Expression of Wild-Type Rp1 Protein in Rp1 Knock-in Mice Rescues the Retinal Degeneration Phenotype

Mutations in the retinitis pigmentosa 1 (RP1) gene are a common cause of autosomal dominant retinitis pigmentosa (adRP), and have also been found to cause autosomal recessive RP (arRP) in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39) are located in the 4(th) and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3(rd) exon of RP1 (c.686delC; p.P229QfsX35) found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled.     

Charakterisierung des Rhodopseudomonas palustris-Bacteriophagen Rp 1

Zusammenfassung RP 1-Phagen sind wenig stabil. Durch Chloroform werden sie rasch inaktiviert, während entsprechende Konzentrationen an Äther ohne Titerverlust toleriert werden; pH-Werte, kleiner als 5 und größer als 8,5 führen zur Inaktivierung, ebenso Temperaturen über 30°C. Die temperaturabhängige Inaktivierungskinetik ist bei 46°C bi-phasisch. Der Phage hat eine Latenzzeit von 90 bis 100 min, eine rise period von 2 Std und eine Wurfgröße von 10–12.Anhand von Ultradünnschnitten konnte gezeigt werden, daß die intracelluläre Phagenentwicklung im peripheren Cytoplasma abläuft. Durch Fluorescenzfärbung und Einbau von ³H-Thymidin konnte 2-DNS als Phagennucleinsäure bestimmt werden.

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Characterization of the Rhodopseudomonas palustris-bacteriophage Rp 1

Summary Isolation and enrichment of the Rp1-bacteriophage of Rhodopseudomonas palustris, strain 1 e5, resulted in a yield of 10 to 20% of the total amount of phage particles in the lysate and a titer of about 10⁹ pfu/ml. The high lost of phage particles may be due to an unspecific adsorption to the membranes of the bacterial cell. The bacteriophage Rp 1 is quickly inactivated at a pH lower than 5 and higher than 8.5 and a temperature higher than 30°C. The inactivation curve at 46°C shows two phases. The phage is rapidly inactivated by chloroform but not by ether. The one-step-growth curve shows a latency period of 90 to 100 min, a rise period of 2 h, and a burst size of 10 to 12 pfu/cell. The development of Rp1 was demonstrated to be in the peripheral region of the cytoplasm. By fluorescence staining and incorporation of ³H-thymidine the nucleic acid of Rp 1 was found to be a double-stranded DNA. The phage is tentatively put into the group C of Bradley.

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Abkürzungen Rps. Rhodopseudomonas - pfu plaque forming units     

Evaluation of chemopreventive agents for genotoxic activity

We conducted genetic toxicity evaluations of 11 candidate chemopreventive agents with the potential for inhibiting carcinogenesis in humans at increased risk of cancer. The compounds were evaluated for bacterial mutagenesis in the Salmonella-E. coli assay, for mammalian mutagenesis in mouse lymphoma cells, for chromosome aberrations in Chinese Hamster Ovary (CHO) cells, and for micronucleus induction in mouse bone marrow. Tested agents were indole 3-carbinol (I3C), bowman-birk inhibitor concentrate (BBIC), black tea polyphenols (BTP), farnesol, geraniol, l-Se-methylselenocysteine (SeMC), 5,6-dihydro-4H-cyclopenta[1,2]-dithiol-3-thione(DC-D3T), 4'-bromoflavone, 2,5,7,8-tetramethyl-(2R-[4R,8R,12-trimethyltridecyl] chroman-6-yloxy) acetic acid (alpha-TEA), SR13668 (2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo[2,3-b] carbazole and SR16157 (3-O-sulfamoyloxy-7alpha-methyl-21-(2-N,N-diethylaminoethoxy)-19-norpregna-1,3,5(10)-triene). All these agents, except I3C and BTP, were negative in the Salmonella-E. coli assay in the presence and absence of metabolic activation (S9). I3C and BTP induced a weak mutagenic response in the presence and absence of S9 with strains TA100 and TA98, respectively. Of the three compounds tested in the mouse lymphoma assay (I3C, BBIC, and BTP), only BTP was mutagenic in the presence of S9. In the chromosomal aberration assay, of the 8 compounds that were tested, 4'-bromoflavone elicited a positive response in the absence of S9 only, while SR16157 was positive in the presence of S9. The results with geraniol remain inconclusive. I3C, BBIC and BTP were not tested in the chromosomal aberration assay. None of the 11 agents induced micronuclei in mouse bone marrow erythrocytes.     

Recombination at the Rp1 locus of maize.

Summary The Rp1 locus of maize determines resistance to races of the maize rust fungus (Puccinia sorghi). Restriction fragment length polymorphism markers that closely flank Rp1 were mapped and used to study the genetic fine structure and role of recombination in the instability of this locus. Susceptible progeny, lacking the resistance of either parent, were obtained from test cross progeny of several Rp1 heterozygotes. These susceptible progeny usually had non-parental genotypes at flanking marker loci, thereby verifying their recombinational origin. Seven of eight Rp1 alleles (or genes) studied were clustered within about 0.2 map units of each other. Rpl ^G, however, mapped from 1–3 map units distal to other Rp1 alleles. Rp5 also mapped distally to most Rp1 alleles. Other aspects of recombination at Rp1 suggested that some alleles carry duplicated sequences, that mispairing can occur, and that unequal crossing-over may be a common phenomenon in this region; susceptible progeny from an Rp1 ^A homozygote had recombinant flanking marker genotypes, and susceptible progeny from an Rp1 ^DlRp1 ^F heterozygote showed both possible nonparental flanking marker genotypes.     

Recombination events generating a novel Rp1 race specificity

Genes at the maize Rp1 rust resistance complex often mispair in meiosis, which allows genes to recombine unequally, creating recombinant haplotypes. Four recombinant haplotypes were identified from progeny of an Rp1-D/Rp1-I heterozygote that conferred a nonparental resistance specificity designated Rp1-I*. Sequence comparisons of paralogs in the recombinant and parental haplotypes demonstrated that all four recombinants were derived from intergenic (between gene) recombination events. The sequence of paralogs in the HRp1-I parental haplotype indicated this haplotype includes 41 or more rp1 genes, at least 31 of which are transcribed. The results indicate that most of the novel resistance specificities that have arisen spontaneously at Rp1 are the result of reassort ment of existing Rp1 genes.     

Recombinant Rp1 genes confer necrotic or nonspecific resistance phenotypes

Genes at the Rp1 rust resistance locus of maize confer race-specific resistance to the common rust fungus Puccinia sorghi. Three variant genes with nonspecific effects (HRp1 -Kr1N, -D*21 and -MD*19) were found to be generated by intragenic crossing over within the LRR region. The LRR region of most NBS-LRR encoding genes is quite variable and codes for one of the regions in resistance gene proteins that controls specificity. Sequence comparisons demonstrated that the Rp1-Kr1N recombinant gene was identical to the N-terminus of the rp1-kp2 gene and C-terminus of another gene from its HRp1-K grandparent. The Rp1-D*21 recombinant gene consists of the N-terminus of the rp1-dp2 gene and C-terminus of the Rp1-D gene from the parental haplotype. Similarly, a recombinant gene from the Rp1-MD*19 haplotype has the N-terminus of an rp1 gene from the HRp1-M parent and C-terminus of the rp1-D19 gene from the HRp1-D parent. The recombinant Rp1 -Kr1N, -D*21 and -MD*19 genes activated defense responses in the absence of their AVR proteins triggering HR (hypersensitive response) in the absence of the pathogen. The results indicate that the frequent intragenic recombination events that occur in the Rp1 gene cluster not only recombine the genes into novel haplotypes, but also create genes with nonspecific effects. Some of these may contribute to nonspecific quantitative resistance but others have severe consequences for the fitness of the plant.     

Conversion of Major Ginsenoside Rb1 to Ginsenoside F2 by Caulobacter leidyia

Ginsenoside Rb₁ is the most predominant ginsenoside in Panax species (ginseng) and the hydrolysis of this ginsenoside produces pharmaceutically active compounds. Caulobacter leidyia GP45, one of the isolates having strong β-glucosidase-producing activity, converted ginsenoside Rb₁ to the active metabolites by 91%. The structures of the resultant metabolites were identified by NMR. Ginsenoside Rb₁ had been consecutively converted to ginsenoside Rd (1), F₂ (2) and compound K (3) via the hydrolyses of 20-C β-(1→6)-glucoside, 3-C β-(1→2)-glucoside, and 3-C β-glucose of ginsenoside Rb₁.     

Untersuchungen zur Adsorption des Bacteriophagen Rp 1 an Rhodopseudomonas palustris 1 e 5

Zusammenfassung Adsorptionskinetiken mit verschiedenen Rhodopseudomonas palustris-Stämmen ergaben, daß Rp 1-Phagen nur an Stamm 1 e 5 adsorbieren. Als Adsorptionskonstante wurde ein Wert von K=3,8·10^-10 ml/min ermittelt. Während des Wachstums der Wirtsbakterien werden die Rp 1-Phagen nur in der Teilungsebene und an einem Zellpol adsorbiert. Die Phagen adsorbieren auch an den intracytoplasmatischen Membranen. Die Spezifität dieser Adsorption wird diskutiert.

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Investigations on the adsorption of the bacteriophage Rp1 to Rhodopseudomonas palustris 1 e 5

Summary The adsorption of the bacteriophage Rp 1 is restricted to cells of the strain 1 e 5 of Rhodopseudomonas palustris. The adsorption constant was found to be K=3.8·10^-10 ml/min. During the growth phase of the host bacterium Rp 1 is only adsorbed to one of the cell poles and the division plain of the cells. In addition, the phage is adsorbed to the intracytoplasmic membranes. The specifity of this adsorption is discussed.

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Abkürzungen pfu plaque forming units - Rps. Rhodopseudomonas - R. Rhodospirillum     

Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway

Context: Ginsenoside Rb1 improves insulin sensitivity and glucose uptake in muscle cells via different signaling pathways; however, it is not clear that it has any effect on leptin signaling in skeletal muscle.

Objectives: The aim of this study was to investigate the effect of ginsenoside Rb1 on leptin receptors expression and main signaling pathways of leptin (STAT3, PI3 kinase and ERK kinase) in C2C12 skeletal muscle cells.

Materials and methods: C2C12 myotubes were incubated with various concentrations of Rb1 (0.1, 1 and 10?μM) for different incubation times (1–12?h). Leptin receptors expression and GLUT-4 translocation were analyzed using realtime PCR and western blot analyses, respectively. PI3 and ERK kinases were blocked using their specific inhibitors (wortmannin and PD98059) in the presence and absence of RB1 to determine the main signaling pathway related to leptin receptor activation in C2C12 cells.

Results: Rb1 could maximally stimulate both leptin receptors (OBRa and OBRb) mRNA and protein expression and phosphorylation of STAT3, PI3K and ERK2 in C2C12 myotubes at 10?μM for 3?h. Rb1 induced GLUT4 translocation was inhibited by the silencing of OBRb mRNA, demonstrated that glucose uptake was mediated via leptin receptor activation. GLUT4 recruitment to the cell surface induced by Rb1 was inhibited by wortmannin, an inhibitor of PI3K in combination with OBRb siRNA, but not by PD98059 an ERK2 kinase-1 inhibitor, indicating that GLUT4 translocation induced by Rb1 was associated with the leptin receptor upregulation and subsequent activation of PI3K.

Conclusions: Our results suggest that Rb1 promote translocation of GLUT4 by upregulation of leptin receptors and activation of PI3K.     

Recombination between paralogues at the Rp1 rust resistance locus in maize

Rp1 is a complex rust resistance locus of maize. The HRp1-D haplotype is composed of Rp1-D and eight paralogues, seven of which also code for predicted nucleotide binding site-leucine rich repeat (NBS-LRR) proteins similar to the Rp1-D gene. The paralogues are polymorphic (DNA identities 91-97%), especially in the C-terminal LRR domain. The remaining family member encodes a truncated protein that has no LRR domain. Seven of the nine family members, including the truncated gene, are transcribed. Sequence comparisons between paralogues provide evidence for past recombination events between paralogues and diversifying selection, particularly in the C-terminal half of the LRR domain. Variants selected for complete or partial loss of Rp1-D resistance can be explained by unequal crossing over that occurred mostly within coding regions. The Rp1-D gene is altered or lost in all variants, the recombination breakpoints occur throughout the genes, and most recombinant events (9/14 examined) involved the same untranscribed paralogue with the Rp1-D gene. One recombinant with a complete LRR from Rp1-D, but the amino-terminal portion from another homologue, conferred the Rp1-D specificity but with a reduced level of resistance.     

Activity of Plasmid Replicons in CAULOBACTER CRESCENTUS : Rp4 and Cole1

The RP4 replicon was detected as covalently-closed circular DNA in Caulobacter crescentus strains into which it had been transferred from Escherichia coli. RP4-mediated transfer of ColE1-associated markers into C. crescentus occurred, but only as the result of transposon-mediated events. Both transposition of a ColE1-associated marker onto RP4 and cointegration of ColE1 with RP4 were observed. Chimeric plasmids containing both a ColE1 and an RP4 origin of replication were stably maintained in C. crescentus , but similar plasmids lacking the RP4 origin of replication were not stably maintained in C. crescentus. Thus we show that the ColE1 replicon cannot be maintained in C. crescentus unless it is covalently linked to another replicon, such as RK2, that can be maintained.     

从西洋参中提取分离纯化人参皂苷R_(b1)和人参皂苷R_e的研究

用大孔吸附树脂从西洋参提取物中富集西洋参总皂苷,再利用丙酮沉淀及重结晶方法获得高纯度的人参皂苷Rb1和Re.可以得到含量大于95%的人参皂苷Rb1(收率2.6%)和92%的人参皂苷Re(收率0.5%),以及纯度为98.5%的人参皂苷Rb1(收率2.0%)和97.8%的人参皂苷Re(收率0.25%).该方法简便、实用,适用于工业大生产,为进一步开发成新药奠定了基础.     

Unequal Exchange and Meiotic Instability of Disease-Resistance Genes in the Rp1 Region of Maize

The Rp1 region of maize was originally characterized as a complex locus which conditions resistance to the fungus Puccinia sorghi, the causal organism in the common rust disease. Some alleles of Rp1 are meiotically unstable, but the mechanism of instability is not known. We have studied the role of recombination in meiotic instability in maize lines homozygous for either Rp1-J or Rp1-G. Test cross progenies derived from a line that was homozygous for Rp1-J, but heterozygous at flanking markers, were screened for susceptible individuals. Five susceptible individuals were derived from 9772 progeny. All five had nonparental combinations of flanking markers; three had one combination of recombinant flanking markers while the other two had the opposite pair. In an identical study with Rp1-G, 20 susceptible seedlings were detected out of 5874 test cross progeny. Nineteen of these were associated with flanking marker exchange, 11 and 8 of each recombinant marker combination. Our results indicate that unequal exchange is the primary mechanism of meiotic instability of Rp1-J and Rp1-G.     

Evaluation of sphinganine and sphingosine as human breast cancer chemotherapeutic and chemopreventive agents

No comparative study of the effects of sphingolipid metabolites on proliferation and differentiation in normal human breast epithelial cells versus stem cells and tumorigenic cells has been reported. The purpose of this study was to evaluate the chemotherapeutic and chemopreventive potential of sphingoid bases (sphingosine and sphinganine) using a novel cell culture system of normal human breast epithelial cells (HBEC) developed from breast tissues of healthy women obtained during reduction mammoplasty (Type I HBEC with stem cell characteristics and Type II HBEC with basal epithelial cell phenotypes) and transformed tumorigenic Type I HBEC. The results show that sphinganine inhibited the growth and induced apoptosis of transformed tumorigenic Type I HBEC more potently than sphingosine (IC(50) for sphinganine 4 microM; sphingosine 6.4 microM). Both sphinganine and sphingosine at high concentrations (8-10 lM) arrested the cell cycle at G(2)/M. Sphinganine inhibited the growth and caused death of Type I HBEC more strongly than sphingosine. In comparison, Type II HBEC (normal differentiated cells) were less sensitive to the growth-inhibitory effects of sphingoid bases than Type I HBEC (stem cells) or transformed tumorigenic Type I HBEC, suggesting that sphingoid bases may serve as chemotherapeutic agents. At concentrations (0.05, 0.1, and 0.5 microM) that are below the growth-inhibitory range, sphingoid bases induced differentiation of Type I HBEC to Type II HBEC, as detected morphologically and via expression of a tumor suppressor protein, maspin, which is a marker of Type II HBEC. Thus, sphingoid bases may function as chemotherapeutic as well as chemopreventive agents by preferentially inhibiting cancer cells and eliminating stem cells from which most breast cancer cells arise.     

人参皂甙Rb1对大鼠脑缺血再灌注神经损伤后的修复作用

通过阻塞大鼠大脑动脉,制备短暂性脑缺血大鼠模型,将出现神经功能缺失症状的大鼠随机分组,实施再灌注后立即按(40mg/kg)进行腹腔注射人参皂甙Rb1。结果发现大鼠脑缺血再灌注后,人参皂甙Rb1通过促进NAIP、Bcl-2表达和抑制Bax表达发挥神经损伤后的修复作用。人参皂甙Rb1给药组在各时间点的胶质细胞源性神经营养因子(GDNF)阳性细胞数远远高于单纯脑缺血再灌注组(P<0.01)。GDNF的表达与缺血性损伤有一定的联系,可认为人参皂甙Rbl对神经系统有一定保护作用。