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At least 19 recordsLinked to original sources
PubMed2025

Identification of Plant Chromatin Interaction Networks Using IP-MS and co-IP.

Proteins often act in concert to perform their function. Thus, the identification of protein complexes is crucial if we want to understand how they work. In this chapter, we present a highly sensitive protocol for the immunoprecipitation of nuclear chromatin-linked proteins in Arabidopsis thaliana that does not rely on time-consuming nuclei extraction. Interaction partners are identified using mass spectrometry and confirmed by co-immunoprecipitation. To help solubilize chromatin-bound proteins and eliminate nonspecific interactions of proteins binding the same DNA stretch, we include an enzymatic digestion step to remove DNA before immunoprecipitation. Our protocol offers a simplified process using optimized buffers, which facilitates quick and effective immunoprecipitation. The outcome is high-quality eluates that are ideal for identifying proteins through MS.

Chromatin↗
PubMed1979

Chemotherapy for murine ovarian cancer: a rationale for ip therapy with adriamycin.

The metastatic spread of a trnasplantable murine ovarian cancer is similar to the spread of ovarian cancer in patients with advanced disease, making it a useful model to investigate novel experimental therapies. The ip inoculation of 10(6) tumor cells into C3HeB/FeJ mice leads to the formation of ascites, sub-diaphragmatic tumor deposits, intra-abdominal tumors, and death within 25 days. Adriamycin (ADR) was found to be an active agent against this murine ovarian cancer. The effects of ADR were dependent upon the route of administration. A single ip LD10 dose of ADR (5 mg/kg) administered 2 days after inoculation with 10(6) tumor cells produced long-term survival (greater than 60 days) in 70% of the mice. An iv LD10 dose had no effect on survival. The survival advantage of ip ADR (compared to the iv route) was found to be related to: (a) a greater suppression of DNA synthesis in the tumor; (b) a rapid penetration of ADR into the nuclei of ascites tumor cells and into sub-diaphragmatic tumor deposits; and (c) significantly higher levels of ADR in tumor cells following ip administration. The ip route may also be less cardiotoxic since the peak levels after an iv dose were three times greater than after an equal ip dose. If local toxicity does not prove to be a major problem, then ip ADR may be a useful mode of therapy in patients with intra-abdominal tumors.

Animals↗
PubMed1975

Synthesis and inducibility of the Uterine estrogen-induced protein, IP, during the rat estrous cycle: clues to uterine estrogen sensitivity.

Analysis of newly labeled soluble proteins in uteri of mature rats throughout the estrous cycle indicates that the relative rate of synthesis of the uterine estrogen-induced protein, IP, is high at proestrus, when endogenous estrogen secretion is maximal; it is not synthesized at detectable levels at estrus and metestrus; and some minimal synthesis is seen in diestrus uteri. Injection of exogenous estrogen results in some increase in the IP synthesis rate at proestrus; slight induction of IP synthesis at estrus; and maximal induction (as great as that induced in the mature, ovariectomized uterus by estrogen) at metestrus and diestrus. Studies in the immature (day 21-24) rat, aimed at determining the possible causes of the uterine recalcitrance to exogenous estrogen seen at estrus, indicate that one can reproduce in the immature uterus a period of feeble responsiveness to a second injection of estrogen after exposure to a first, high dose of estrogen. After a single injection of estrogen, there is a lag between the gradual return of full IP inducibility (requiring over 40 hours to reach the control level) and the return of nuclear-translocatable receptor (at control level by 24 hr). This suggests that in addition to the presence of nuclear-translocatable receptor, the response to a second injection of estrogen is dependent upon other factors whose replenishment and/or reactivation is slower than that of the receptor.

Age Factors↗
PubMed1976

Androgen-uterine interaction: nuclear translocation of the estrogen receptor and induction of the synthesis of the uterine-induced protein (IP) by high concentrations of androgens in vitro but not in vivo.

High concentrations of androgens in vitro [10(-6) and 10(-7)M 5alpha-dihydrotestosterone (DHT) and testosterone (T)] translocate the estrogen receptor from cytoplasmic to the nuclear fraction of the immature rat uterus, and the androgen translocated sites are capable of eliciting the synthesis of the specific uterine "induced protein" (IP), formerly attributed to estrogenic compounds only. The magnitude of receptor translocation and IP synthesis induction is related to the concentration of androgen, and, at equal concentrations, DHT is more effective than T. Competitive protein-binding asssays with cell-free uterine cytosol indicate that DHT and T bind with barely detectable affinity to the cytosol estrogen receptor detectable affinity to the cytosol estrogen receptor [relative binding ability ca. 0.001% that of estradiol (E2)], and radioactive E2 uptake into whole uterus after pretreatment with androgens indicates that the androgen-translocated nuclear sites are readily filled by E2. DHT and T translocate the estrogen receptor to the nucleus in vitro, and the salt-extracted nuclear receptor is present as a 5 S ("transformed") receptor; however, concentrations of DHT and T that effect translocation are unable to elicit the heat-activated transformation of the estrogen receptor in cell free cytosol under conditions in which low concentrations of E2 fully transform the receptor. Under in vivo conditions, high levels of androgens (5--2,000 mug DHT or T) do not evoke any detectable translocation of the estrogen receptor and do not elicit any IP synthesis induction although uterine weight is increased (greater than 200 mug androgen) in long-term (2--3 day) assays. The analysis of androgen uptake and metabolism indicates that 500 mug or higher doses of DHT or T in vivo result in uterine concentrations of unmetabolized DHT or T equal to those seen after exposure to 5 X 10(-7) - 1 X 10(-6)M DHT or T in vitro. Hence, under conditions where in vivo and in vitro tissue uptake of androgen is equivalent, in vivo androgens are unable to affect the estrogen receptor system as is seen in vitro. These studies indicate that the in vitro and in vivo effects of androgens on the uterus are clearly different and suggest that the actions of androgens on the uterus in vivo are probably not directly mediated through the estrogen receptor system.

Animals↗
PubMed1978

Behavior of axenic IP-106 strain of Entamoeba histolytica in the golden hamster.

Strain IP-106 of Entamoeba histolytica was isolated 12 yr ago from a case of amebic dysentery and has been maintained in axenic culture for the past 11 yr. Hamsters were inoculated intrahepatically, intraperitoneally, and intracecally with 6.5, 5.0, and 8.0 X 10(4) axenic trophozoites, respectively. With the first two routes of inoculation all the animals developed liver abscesses, and most developed amebic metastases to other sites. After intracecal inoculation only 3 out of 6 animals developed abscesses at the primary site and metastases to other sites. Most numerous metastases were obtained following intrahepatic inoculation. Intrahepatic inoculation with 1 and 2 X 10(4) axenic amebae resulted in an increase in the size of the liver abscess for a period of 2 wk and its apparent total resorption by day 17 post-inoculation, accompanied by a corresponding gain of body weight. With 4 and 6 X 10(4) amebae there was a steady increase of the size of the liver abscess and corresponding loss of body weight. High antiamebic indirect hemagglutination titers were observed only on day 17 post infection in animals inoculated with 2, 4, and 6 X 10(4) amebae. The extent of amebic metastases was roughly proportional to the size of the inoculum.

Animals↗
PubMed2026

Generation of isogenic gene-corrected cell lines from a USH2A-RP patient-derived iPS cell line.

Comparative studies using induced pluripotent stem cells (iPSCs) from patients with those from healthy individuals as controls are flawed by genetic background contribution to disease phenotype. Here, we used precise gene editing to generate gene-corrected isogenic control lines for a single pathogenic variant in the USH2A gene (c.2276G > T) associated with retinitis pigmentosa (RP). Both homozygously and heterozygously corrected cell lines were successfully generated. These cell lines will serve to unravel RP phenotype differences specific to the USH2A mutation upon their conversion into disease relevant cell types.

Journal Article↗
PubMed1976

Control of inositol biosynthesis in Saccharomyces cerevisiae; inositol-phosphate synthetase mutants.

Inositol-requiring mutants of Saacharomyces cerevisiae were tested in cell extracts for the ability to convert glucose-6-phosphate to inositol-phosphate (IP synthetase) and inositol (IP phosphatase). Mutants representing any one of 10 unlinked loci conferring the inositol requirement were unable to synthesize either compound in an assay with glucose-6-phosphate as the substrate. These results indicate that the mutants lack IP synthetase activity and that at least 10 genes control the conversion of glucose-6-phosphate to inositol-phosphate. In addition, a mutation known to be unlinked with the ino1 locus interacts with a leaky ino1 allele and may play a role in the regulation of IP synthetase. This mutation causes a 47% reduction in wild-type IP synthetase activity and, when combined in a haploid strain with the leaky ino1 allele, it reduced IP synthetase activity to a level below that which is growth supporting. Wild-type and IP synthetase-deficient strains were tested for reduced nicotinamide adenine dinucleotide (NADH) accumulation, since NAD+ is required in the conversion of glucose-6-phosphate to inositol. No detectable accumulation of NADH was observed in the wild-type strain, presumably because the NADH generated is rapidly oxidized during subsequent partial reactions of IP synthetase. Mutants representing three different loci accumulate NADH and may, therefore, lack the NADH-mediated reductase activity of IP synthetase. Other mutants tested fail to accumulate NADH and may, therefore, lack the NAD+-mediated oxidase activity of IP synthetase. Phospholipid synthesis was studied by 32P pulse labeling in one mutant under conditions of inositol supplementation and starvation. Starved cells incorporate 32P into phospholipids normally for 2 h, followed by a period in which the rate of phosphatidylinositol synthesis decreases and the rate of phosphatidylcholine synthesis increases. After 5 to 6 h starvation, all cellular phospholipid synthesis ceases.

Carbohydrate Epimerases↗
PubMed1975

Androgen induction of a specific uterine protein.

Studies were conducted to determine the ability of androgens in vitro to elicit the induction of a specific uterine protein (IP) normally attributed to estrogens. Both 5alpha-dihydrotestosterone (5alpha-DHT) and testosterone were effective in stimulating IP synthesis in rat uterine tissue in a concentration dependent manner (0.1 muM to 50 muM). 5alpha-DHT was more effective than testosterone and reached approximately 85% of the estradiol stimulated IP response at 10 muM and 50 muM; whereas testosterone was only able to achieve about a 70% IP response at 50 muM. This androgen stimulated IP synthesis was stereospecific since cis-testosterone and 5beta-DHT, inactive androgen isomers, were unable to evoke a detectable IP response at any concentration studied. Antiandrogens were unable to inhibit the 5alpha-DHT stimulated IP synthesis but antiestrogens were able to greatly inhibit the 5alpha-DHT and testosterone stimulated IP responses in a concentration dependent manner. The anti-estrogens, themselves, were very weak inducers of the IP response. The nuclear accumulation of the estrogen receptor by various androgens and inactive androgen isomers was also determined. Approximately 100% nuclear accumulation of receptor was attained with 1 muM 5alpha-DHT, whereas 50 muM testosterone was needed for 100% uptake. 5beta-DHT was unable to translocate the receptor at the lower concentrations tested, but caused a significant nuclear accumulation of 50 muM. Cis-testosterone was unable to cause the nuclear accumulation of the estrogen receptor at all concentrations studied. These studies suggest that some of the estrogen receptors accumulated in the nuclei by androgens, inactive androgen isomers, or antiestrogens may not be capable of eliciting a biological response.

Animals↗
PubMed1979

Toxicity of Secalonic acid D.

Toxicity of secalonic acid D was examined by using lethality, growth retardation, and histopathology as indexes. The ip LD50 values of 37, 31, and 27 mg/kg were obtained for Charles River CD-1, Texas (ICR), and Sprague-Dawley (CF-1) strains of mice, respectively. The ip LD50 was 52 mg/kg in female CD-1 mice. The iv LD50 was 25 mg/kg in CD-1 male mice. Oral LD50 values of 400 mg/kg in male CD-1 mice and 25 and greater than 400 mg/kg in Sprague-Dawley day-old and weanling (21 d) rats of both sexes, respectively, were obtained. Doses of 20 mg/kg or more ip retarded growth and doses of 30 mg/kg or more ip were lethal to CD-1 mice. Oral doses required to produce such effects in day-old rats were 5 and 20 mg/kg (or higher), respectively. All ip doses of secalonic acid D caused pulmonary atelectases and foccal peritonitis in male CD-1 mice. The latter involved surfaces of abdominal viscera and produced limited subcapsular necrosis of hepatic parenchyma. Exposure to a single lethal dose iv (25 mg/kg or more) of secalonic acid D caused limited hepatic portal necrosis but no peritonitis or other associated local effects observed in CD-1 male mice after ip exposure. Cytoplasmic liposis and loss of glycogen and RNA from hepatocytes were observed in a single mouse receiving 50 mg/kg iv. Death resulting from cardiac and/or pulmonary insufficiency was suggested by atelectasis, pulmonary hemorrhages and edema, and massive atrial dilation in mice that died after lethal ip or iv doses of secalonic acid D. Five daily sublethal ip doses in CD-1 male mice resulted in dose-dependent mortality (LD50, 11.5 mg/kg) indicating cumulative effects.

Animals↗
PubMed1979

Comparison between the action of estradiol and that of the antiestrogen U 11-100 A on the induction in the rat uterus of a specific protein (the induced protein).

When measured by an in vitro approach, involving incubation in the presence of labeled leucine, the rate of synthesis of the specific estrogen-induced protein (IP) after in vivo stimulation of the rat uterus by the antiestrogens U 11-100 A (UA; 1-(2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphtyl)-phenoxy]ethyl)pyrrolidine hydrochloride) or CI-628 (alpha-[4-pyrrolidinoethyoxy]phenyl-4-methoxy-d-nitrostilbene) was very low compared to the response measured under the same conditions after in vivo stimulation with 17 beta-estradiol (E2). In the course of investigations aimed at clarifying the role of IP in estrogen action, we have conducted similar experiments, but the labeling step aimed at detecting and measuring IP synthesis was carried out in vivo. We have observed that UA promoted a full IP response which is lost or missed in incubated uteri. Similar results were obtained with CI-628 and tamoxifen. A comparison between IP responses obtained and measured in vivo after E2 and those after UA action revealed that the responses paralleled the number of receptors that are translocated to the nucleus by each compound. Thus, while transient after E2 treatment, the IP (or IP-like) response was maintained for as long as 12 h (as is the nuclear receptor occupancy) when UA was used as inducer. Several explanations for the disappearance of the UA-induced IP response under in vitro conditions are considered.

Animals↗
PubMed1977

Lipid hemodialysis versus charcoal hemoperfusion in imipramine poisoning.

Previous experimental results have demonstrated the possibility of eliminating imipramine (14 C-IP) by hemodialysis. A simultaneous uptake of the substance by the polyvinyl chloride extracorporeal blood lines could be shown. Based on these results the imipramine absorption capacity of the blood lines and of the artificial kidney (Hollow Fiber Artificial Kidney, HFAK, Model 4) were studied. Imipramine (IP) absorption capacity of a usual blood-line set (arterial and venous, surface area 86,000 mm) was estimated to be 43 mg, and that of the HFAK to be 207 mg. Charcoal hemoperfusion (300 gm of coated activated coconut charcoal) eliminated more than 90% of IP from the blood within 3 hr if the initial IP blood concentration was 2 mg/ml. In comparison, lipid hemodialysis using 20% soybean oil as dialysate eliminated 95% of IP from the blood when the initial IP blood concentration was 1 mg/ml and 98% when the initial IP blood concentration was 2 mg/ml. In vivo studies on the elimination of IP by lipid (10%) hemodialysis demonstrated a substantial removal of the substance. Within 2 hr of treatment, 12% of the administered dose (75% of the LD50) was eliminated.

Adsorption↗
PubMedDate not supplied

Taurine, analogues and ethanol elicited responses.

The effect of taurine, of some of its precursors and major metabolic products on spontaneous locomotor activity were studied in mice. The effect of taurine and some analogues on certain ethanol-mediated responses were observed. Administration of taurine, 50 mg/kg, IP, did not significantly alter motility in experimental animals compared to controls. Behavioral depression was evident subsequent to injection of cysteine hydrochloride or taurocholic acid (50 mg/kg). Administration of taurocholic acid, 50 mg/kg, IP, 30 min prior to a narcotic dose of ethanol, 5 g/kg, IP, reduced the time required for the onset of ethanol-narcosis. Pretreatment with cysteic acid, 50 mg/kg, IP, prolonged ethanol-produced narcosis. Treatment with cysteic acid 30 min prior to ethanol, 2.5 g/kg, IP, was found to decrease whole blood ethanol concentration as compared to the respective controls without a concomitant changes in brain ethanol levels. Administration of taurocholic acid, 100 mg/kg, IP, decreased the intake of an ethanol solution in rats preferring 5% ethanol solution over water as the drinking fluid of choice. None of the compounds tested altered endogenous specific activity of mouse liver alcohol dehydrogenase when given once daily (50 mg/kg, IP) for 10 consecutive days. The results suggest that both taurocholic acid and cysteic acid exert additive action to some ethanol-elicited responses studied.

Alcohol Drinking↗
PubMed1978

Comparison of immunofluorescence and immunoperoxidase staining for identification of rubella virus isolates.

To explore possible advantages which immunoperoxidase (IP) staining might have over immunofluorescence (IF) staining for identifying rubella virus isolates, direct comparative studies were done on the same coded clinical materials using the same rubella immune rabbit serum as the primary antiserum in both systems. The rubella immune rabbit serum and conjugated anti-rabbit immune globulins could be used more dilute in the IP system than in the IF system. Both IP and IF staining detected rubella antigen in all specimens which were positive by interference. IP staining also detected low levels of rubella antigen in a few additional specimens which had originally been positive for rubella virus, but which on retesting were negative by interference and IF staining. With second-cell-culture-passage material, IP and IF staining showed comparable specificity, and the few specimens which reacted nonspecifically generally did so in both systems. Cell cultures inoculated directly with urine specimens showed greater nonspecificity by IP than by IF, but this activity could be abolished by pretreatment with sodium azide and peroxide; other methods tried for inactivating endogenous peroxidase activity destroyed rubella antigen as well. The intensity of staining for positive specimens was comparable in the two systems. However, more antigen was demonstrable in both systems when BHK-21 cells were inoculated as a cell suspension and then permitted to grow into monolayers than when the same specimens were inoculated into preformed monolayers. IP staining was considered to be a highly satisfactory alternative to IF staining for identification of rubella virus isolates.

Animals↗