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At least 217 records · Page 12Linked to original sources
PubMed1991

Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro.

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10 micrograms follicle-stimulating hormone (FSH)/ml and 10 micrograms luteinizing hormone (LH)/ml, 1 microgram oestradiol/ml, and 1 x 10(6) granulosa cells at 39 degrees C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100 micrograms/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P less than 0.01), development to blastocysts (P less than 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P less than 0.001). FCS was superior to HS for development to blastocysts (P less than 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P less than 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P less than 0.01). In the presence of cells, ova preferred FCS, in their absence, serum had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Role of platelet-derived growth factor in development of in vitro matured and in vitro fertilized bovine embryos.

This experiment was designed to determine whether the stimulatory effects of bovine oviductal epithelial cells (BOEC) on development of early bovine embryos are due to platelet-derived growth factor (PDGF). Four hundred and twenty five 8-cell bovine embryos derived from in vitro maturation and in vitro fertilization procedures were equally and randomly allotted to one of the following culture treatment groups: control medium alone (Menezo's B2 medium; MB2), MB2 with 1 ng PDGF ml-1 (PDGF), 1 ng PDGF ml-1 and 10 micrograms anti-PDGF antibody ml-1 (PDGF + Ab), BOEC or BOEC and 10 micrograms anti-PDGF antibody ml-1 (BOEC + Ab). All embryos were cultured in 100 microliters of serum-free MB2 medium supplemented with 2 mg fatty-acid-free bovine serum albumin ml-1. Embryos for all treatment groups were incubated at 39 degrees C and 5% CO2 in humidified air in groups of five embryos per well in 96-well culture plates until 7 days after in vitro insemination. A higher proportion of embryos developed to > 8-cell and to the morula stage following culture with PDGF, BOEC or BOEC+Ab than with MB2 alone. Incubation of PDGF and BOEC-treated embryos with anti-PDGF reduced development to the morula and blastocyst stages. However, anti-PDGF did not completely inhibit blastocyst development when added to BOEC. In addition, embryos incubated with BOEC and anti-PDGF contained a reduced number of inner cell mass cells compared with embryos incubated with BOEC alone. These results indicate that PDGF provides a developmental stimulus similar to BOEC for bovine embryos at the fourth cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro.

This study was carried out to determine the effects of beta-mercaptoethanol (bME) during a transient co-culture of gametes for 10 min, and/or the following culture until 6-9 h after insemination, on sperm penetration of porcine in vitro maturation (IVM) oocytes and the early development in vitro. When fresh spermatozoa were cultured in various concentrations of bME for 2 h, bME neutralized the stimulatory effect of caffeine-benzoate on sperm capacitation and the spontaneous acrosome reaction at 50-250 micromol/l. When 50 micromol/l bME were added during a transient co-culture of gametes for 10 min, the sperm penetration rate was reduced 9 h after insemination (70.5-82.0% vs 90.5-94.0% in the absence of bME), but the incidence of monospermic penetration was not affected. When 50 micromol/l bME were supplemented during culture after a transient co-culture, the sperm penetration rate was not affected, but the incidence of monospermy oocytes was increased (43.9-45.8% vs 31.7-34.3% in the absence of bME). The presence of bME following a transient co-culture minimized a decrease of oocyte glutathione content at 6 h after insemination (7.9 pmol/oocyte before in vitro fertilization (IVF), 6.7 pmol/oocyte in the presence of bME vs 5.5 pmol/oocyte in the absence of bME). When the distribution of cortical granules was evaluated 1 h after activation with calcium ionophore, mean pixel intensity of fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) at the cortex region was lower in the oocytes activated and cultured in the presence of 50 micromol/l bME. Although the presence of 50 micromol/l bME during a transient co-culture for 10 min and the following culture did not increased blastocyst formation (29.6-37.7%), 50 micromol/l bME during the following culture significantly increased the mean cell numbers per blastocyst (73.3-76.4 vs 51.2 in the presence and absence of bME respectively). These results demonstrate that supplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.

Animals↗
PubMed1983

In vitro fertilization of bovine oocytes by spermatozoa capacitated in vitro.

In vitro fertilization of ovulated bovine oocytes was attempted with fresh or frozen semen after capacitation in vitro. Sperm incubations and oocyte cultures were performed in a Krebs Ringer bicarbonate medium containing Na pyruvate, glucose and .3% bovine serum albumin. High ionic strength (HIS) medium was prepared by adding NaCl to provide an osmolarity of 370 to 380 nOsmol/kg. Donor cows were treated with prostaglandin F2 alpha and a series of follicle-stimulating hormone injections and ovulated oocytes were recovered 72 h after prostaglandin treatment. Fresh or frozen semen was: 1) placed directly into a microdrop of standard medium (SM) under oil; 2) washed by centrifugation with SM and placed in a microdrop of SM or 3) pretreated with HIS for 10 min, washed and placed into a microdrop of SM. In all cases, spermatozoa were preincubated for 3 h at a concentration of approximately 10(6) cells/ml before addition of oocytes. Oocytes were incubated with spermatozoa for 6 h, transferred to fresh medium and cultured for 24 h. When spermatozoa were placed directly into a microdrop, three of 34 (9%) oocytes were penetrated, but none divided. Spermatozoa washed with SM penetrated 20 of 45 (44%) oocytes and three (7%) divided. Spermatozoa incubated in HIS penetrated 14 of 47 (30%) oocytes and five (11%) divided. The washing of spermatozoa with standard medium was equally as effective as incubation with high ionic strength medium in inducing in vitro capacitation of bovine spermatozoa.

Animals↗
PubMed1989

In vitro fertilization and development of in vitro matured bovine follicular oocytes.

Bovine follicular oocytes matured in vitro were fertilized in vitro using epididymal spermatozoa from five different bulls and then cultured to the blastocyst stage in vitro. The fertilization rate, based on one pair of pronuclei and presence of one sperm tail, ranged from 55.2 to 64.3%. Embryo development (cleavage to blastocyst stage) ranged from 21.4 to 31.0% of the cultured ova reaching 8 cells at 3 to 4 d after insemination to 1.3 to 3.7% reaching hatched blastocysts at 9 to 10 d. It is concluded that individual variation among bulls is not a significant factor in fertilization and development rates of bovine follicular oocytes when epididymal spermatozoa are used.

Animals↗
PubMedDate not supplied

[In vitro fertilization of bovine oocytes in in-vitro protein-free culture system].

We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts.

Animals↗
PubMed1991

Birth of the first 100 babies in Hawaii after conception in-vitro: experience at the Pacific In-Vitro Fertilization Institute.

This paper reports on the first 100 infants delivered consecutively the result of 63 pregnancies after conception in-vitro at the Pacific In-Vitro Fertilization (IVF) Institute. These pregnancies were established prior to the introduction of embryo-cryopreservation into the program. Ninety-seven percent (97%) of singleton pregnancies delivered after 36 weeks; only 5/31 (16%) required tocolytic therapy. The overall multiple gestation rate was 51%, higher than in many other IVF centers. Maternal complications and neonatal morbidity were associated primarily with these multiple gestations. Forty-one percent (41%) delivered in less than 36 weeks, and tocolytics were administered in 22/27 (81%) of twin and 5/5 (100%) of triplet gestations. Neonatal morbidity was noted in 39 infants (33 from multiple gestations); 28 neonates were admitted to the intensive care unit (27 from multiple gestations). Nine births had or developed minor abnormalities (hernias, pyloric stenosis). One case of Trisomy 21, and one infant death at 11 months occurred in the group. The caesarean section rate for all IVF deliveries was 65%. It is concluded that IVF is clearly established in Hawaii for infertility refractory to other methods of treatment. The risk associated with IVF stem from the possibility of multiple gestations, not whether the pregnancy was initiated in-vitro. In the future, embryo cryopreservation may help to reduce the risk of multiple pregnancy.

Adult↗
PubMed1988

In-vitro fertilization pregnancies in Australia and New Zealand, 1979-1985. Australian In-Vitro Fertilization Collaborative Group.

We report here data on 1510 pregnancies that resulted from in-vitro fertilization in 12 in-vitro fertilization units in Australia and in one unit in New Zealand in the period 1979 to 1985. Live births occurred in 57.5% of all pregnancies (including preclinical abortions) and in 69.0% of clinical pregnancies. There was a high incidence of ectopic pregnancies (5.2% of clinical pregnancies) and spontaneous abortions (24.3% of intrauterine pregnancies). Of 902 viable pregnancies, 202 (22.4%) pregnancies resulted in multiple births. There were 700 infants who were born after singleton births and 438 infants who were born after multiple births. The over-all incidence of preterm delivery was 27.0%, with an incidence of 18.5% of singleton pregnancies. Low birth weight occurred in 34.8% of infants. The perinatal death rate was 47.5 deaths per 1000 births; still births accounted for three-quarters of these deaths. These findings can be used to counsel infertile couples about the risks of reproductive losses after treatment by in-vitro fertilization. National health surveys should include questions that relate to the prevalence of infertility to encourage studies of the prevention of infertility and to assess the requirements for clinical services.

Adult↗
PubMed1994

Influence of in vitro methods, receptor fluids on percutaneous absorption and validation of a novel in vitro method.

In vitro experiment using excised skin has been valuable for studying the mechanism of percutaneous absorption. Based on previously established static diffusion cell system in this laboratory, a novel model-perfused glass diffusion cell system is designed. The results of initial comparative study on percutaneous absorption between glass perfused diffusion cell and static diffusion cell, in vitro and in vivo permeation as well as factors affecting permeation with seven radiolabelled chemicals are presented. The results demonstrate that the perfused diffusion cell system, which used a perfusion fluid below the surface of skin to take up the materials which penetrated the skin, is more similar to physiologic condition, convenient and automatic than that of the static cell. It well predicts the in vivo percutaneous absorption if appropriate receptor fluid is chosen. The results also show that the selection of receptor fluid is critical for in vitro permeation of chemicals with different solubility.

Animals↗
PubMed1997

Genistein inhibits growth of B16 melanoma cells in vivo and in vitro and promotes differentiation in vitro.

Consumption of soy products has been linked to a reduced mortality and morbidity from a number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumour cell lines in vitro; however, a role of genistein in retarding tumour growth in vivo is less well documented. In this study, in addition to examining the effects of genistein on the growth of murine B16 melanoma cells in vitro, we have examined the effects of feeding a genistein-rich diet on s.c. growth of these tumour cells in mice. In vitro, the melanoma cells showed an increase in sensitivity to genistein with increasing time of exposure, culminating in a 50% growth inhibition (IC50) at 12.5 microM after 7 days. Genistein at 25 microM induced micronucleus formation after 24 hr and at concentrations as low as 2.5 microM induced morphological changes indicative of differentiation. Growth of solid tumours implanted into female C57BL/6J mice was inhibited by 50% when mice were fed genistein for 1 week before and for 1 week after inoculation with B16 melanoma cells. Plasma genistein concentrations at the time of tumour removal were 1.1 microM, which is similar to levels reported in humans consuming diets high in soybeans or soybean products, while control animals had no detectable genistein in plasma. Our results provide additional in vivo evidence suggesting that genistein retards the growth of implanted tumours, adding further to studies suggesting that this isoflavonoid is a biologically active component of soy foods.

Animals↗
PubMed1993

Analysis of the six additional chemicals for in vitro assays of the European Economic Communities' EEC aneuploidy programme using Saccharomyces cerevisiae D61.M and the in vitro porcine brain tubulin assembly assay.

We tested six additional chemicals (acetaldehyde, benomyl, diethylstilboestrol, diethylstilboestrol dipropionate, griseofulvin, and mercaptoethanol) for in vitro systems of the coordinated programme to study aneuploidy induction sponsored by the Commission of the European Communities in two in vitro test systems. Using Saccharomyces cerevisiae D61.M (mitotic chromosomal malsegregation assay), benomyl showed a dose-dependent increase in the frequency of chromosomal malsegregation with a lowest effective dose tested (LEDT) of 30 micrograms/ml (0.1 mM). Diethylstilboestrol (DES) showed solvent-dependent effects. DES dissolved in ethanol induced an increase in chromosomal malsegregation as well as in the frequency of total resistant colonies (mutations and recombinations) with a LEDT around 13 micrograms/ml (0.048 mM). Using dimethylsulfoxide as the solvent, no increases were observed with DES up to 333 micrograms/ml (1.24 mM). Acetaldehyde induced an increase in chromosomal malsegregation with the cold treatment protocol (LEDT: 1.25 microliters/ml (21 mM) and 0.75 microliters/ml (13 mM), respectively) but no increase with the overnight protocol (highest dose tested (HDT): 1.75 microliters/ml; 30 mM). Concerning the frequency of total cycloheximide-resistant colonies (mutations and recombinations) increases were obtained with both protocols. The other three compounds were negative when tested up to toxic doses (survival below 10%), up to the maximum solubility in the solvent used or up to heavy precipitation in the incubation mix. The HDT were 333 micrograms/ml (0.88 mM) for diethylstilboestrol dipropionate, 1,600 micrograms/ml (4.5 mM) for griseofulvin and 0.5 microliters/ml (7 mM) for mercaptoethanol. Concerning effects on porcine brain tubulin assembly in vitro, diethylstilboestrol and griseofulvin inhibited the assembly process. The IC30% (30% inhibition concentration) values were 12.5 microM and 100 microM for DES and griseofulvin, respectively. Mercaptoethanol showed no effects up to 50 mM.

Acetaldehyde↗
PubMed1989

Possible involvement of chromosome 1 in in vitro immortalization: evidence from progression of a human adenoma-derived cell line in vitro.

We have previously reported that continuous in vitro passage in the presence of 3T3 feeders of a non-tumorigenic adenoma-derived epithelial cell line, designated PC/AA, resulted in its becoming immortal. At early passage PC/AA was normal diploid, whereas every cell of PC/AA late passage had an isochromosome 1(q) which led us to suggest that abnormalities of chromosome 1 may be involved in tumour progression. We now report the isolation of a 3T3-feeder-independent variant of early-passage PC/AA, designated PC/AA/FI, which was immortal in vitro and remained non-tumorigenic. Each cell of PC/AA/FI again has an isochromosome 1(q), like the late-passage PC/AA. However, with PC/AA/FI it is the other chromosome 1 of the homologous pair which is involved in the formation of the isochromosome 1(q). This is possible to determine because of the polymorphic centromeric heterochromatin on chromosome 1 of the early-passage PC/AA. With the late-passage PC/AA (grown with 3T3 feeders) the homologue with the large C-band has given rise to an isochromosome 1(q) whereas with PC/AA/FI it is the other homologue with the smaller C-band which has given rise to this isochromosome. Both the immortal PC/AA/FI and the immortal PC/AA late passage, therefore, have independent abnormalities involving chromosome 1. These results indicate that chromosome 1 may be involved in in vitro immortalization.

Adenoma↗
PubMed2007

Follicular growth in vitro: detection of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) during in vitro culture of ovine cortical slices.

Primordial follicles from different mammal species can survive and enter the growth phase in vitro but do not develop beyond the primary stage. The hypothesis was that, in sheep, in vitro follicular growth is arrested because of a lack of secretion of GDF9 and/or BMP15. Cortical slices of 0.3-0.5 mm thickness issued from 5- to 6-month-old lambs were cultured for 15 days. The pieces were fixed on days 0, 2, 4, 7, 10, and 15 of culture. Follicle morphology, RT-PCR exploration of GDF9 and BMP15 mRNA, immunohistochemical location of their proteins and their receptor BMPRIB and BMPRII were assessed at different time of culture. The mean percentage of primordial follicles decreased from 58.6% (day 0) to 13.4% (day 15) (P<0.01), whereas that of primary follicles increased from 3.2% (day 0) to 31.5% on day 4 (P<0.01), then remained stable until day 15 (35.6%). The percentage of atretic follicles increased from 14.7% (day 0) to 27.1% (day 15) (P<0.05). A few secondary follicles were observed on days 4 and 10, representing 1.0%, and 2.1% of the total number of follicles. GDF9 and BMP15 mRNAs were detected from harvesting (day 0) up to day 15 following culture. At the same time, positive immunoreactions for GDF9, BMP15 and for BMPRIB and BMPRII were also found in oocyte cytoplasm. In conclusion, expression of GDF9, BMP15 and their receptors BMPRIB and BMPRII are detected during in vitro culture of ovine cortical slices.

Animals↗
PubMed2004

Elemental sulfur: toxicity in vivo and in vitro to bacterial luciferase, in vitro yeast alcohol dehydrogenase, and bovine liver catalase.

The aim of this research was to analyze the effects and the modes of action of elemental sulfur (S(0)) in bioluminescence and respiration of Vibrio fischeri cells and the enzymes crude luciferase, pure catalase, and alcohol dehydrogenase (ADH). Metallic copper removed sulfur and reduced the toxicity of acetone extracts of sediment samples analyzed in the bioluminescence test. The sulfur inhibition of cell bioluminescence was noncompetitive with decanal, the luciferase substrate; reversible, with maximum toxicity after 15 min (EC(50) = 11.8 microg/L); and almost totally recovered after 2 h. In vitro preincubation of crude luciferase extract with sulfur (0.28 ppm) weakly inhibited bioluminescence at 5 min, but at 30 min the inhibition reached 60%. Increasing the concentration of sulfur in the parts per million concentration range in vitro decreased bioluminescence, which was not constant, but depended on exposure time, and no dead-end/total inhibition was observed. The redox state of enzymes in the in vitro system significantly affected inhibition. Hydrogen peroxide restored fully and the reducing agent dithiothreitol, itself toxic, restored only partially luciferase activity in the presence of sulfur. Sulfur (5.5 ppm) slightly inhibited ADH and catalase, and dithiothreitol enhanced sulfur inhibition. High sulfur concentrations (2.2 ppm) inhibited the bioluminescence and enhanced the respiration rate of V. fischeri cells. Elemental sulfur data were interpreted to show that sulfur acted on at least a few V. fischeri cell sites: reversibly modifying luciferase at sites sensitive to/protected by oxidative and reducing agents and by affecting electron transport processes, resulting in enhanced oxygen consumption. Sulfur together with an enzyme reducing agent inhibited the oxidoreductive enzymes ADH and catalase, which have --SH groups, metal ion cofactors, or heme, respectively, in their active centers.

Alcohol Dehydrogenase↗
PubMed1995

In vitro assembly of cowpea chlorotic mottle virus from coat protein expressed in Escherichia coli and in vitro-transcribed viral cDNA.

The small spherical plant virus, cowpea chlorotic mottle virus (CCMV), provides an ideal system to examine spherical virus assembly. We have modified the CCMV in vitro assembly system to produce virions from coat protein expressed in Escherichia coli and viral RNA transcribed in vitro from full-length cDNAs. Examination of the in vitro-assembled particles with cryoelectron microscopy and image reconstruction techniques demonstrates that the particles are indistinguishable from plant purified particles at 2.5 nm resolution. Mutational analysis of the coat protein N- and C-terminal extensions demonstrate their respective roles in virus assembly. The N-terminus is required for assembly of RNA containing particles but not for the assembly of empty virions. The C-terminus is essential for coat protein dimer formation and particle assembly.

Base Sequence↗
PubMed1988

Characterization and in vitro expression of the cytochrome b-559 genes of barley. II. In vitro transcription and translation.

The two cytochrome b-559 apoproteins of 9.4 kD and 4.5 kD molecular weight have been expressed in vitro using DNA templates containing either the two genes psbE and psbF in tandem or the individual genes. Transcription with E. coli RNA-polymerase or SP6 RNA-polymerase has been followed by translation in E. coli derived lysates. Simultaneous as well as independent synthesis of the apoproteins is possible. A 9.4 kD in vitro translation product has been identified as apoprotein I by immunoprecipitation with a monoclonal antibody specific for the C-terminal part of the 9.4 kD apoprotein of cytochrome b-559. The isolated psbF gene directs the synthesis of a translation product with a molecular weight of 4.5 kD corresponding to apoprotein II. Expression of the psbE gene requires the presence of endogenous regulatory sequences 5' upstream of psbE, while this is not the case for psbF. Additional in vitro translation products of 5.7 and 2.4 kD molecular weights are synthesized and probably translated from two reading frames starting with two different out-of-phase ATG codons in the nucleotide sequence of the psbE gene.

Base Sequence↗
PubMed2002

Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoeophytic woody medicinal plant.

Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l(-1 )N(6)-benzylaminopurine (BAP) and 0.5 mg l(-1 )indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l(-1 )BAP and 0.5 mg l(-1 )kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l(-1 )naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l(-1)) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.

Boraginaceae↗
PubMed1982

Formation of 6-dimethylcarbamyloxy-dGuo, 6-dimethylamino-dGuo and 4-dimethylamino-dThd following in vitro reaction of dimethylcarbamyl chloride with calf thymus DNA and 6-diethylcarbamyloxy-dGuo following in vitro reaction of diethylcarbamyl chloride with calf thymus DNA.

The rodent carcinogens dimethylcarbamyl chloride (DMCC) and diethylcarbamyl chloride (DECC) react with dGuo (pH 7.0-7.5, 37 degrees C, 4 h) to form the O6-acyl derivatives 6-dimethylcarbamyloxy-2'-deoxyguanosine (6-DMC-dGuo) and 6-diethylcarbamyloxy-2'-deoxyguanosine (6-DEC-dGuo), respectively. Reaction of DMCC with dThd under identical conditions yielded 4-dimethylamino-thymidine (4-DMA-dThd). Compounds 6-DMC-dGuo and 6-DEC-dGuo undergo a nucleophilic aromatic substitution reaction with dimethylamine (DMA) to form 6-dimethylamino-2'-deoxyguanosine (6-DMA-dGuo) via displacement of the C-6 dialkylcarbamyloxy moiety. The substitution reaction did not take place when diethylamine or NH3 were substituted for DMA. The structures of the new compounds 6-DMC-dGuo, 6-DEC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were deduced from chemical analyses and syntheses, UV and nuclear magnetic resonance (NMR) spectra and electron impact, isobutane chemical ionization and source insertion isobutane chemical ionization mass spectra. It was postulated that 4-DMA-dThd was formed following reaction of the transient intermediate 4-DMC-dThd with DMA formed by hydrolysis of DMCC. Calf thymus DNA was reacted in vitro with DMCC (pH 7.0-7.5, 37 degrees C, 4 h) and the modified DNA hydrolyzed enzymatically to 2'-deoxynucleosides. Compounds 6-DMC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were identified in the hydrolysate by high-pressure liquid chromatography (HPLC). In an identical manner 6-DEC-dGuo was identified following in vitro reaction of DECC with calf thymus DNA. Compounds 6-DEC-dGuo and 6-DMC-dGuo possess novel structures with respect to the types of adducts known to be formed between carcinogens and bases in DNA. The implications of these findings with respect to chemical mutagenesis and carcinogenesis is discussed. The structural relationship between N4-dimethyl-5-methylcytosine (4-dimethylamino-Thy) formed in DNA following in vitro reaction with DMCC and 5-methylcytosine, the only modified base found in vertebrate DNA is noted.

Animals↗