按照1∶2的比例配制而成的一种用于鼠类不育控制的激素类复合不育剂,对鼠类的繁殖及繁殖行为等有一定影响,但对鼠类空间学习与记忆、焦虑行为等非繁殖行为的影响还没有报道。为此,用0(对照)、1.0、2.0、3.0 mg/kg剂量的EP-1对昆明小鼠(Mus musculus)进行灌胃处理,然后用Morris水迷宫和高架十字迷宫分别测定其空间记忆、焦虑行为。结果发现灌胃后15 d,剂量为2.0 mg/kg的EP-1使小鼠空间记忆能力显著下降,但30 d后其空间记忆能力有所恢复,表明2.0 mg/kg剂量的EP-1可以在一定时间范围内降低小鼠的空间记忆能力。但不同剂量的EP-1对小鼠焦虑行为无显著影响。该结果可以为从对非繁殖行为的影响的角度研究EP-1对鼠类的作用提供一定启示。     

Linkage of the locus for conversion of albumin (Acf-1)in the house mouse,Mus musculus

The linkage of the locus for conversion of albumin (Acf-1) has been established on chromosome 1 with the following gene order and recombination percentages: Id-1 19.3±5.2% Acf-1 4.2±1.7% Dip-1 18.4±4.2% Lp.This work was supported by NIH Postdoctoral Fellowship 1F32 GM0527701, Grant BMS75-03397 from the National Science Foundation, Grant ACS VC-17-R from the American Cancer Society, and Contract NO1-ES42159 from the National Institute of Environmental Health Sciences. The Jackson Laboratory is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.     

Oestrous females investigate the unfamiliar male more than the familiar male in both commensal and non-commensal populations of house mice

We studied female preferences for familiar and unfamiliar males. The subjects were laboratory-born house mice: (1) non-commensal Mus musculus domesticus from the eastern part of Syria along the Euphrates River; and (2) commensal M. m. musculus from the Czech Republic. Pair-choice preference tests have revealed that oestrous females of both populations sniffed towards unfamiliar males more than familiar males. In the case of females exhibiting postpartum oestrus, this preference was less pronounced and statistically not significant. Thus, our mice clearly exhibited the behavioural pattern known from commensal populations of polygynous and/or promiscuous M. m. domesticus. We found no inverse tendency to seek proximity to the familiar male that has been previously reported from closely related and presumably monogamous aboriginal mouse Mus spicilegus. We conclude that neither commensal M. m. musculus, nor non-commensal M. m. domesticus, are likely to share a monogamous mating system with mound-building mice.     

Morphometric variation in nearly unstudied populations of the most studied mammal: The non-commensal house mouse (Mus musculus domesticus) in the Near East and Northern Africa

The phenotypic consequences in the house mouse (Mus musculus domesticus) of the transition from an ordinary field-dwelling rodent to a species that is dependent on human populations was studied by investigating the morphometric variation of non-commensal populations of M. m. domesticus from Syria, Jordan, SW Iran, and Libya and comparing them with that of conspecific commensal populations from Eastern Turkey, Greece, and Bulgaria. Commensal populations of M. musculus musculus from the Czech Republic were used as an outgroup. About 849 adult specimens of M. musculus were analysed by multivariate procedures based on standard molar, skull and body measurements. As expected, there was considerable variation among the studied populations and a good correspondence between morphometric and geographic distances. The resulting morphometric tree was consistent with the hypothesis that the original radiation of M. m. domesticus took place somewhere in the Near East. Commensal populations of M. m. domesticus form a single derived branch. Specimens originating from four different sites in eastern Syria showed the greatest similarity to one another and possessed relatively bigger molars than the other studied populations. Commensal populations were characterised by longer tails when compared to non-commensal populations, which suggests an adaptation for living in a more three-dimensionally heterogeneous environment for commensal populations.     

Chromosomal location of soluble glutamic-pyruvic transaminase-1 (Gpt-1) in the mouse

Three alleles at the Gpt-1 (glutamic-pyruvic transaminase-1) locus in the mouse, as identified by electrophoresis on cellulose acetate, and their distribution among inbred mouse strains and wild stocks are described. The Gpt-1 locus was shown to control the soluble form of the enzyme. Three-point linkage analysis established the location of Gpt-1 on chromosome 15 between uw and bt. In addition, a new staining procedure is described that allows the visualization of GPT activity on gels by the deposition of formazan. This is an improvement over previous methods that produced bands of nonfluorescence against a fluorescent background.This investigation was supported in part by Research Grant GM 20919 from the National Institute of General Medical Sciences, and by contract NO1-ES-4-2159 with the National Institute of Environmental Health Sciences. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals

Seed oil from castor bean (Ricinus communis) contains high amounts of hydroxy fatty acid rich triacylglycerols (TAGs) that can serve as raw material for production of bio-based products such as nylon, cosmetics, lubricants, foams, and surfactants. Diacylglycerol acyltransferase (DGAT) catalyses the terminal reaction in the acyl-CoA dependent Kennedy pathway of triglyceride biosynthesis. There is still some debate whether there are three or four enzymes in yeast that have DGAT activity and catalyse the synthesis of TAG but of these the DGAT2 homologue Dga1 contributes in a major way to TAG biosynthesis. Here we report on the cloning of a cDNA for DGAT2 from castor bean and prove its biological activity following expression in yeast and enzymatic assays using diricinolein as the acceptor and ricinoleoyl-CoA as the donor. Previous reports of DGAT in castor have focussed on DGAT1 which has little amino acid sequence homology to DGAT2. Expressional studies demonstrate that DGAT2 is 18-fold more highly expressed in seeds than in leaves and shows temporal specific expression during seed development. In contrast, DGAT1 shows little difference in expression in seeds versus leaves. We conclude that in castor bean DGAT2 is more likely to play a major role in seed TAG biosynthesis than DGAT1.     

Hoechst 33258 binds to G-quadruplex in the promoter region of human c-myc

In vitro binding of Hoechst 33258 to the promoter region of human c-myc, d(GG GGAGGG TGG GGA GGG TGG GGA AGG TGG GG) which forms G-quadruplex, both in vitro and in vivo in the presence of metal ions, was investigated by equilibrium absorption, fluorescence, and kinetic surface plasmon resonance methods. Hypochromic effect in UV absorption spectra and blue shift in fluorescence emission maxima of Hoechst in the presence of quadruplex revealed that Hoechst binds to the quadruplex. Analysis of UV and fluorescence titration data revealed that Hoechst binds to quadruplex with binding affinity of the order of 10(6). Anisotropy measurements and higher lifetime obtained from time-resolved decay experiments revealed that quadruplex-bound Hoechst is rotationally restricted in a less polar environment than the bulk buffer medium. From surface plasmon resonance studies, we obtained kinetic association (k(a)) and dissociation (k(d)) of 1.23+/-0.04 x 10(5)M(-1)s(-1) and 0.686+/-0.009 s(-1), respectively. As Hoechst is known to bind A-T-rich region of duplex DNA, here we propose the likelihood of Hoechst interacting with the AAGGT loop of the quadruplex.     

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.     

En1 and Wnt7a interact with Dkk1 during limb development in the mouse

Wnt signaling plays an essential role in induction and development of the limb. Missing digits are one consequence of the reduced Wnt signaling in Wnt7a null mice, while extra digits result from excess Wnt signaling in mice null for the Wnt antagonist Dkk1. The extra digits and expanded apical ectodermal ridge (AER) of Dkk1-deficient mice closely resemble En1 null mice. To evaluate the in vivo interaction between En1 and the canonical Wnt signaling pathway, we generated double and triple mutants combining the hypomorphic doubleridge allele of Dkk1 with null alleles of En1 and Wnt7a. Reducing Dkk1 expression in Dkk1d/+Wnt7a-/- double mutants prevented digit loss, indicating that Wnt7a acts through the canonical pathway during limb development. Reducing Dkk1 levels in Dkk1d/dEn1-/- double mutants resulted in severe phenotypes not seen in either single mutant, including fused bones in the autopod, extensive defects of the zeugopod, and loss of the ischial bone. The subsequent elimination of Wnt7a in Dkk1d/dEn1-/-Wnt7a-/- triple mutants resulted in correction of most, but not all, of these defects. The failure of Wnt7a inactivation to completely correct the limb defects of Dkk1d/dEn1-/- double mutants indicates that Wnt7a is not the only gene regulated by En1 during development of the mouse limb.     

Cloning and functional analysis of two type 1 diacylglycerol acyltransferases from Vernonia galamensis

Vernonia galamensis accumulates vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid) as the major fatty acid in its seed oil. Such epoxy fatty acids are useful in a number of industrial applications. Successful genetic engineering of commercial oilseed crops to produce high levels of vernolic acid depends on a better understanding of the source plant enzymes for vernolic acid accumulation. Developing V. galamensis seed microsome assays demonstrate that diacylglycerol acyltransferase (DGAT), an enzyme for the final step of triacylglycerol synthesis, has a strong substrate preference for vernolic acid bearing substrates including acyl-CoA and diacylglycerol. There are two classes of DGATs known as DGAT1 and DGAT2. Here we report on the isolation, characterization, and functional analysis of two DGAT1 cDNAs from V. galamensis (VgDGAT1a and VgDGAT1b). VgDGAT1a and VgDGAT1b are expressed in all plant tissues examined with highest expression in developing seeds. Enzymatic assays using isolated microsomes from transformed yeast show that VgDGAT1a and VgDGAT1b have the same DGAT activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-dioleoylglycerol are preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. This data indicates that the two VgDGAT1s are functional, but not likely to be responsible for the selective accumulation of vernolic acid in V. galamensis seed oil.     

Penetration and maturation of Schistosoma mansoni in suckling and adult Swiss Webster and DBA/2 mice

Murine schistosomosis is a widely used experimental model of the human disease. Different methods have been employed to infect mice with Schistosoma mansoni cercariae, such as subcutaneous or intraperitoneal injections, the tail immersion technique, and the use of a metal ring placed on the abdominal skin of anaesthetized animals. An alternative method of infection that requires no anaesthesia and no restraint is to place suckling mice (10 days old) on a Petri dish with a small volume of water containing cercariae. In this study, we compared the penetration and maturation of S. mansoni in mice infected by this alternative method with those noted in mice infected at an older age (45 days) by the tail immersion technique. Besides evaluating the effects of age and method of infection, we also compared the susceptibility of two strains of mice (Swiss Webster (SW) and DBA/2). Mice were exposed to 100 cercariae and worms (by portal-hepatic perfusion) as well as eggs were recovered in the liver and intestines on postinfection (PI) days 35, 55 and 90. Skin penetration was very efficient (about 100%) irrespective of the mouse strain, sex, age and method of infection. Worm and egg recoveries were higher in SW mice at any PI interval, but strain differences tended to be less pronounced on PI day 90. In both strains, recoveries of worms and eggs were clearly higher in mice infected at a younger age (10 days old). This study thus suggests that infection of free-moving suckling mice is a suitable alternative to other methods of infection with S. mansoni.     

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.