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PubMed2002

Analysis of the conjugal transfer system of the pheromone-independent highly transferable Enterococcus plasmid pMG1: identification of a tra gene (traA) up-regulated during conjugation.

pMG1 (65.1 kbp) is a pheromone-independent Enterococcus faecium conjugative plasmid conferring gentamicin resistance. pMG1 is able to transfer to enterococci at a high frequency in broth mating experiments, and it does not respond to the sex pheromones which are involved in the high-frequency transfer system of some conjugative plasmids in Enterococcus faecalis. To analyze regulation of tra gene expression in pMG1, transcripts of pMG1 were examined during conjugation. RNA samples were prepared from mating mixtures 20, 40, 80, and 160 min after initiation of mating and were subsequently analyzed by Northern hybridization by using a variety of pMG1 DNA fragments as probes. One transcript of gene 71ORF2 increased to the maximal level 20 min after the start of mating. The level of this transcript decreased after 40 min of mating to the same level as the level in the control donor culture. The increase was not observed in cultures of the donor cells or recipient cells. These findings suggested that the increase was a conjugation-specific event. The 71ORF2 gene of pMG1 was disrupted by using the suicide vector pMG226 carrying an erythromycin resistance gene that is expressed in enterococci. The transfer frequencies of mutant plasmid pMG229, which had a disrupted 71ORF2 gene in pMG1, and the parent plasmid pMG1 were 1.6 x 10(-7) and 1.1 x 10(-3) per donor cell, respectively, in broth mating experiments, and the transfer frequencies of pMG229 and pMG1 were 2.7 x 10(-3) and 8.5 x 10(-2) per donor cell, respectively, in filter mating experiments. This indicated that the transfer frequency of plasmid pMG229 was reduced during broth mating and was not altered during filter mating. 71ORF2, which is designated traA, is a gene involved in the tra gene system for conjugation, and the product of this gene is associated with the formation or stabilization of mating aggregates during broth mating.

Base Sequence↗

PubMed1982

Measurement of transcribed human X-chromosomal DNA sequences transferred to rodent cells by chromosome-mediated gene transfer.

Transfer of genetic information can be effected by incubation of cultured eucaryotic cells with isolated metaphase chromosomes. In most cases, a resulting transformed cell contains only a fragment of a donor chromosome. The amount of transferred donor DNA has been quantified in 11 independent mouse A9 transformants by nucleic acid hybridization analysis. Each transformant had been selected for hprt (hypoxanthine phosphoribosyltransferase; EC 2.4.2.8) transfer and contained part of the human X chromosome. A labeled probe of transcribed human X-chromosomal DNA was prepared by hybridization of nick-translated unique-sequence human DNA with whole cellular RNA from a human-mouse hybrid cell line, A9/HRBC2-A, containing a single human chromosome., X. The amount of human X-chromosomal DNA in the transformants was quantitated by comparing the hybridization of this probe with transformant and A9/HRBC2-A DNAs. Two unstable transformants which had a microscopically detectable donor chromosome fragment contained 15% of the human X-chromosomal single-copy DNA. Four other unstable transformants contained 4 to 7% of human X-chromosomal DNA sequences. The transferred DNA was below the level of detection in three other unstable and in all three stable transformants. We conclude that the initial transfer event can introduce a substantial amount of genetic information but only smaller amounts of DNA are stably incorporated by integration.

Animals↗

PubMed1975

[Caffeine as an inhibitor of the conjugation transfer of R-factors. A study of the quantitative effect of caffeine on the conjugation transfer of R-factors].

The effect of caffeine on the conjugation transfer of R-factors was studied using a standartized kinetic conjugation system described earlier. It was shown that caffeine had a pronounced inhibitory effect on R-factor transfer. The inhibition coefficients at a caffeine concentration of 2000 gamma/ml were from 2.5 (when using strain CSH-2 R222 of E. coli as the donor) to 13.8 (when using strain J5-3 RI drd of E. coli as the donor). Higher concentrations of caffeine induced a significant increase in the coefficient. Since R-transfer inhibition was induced by rather high concentrations of caffeine, the effect of equivalent concentrations of some normal metabolites of the purine and pyrimidine series, such as ATP, guanine, cytosine and thymine was studied. It was shown that the substances had no inhibitory effect on R-factor transfer. Inhibition of P-transfer by caffeine did not depend on either the type of R-factor or the microbial host.

Bacteriological Techniques↗

PubMed1985

[Comparative study of lipid transfer between cell organelles using lipid-transfer proteins in vitro].

The transfer of de novo synthesized lipids from microsomes to lipid non-synthesizing membranes was studied in vivo and in vitro from the ratios of specific radioactivities of [14C]cholesterol, [14C] and [32P]phosphatidylcholine and [32P]phosphatidylethanolamine in the nuclei and mitochondria to that in microsomes. The radioactivity of lipids transferred from microsomes to mitochondria and nuclei was identical both in vitro and in vivo and when the lipid-exchange protein of the 105 000 g supernatant was used. Acceleration of lipid metabolism in the liver of gamma-irradiated rats was concomitant with the increase in the rate of labeled cholesterol transfer cation to liver cell nuclei and mitochondria, but remained unchanged in in vitro studies involving lipid-exchange protein. The reduction of phosphatidylethanolamine transfer to the nuclei in vitro and in vivo diminished in the same way. The existence in the cell of mechanisms of transfer of de novo synthesized cholesterol other than lipid-exchange protein is postulated.

Animals↗

PubMed1994

Efficacy of intravenous plasma to transfer passive immunity in clinically healthy and clinically ill equine neonates with failure of passive transfer.

The efficacy intravenous plasma to transfer passive immunity to clinically healthy colostrum-deprived and clinically ill foals with failure of passive transfer was investigated. Efficacy of transfer was evaluated by the elevation of serum IgG per gram of IgG administered as a function of body weight. Colostrum deprived healthy foals had a significantly greater increase in serum IgG than did clinically ill foals with failure of passive transfer. Knowledge of the IgG content of plasma to be administered and the health status of a foal with failure of passive transfer should allow more accurate prediction of an individual foal's response to treatment.

Animals↗

PubMed1993

Lipid transfer particle in the hemolymph of the American cockroach: evidence for its capacity to transfer hydrocarbons between lipophorin particles.

A lipid transfer particle (LTP) was isolated and purified from the hemolymph of the adult male American cockroach, Periplaneta americana, essentially according to the method previously developed for the purification of LTP from locust hemolymph. Fast protein liquid chromatography (FPLC) on a Mono Q column was used as the additional step to obtain pure LTP. The electron micrograph of purified cockroach LTP exhibited an unusual and asymmetric shape essentially similar to that reported for Manduca sexta LTP (Ryan et al. 1990. J. Lipid Res. 31:871-879). The cockroach LTP was also found to be basically similar to that of M. sexta and locust in terms of subunit structure and lipid composition, although there were significant differences particularly in the contents of hydrocarbons and diacylglycerol. A simple method for assaying LTP activity was developed, based on the finding that cockroach LTP can catalyze the transfer of labeled hydrocarbons (or diacylglycerol) from labeled high density lipophorin (HDLp) bound with a transfer membrane (Immoblion) to unlabeled HDLp dissolved in saline. This finding reveals that cockroach LTP has the capacity to transfer and/or exchange lipids between lipophorin particles with the same density. It was also demonstrated that cockroach LTP has the capacity to catalyze the transfer and/or exchange of hydrocarbons, in addition to diacylglycerol, between cockroach HDLp and locust low density lipophorin (LDLp).

Animals↗

PubMed1993

Nuclear transfer in cattle: effect of nuclear donor cells, cytoplast age, co-culture, and embryo transfer.

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20-22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8-10 hr or 18-20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P > 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18-20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen of IVF embryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology.

Animals↗

PubMed2006

Plasma cholesteryl ester transfer protein mass and phospholipid transfer protein activity are associated with leptin in type 2 diabetes mellitus.

Adipose tissue contributes to plasma levels of lipid transfer proteins and is also the major source of plasma adipokines. We hypothesized that plasma cholesteryl ester transfer protein (CETP) mass, phospholipid transfer protein (PLTP) activity and cholesteryl ester transfer (CET, a measure of CETP action) are determined by adipokine levels. In this study, relationships of plasma CETP mass, PLTP activity and CET with leptin, resistin and adiponectin were analyzed in type 2 diabetic patients and control subjects. Plasma PLTP activity (P<0.001), CET (P<0.001), leptin (P=0.003), resistin (P<0.001), high sensitive C-reactive protein (P=0.005), and insulin resistance (HOMA(ir)) (P<0.001) were higher, whereas HDL cholesterol (P<0.001) and plasma adiponectin (P<0.001) were lower in 83 type 2 diabetic patients (32 females) than in 83 sex-matched control subjects. Multiple linear regression analysis demonstrated that in diabetic patients plasma leptin levels were related to plasma CETP mass (P=0.018) and PLTP activity (P<0.001), but not to the other adipokines measured. Plasma CET was inversely correlated with adiponectin in univariate analysis, but this association disappeared in multivariate models that included plasma lipids and CETP. In conclusion, both plasma CETP mass and PLTP activity are associated with plasma leptin in type 2 diabetes. The elevated CET in these patients is not independently related to any of the measured plasma adipokines.

Blood Proteins↗

PubMed1991

The incidence of multiple pregnancy after in vitro fertilization and embryo transfer, gamete, or zygote intrafallopian transfer.

This retrospective study concerns the incidence of multiple pregnancy after the replacement of three conceptus using different techniques of assisted reproduction. During a 2-year period, 713 in vitro fertilization-embryo transfers (IVF-ETs) with three embryos, 190 gamete intrafallopian transfers (GIFT) with three oocytes, and 161 zygote intrafallopian transfers (ZIFT) with three zygotes were performed. Although we observed significant differences in implantation and pregnancy rates (PRs), the three techniques resulted in high multiple PR. At 20 weeks, 16% of GIFT pregnancies, 27% of ZIFT pregnancies, and 32% of IVF-ET pregnancies were multiple. Therefore we recommend to limit the number of conceptus transferred to a maximum of three in all cases.

Adult↗

PubMed2004

Type 2 diabetes mellitus is associated with differential effects on plasma cholesteryl ester transfer protein and phospholipid transfer protein activities and concentrations.

BACKGROUND: Human plasma contains two lipid transfer proteins, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), which are crucial in reverse cholesterol transport. METHODS: Plasma CETP and PLTP activity levels and concentrations in 16 type 2 diabetic patients and 16 matched healthy subjects were determined, and these data were correlated to clinical variables, including insulin sensitivity and lipid levels. RESULTS: Plasma triglycerides were higher (p<0.02) and high-density lipoprotein (HDL) cholesterol (p<0.02) was lower in diabetic patients. Plasma CETP activity and concentrations were not significantly different between diabetic and healthy subjects, but CETP specific activity was lower in diabetic patients (p<0.001). Multiple regression analysis showed that plasma CETP activity was positively related to CETP concentration (p=0.0001) and negatively to the diabetic state (p<0.002) or to HbA1c (p<0.02). PLTP activity (p<0.05) and specific activity were higher (p<0.05), whereas there was no difference in PLTP concentration between the two groups. There was no significant bivariate correlation between PLTP concentration and activity, in either healthy or diabetic subjects. Multiple regression analysis did disclose positive relationships of PLTP activity with PLTP concentration (p=0.0001), plasma triglycerides (p=0.0001) and waist/hip ratio (p=0.0001), but not with the diabetic state or HbA1c. CONCLUSIONS: Neither CETP nor PLTP activity was independently associated with insulin sensitivity. Specific CETP activity is decreased in type 2 diabetes mellitus. In contrast, specific PLTP activity is higher in diabetes, as a result of the association of plasma PLTP activity with plasma triglycerides and obesity. Measurement of both plasma lipid transfer protein activity and mass levels may thus provide extra information in diabetes mellitus.

Apolipoproteins↗

PubMed1992

Transvaginal gamete intra-fallopian transfer and embryo intra-fallopian transfer.

This study describes the conduct and results of a recently developed technique for transvaginal catheterization of the Fallopian tube in order to transfer gametes or early embryos. Transvaginal gamete intra-Fallopian transfer (TV-GIFT) was performed in 46 patients after stimulation with human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) and transvaginal oocyte retrieval. This resulted in 11 (23.9%) pregnancies. Eight patients delivered healthy children, including one set of twins. Two patients had abortions at 8 and 11 weeks of gestation and one had an ectopic pregnancy. In a first series of 11 women, oocytes were fertilized in vitro and a maximum of three embryos at the 2- to 8-cell stages were transferred into one tube. Three of the 11 cycles with tubal embryo-stage transfer' (TV-TEST) resulted in clinical pregnancy.

Catheterization↗

PubMed1990

Assisted fertilization in infertile women with patent fallopian tubes. A comparison of in-vitro fertilization, gamete intra-fallopian transfer and tubal embryo stage transfer.

A comparison of the relative efficacy of in-vitro fertilization with uterine embryo transfer (IVF), tubal embryo stage transfer (TEST) and gamete intra-Fallopian transfer (GIFT) was performed in infertile patients with patent Fallopian tubes. A total of 150 couples with unexplained infertility, peritoneal endometriosis or reduced semen quality were included in the study. The three groups were comparable with regard to age distribution, indications, semen parameters, stimulation regimens, response to stimulation and numbers of oocytes retrieved. In the IVF and TEST groups there was no cleavage in 24% and a cleavage rate of only 47.6%. The highest cleavage rate was obtained in the endometriosis patients. The pregnancy rate was highest in the two groups in which in-vitro fertilization was performed, IVF = 45.7%, TEST = 37.9%, GIFT = 26.2%. To obtain one live intrauterine fetus, more oocytes had to be transferred in the GIFT group compared to the number of embryos in the IVF group, 14.4 versus 6.2, P less than 0.05. Due to a high success rate of IVF but at the same time a high frequency of no cleavage in cases of unexplained infertility or male subfertility, we recommend IVF as the primary procedure in infertile couples with patent Fallopian tubes.

Adult↗

PubMed1994

The fluid dynamics of injection: variables as they relate to transvaginal gamete intra-fallopian transfer and tubal embryo transfer.

Transvaginal gamete intra-Fallopian transfer (GIFT) and tubal embryo transfer have not, in most cases, produced pregnancy rates as high as the equivalent transabdominal methods. We postulated that two parameters, i.e. wide dispersion of the gametes and peritoneal spill of gametes or embryos, are of prime importance in explaining this difference. We designed this study to analyse the effect of varying the rate of injection (microliter/min) of radio-opaque contrast media and the distance the transfer catheter is placed into the tubal isthmus upon these two parameters. Selective salpingography was performed on 30 patients who were allocated to one of two experiments. In the first experiment (flow rate), 20 patients were divided into four groups so that we could analyse four sets of 10 Fallopian tubes. Each group was subjected to injection of contrast at different flow rates: 100, 50, 25 or 12.5 microliters/min. Peritoneal spill occurred from 4, 2, 0 and 0 tubes and wide dispersion (> 4 cm) of contrast occurred in 6, 5, 1 and 0 tubes at the respective injection rates of 100, 50, 25 and 12.5 microliters/min. At 12.5 microliters/min contrast was localized to < 2 cm of tubal ampulla in all cases. In the second experiment (catheter placement), the remaining 10 patients were divided to allow analysis of two groups of 10 Fallopian tubes. The transfer catheter was placed either 1.5 cm or 5 cm into the tube and the radio-opaque contrast was injected at 12.5 microliters/min. No difference in the degree of dispersion of contrast along the Fallopian tube was observed. No backflow of contrast into the uterine cavity was observed in any patient following removal of the catheter.(ABSTRACT TRUNCATED AT 250 WORDS)

Biophysical Phenomena↗

PubMed1994

Stage-specific appearance of the mouse antigen TEC-3 in normal and nuclear transfer bovine embryos: re-expression after nuclear transfer.

Bovine embryos, recovered from the uterus in vivo or derived from in vitro matured and in vitro fertilized oocytes, were investigated for the presence of the developmentally regulated mouse antigen TEC-3 by indirect immunofluorescence. During preimplantation embryo development TEC-3 is expressed on bovine morulae and blastocysts. It is absent from unfertilized and fertilized oocytes, and from all stages before the 32-cell stage. The finding that TEC-3 is not expressed before the onset of embryonic transcription, which occurs at the eight-cell stage in the bovine, but only when the embryonic genome is active, makes it a potential marker for studying nuclear reprogramming after nuclear transfer. Nuclear transfer embryos were made by electrical fusion of blastomeres from morulae derived in vivo with enucleated metaphase II oocytes. Indirect immunofluorescence with the TEC-03 antibody showed that the TEC-3 antigen, present on blastomeres of the morula stage embryo, disappeared after fusion and was expressed again when the nuclear transfer embryos developed to the morula and blastocyst stage. These data suggest that the bovine embryonic nucleus may be able to revert to the equivalent of an earlier developmental stage when transferred to ooplasm, and is then capable of following the normal developmental program.

Animals↗

PubMed1991

Comparison of unilateral and bilateral tubal transfer in gamete intrafallopian transfer (GIFT).

Gamete intrafallopian transfer (GIFT) is traditionally performed by delivering gametes into the ampullary region of either one or two fallopian tubes. The choice is made by the surgeon at the time of laparoscopy based upon the patient's anatomy, the number of oocytes available, and clinical judgment. In this nonrandomized, retrospective review, 399 tubal gamete transfers were performed over a period of 18 months, 133 to a single tube and 266 to both tubes. A clinical pregnancy rate of approximately 24% was observed in each modality. The multiple pregnancy rate of 31.3% for one tube was not significantly different from the 25% seen for two tubes. Unilateral tubal transfer offers the distinct advantages of less gamete and tissue handling. This, along with the apparent same outcome parameters, makes unilateral tubal transfer the preferred method of returning gametes at GIFT.

Fallopian Tubes↗

PubMed1994

Relationship between the day of embryo transfer and the outcome in human in vitro fertilization and embryo transfer.

PURPOSE: To clarify the optimal date of embryo transfer (ET), we retrospectively analyzed the relationship between the day of ET and the outcome in human in vitro fertilization and embryo transfer (IVF-ET). METHOD: Of a total of 307 human IVF-ET cycles performed at Kyoto University Hospital between January 1990 and March 1994, we focused on 207 cases of IVF-ET cycles in which two or three good-quality embryos were transferred. These 207 IVF-ET cycles consisted of 54 Day 2 ET cycles, 79 Day 3 ET cycles, 46 Day 4 ET cycles, and 28 Day 5 ET cycles. We compared the pregnancy and live-birth (plus ongoing pregnancy) rates among these four ET groups. RESULTS: The pregnancy rates of ET on Days 2 to 4 were not significantly different, whereas Day 5 ET produced a significantly lower pregnancy rate (Day 2, 29.6%; Day 3, 32.9%; Day 4, 30.4%; Day 5, 10.7%). Similar results were obtained for the live-birth (plus ongoing pregnancy) rates (Day 2, 20.3%; Day 3, 18.9%; Day 4, 17.9%; Day 5, 7.1%). CONCLUSIONS: These results suggest that the day of ET does not fundamentally affect the pregnancy rate in human IVF-ET provided that transfer is made before Day 5.

Embryo Transfer↗

PubMed2004

[German Database of Gene Transfer Clinical Trials. Implementation of a central registry of gene transfer clinical trials in Germany].

Continuous biotechnical development and rapid increase in knowledge in the field of research on human gene transfer raise scientific, medical, and ethical considerations in the scientific community and general public. Dissemination of new scientific findings is needed. Gene transfer studies today not only focus on monogenic diseases or life-threatening conditions such as cancer, but also comprise trials for diagnosis and prophylaxis and common medical conditions such as cardiovascular disease. This raises special attention and public discussion and requires even more the systematic monitoring and assessment of clinical trails being conducted. Whereas the number of gene transfer trials in the USA can be clearly specified, this had not been possible in Germany prior to the establishment of the DeReG database. The aim is implementation of a permanent registry comprehending all clinical gene transfer trials being conducted in Germany. Only a complete and updated database serves as a common foundation of knowledge for various groups and facilitates addressing scientific projects.

Clinical Trials as Topic↗

PubMed1993

Unidirectional transfer in vivo of high-density lipoprotein cholesteryl esters to lower-density lipoproteins in the pig, an animal species without plasma cholesteryl ester transfer activity.

The metabolism of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesteryl esters (CE) was studied in the pig, an animal species without plasma cholesteryl ester transfer activity (CETA). In the first series of experiments, LDL and HDL from normocholesterolemic pigs were radiolabeled with cholesteryl (1-14C)oleate and intravenously administered to two groups of four normocholesterolemic pigs. Radioactive tracer in LDL remained associated with the LDL fraction, and there was no transfer of LDL-CE to HDL. The transport rate (which represents the production and disposal rate) of LDL-CE in normocholesterolemic pigs was 39 mumol CE/h/L. However, radiolabeled HDL-CE were transferred to LDL (25%), and 36% of the LDL-CE mass was derived from the HDL. The transport rate of HDL-CE was 54 mumol CE/h/L, and the flux of HDL-CE to LDL was 14 mumol CE/h/L. There was no accumulation of radiolabeled HDL-CE in very-low-density lipoprotein (VLDL), which suggests that there was no transfer to VLDL. However, this does not rule out the possibility that either the very low levels of VLDL-CE (< 0.09 mmol/L) or the rapid turnover rate of the VLDL pool might have prevented the accumulation of substantial amounts of tracer in VLDL. Therefore, in a second set of experiments, the kinetics of HDL-CE were studied in high-fat-and high-cholesterol-fed pigs with elevated VLDL-CE concentrations (1.92 mmol/L). Hypercholesterolemia was associated with increased transport rates of LDL-CE (165 mumol/h/L) and HDL-CE (78 mumol/h/L) and with an increased flux of HDL-CE to LDL (78 mumol/h/L).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗