MicroRNA-193b-3p acts as a tumor suppressor by targeting the MYB oncogene in T-cell acute lymphoblastic leukemia.

The MYB oncogene is a leucine zipper transcription factor essential for normal and malignant hematopoiesis. In T-cell acute lymphoblastic leukemia (T-ALL), elevated MYB levels can arise directly through T-cell receptor-mediated MYB translocations, genomic MYB duplications or enhanced TAL1 complex binding at the MYB locus or indirectly through the TAL1/miR-223/FBXW7 regulatory axis. In this study, we used an unbiased MYB 3'untranslated region-microRNA (miRNA) library screen and identified 33 putative MYB-targeting miRNAs. Subsequently, transcriptome data from two independent T-ALL cohorts and different subsets of normal T-cells were used to select miRNAs with relevance in the context of normal and malignant T-cell transformation. Hereby, miR-193b-3p was identified as a novel bona fide tumor-suppressor miRNA that targets MYB during malignant T-cell transformation thereby offering an entry point for efficient MYB targeting-oriented therapies for human T-ALL.

A phase IB trial of 24-hour intravenous PX-12, a thioredoxin-1 inhibitor, in patients with advanced gastrointestinal cancers.

We investigated the safety, pharmacokinetics, and pharmacodynamics of PX-12, a thioredoxin-1 (Trx-1) inhibitor, administered as a 24-hour infusion every 7 or 14 days in patients with gastrointestinal malignancies. PX-12 is the first Trx-1 inhibitor to undergo clinical development. The first Phase 1 study of PX-12 demonstrated promising clinical activity, but the 1 and 3 hour-infusion schedules investigated were associated with a strong and irritating odor due to exhalation of one of its metabolites, 2-butanethiol. In an effort to achieve tolerability and achieve a drug exposure level necessary for biological activity, the current study was undertaken. While the maximally tolerated dose was estimated to be 300 mg/m(2) /24 h once a week as the 2-butanethiol expirate was tolerable at that dose level, no evidence of clinical activity was observed. Pharmacokinetic studies of the parent compound PX-12 demonstrated rapid, irreversible binding to plasma components, resulting in low (ng/ml) peak plasma concentrations of non-bound PX-12 during infusion. DCE-MRI was performed pre-and post-infusion in three patients. There were no significant trends observed in changes in plasma Trx-1, vascular endothelial growth factor (VEGF), or beta fibroblast growth factor (FGF-2) pre- or post-treatment. However, there was a trend for a decrease in circulating Trx-1 during the first four PX-12 treatment cycles in patients that had a Trx-1 baseline level >18 ng/mL. Aggregate clinical trial results suggest that further clinical development of PX-12, as an intravenous infusion, is not feasible. However, the Trx-1 pathway remains a target of interest in patients with gastrointestinal malignancies.

Chronic illness in adolescence.

Adolescents experience numerous changes. Coping with a chronic illness adds yet another dimension to their lives. This paper reviews the literature on chronically ill adolescents, addressing social, biological, psychological, cognitive, and cultural issues.

In vitro development of embryos from Sinclair miniature pigs: a preliminary report.

The goal of this project was to identify conditions that result in development from the zygote or the 2-cell stage Sinclair miniature pig embryos to the blastocyst stage. Four media were selected, 2 that have been shown to result in in vitro development in domestic pigs (Hepes buffered Tyrode's medium and Whitten's medium), 1 that is compatible with similar development in the cow (CR-1), and 1 that is compatible with development in the mouse (CZB). One- and two-cell stage embryos from Sinclair miniature pigs were flushed from oviducts in Hepes buffered Tyrode's medium, allocated to 1 of the 4 media and cultured for 120 h. At the end of the culture period, embryos were morphologically scored and nuclei were counted. Morphology scores were lowest for Hepes buffered Tyrode's medium but were not different for Whitten's medium, CZB or CR-1. The highest (P < 0.07) number of nuclei was present in the oocytes cultured in Whitten's medium (21.3), with CR-1 (15.7) and CZB (16.5) not differing significantly. Similar to the morphology scores, Hepes buffered Tyrode's medium resulted in the lowest number nuclei (5.5). In a parallel experiment, domestic pig embryos were cultured in Hepes buffered Tyrode's medium versus Whitten's medium. The domestic pig embryos, while also developing better in Whitten's Medium, developed better in the Hepes buffered Tyrode's medium than did the embryos from Sinclair pigs. Thus, the Sinclair pig embryo develops best if placed in Whitten's Medium.

Immunocytochemical analysis of the association of bovine oviduct-specific glycoproteins with early embryos.

The bovine oviductal epithelium synthesizes and secretes a class of oviduct-specific glycoproteins that is present in the luminal fluid when fertilization and early embryonic development occur. The objective of this study was to determine if these characterized glycoproteins become associated with oviductal embryos. Ovarian ova and oviductal embryos were recovered from super-ovulated cows at 72 h after onset of estrus. Eggs were fixed in 3% paraformaldehyde-1% glutaraldehyde and subsequently embedded in Lowicryl K4M. Sections (1 micron) were processed for peroxidase-antiperoxidase immunocytochemistry. Immunolabeling was not detected in any region of ovarian ova. Oviductal embryos, regardless of cleavage stage, exhibited immunoperoxidase staining localized within their zona pellucidae. Sections (100 nm) obtained from a 4- and an 8-cell embryo were also subjected to colloidal gold immunoelectron microscopy to determine conclusively the subcellular distribution of the oviduct-specific glycoproteins. Gold particles were distributed uniformly throughout the width of the zona pellucida. Also, immunoreactivity was observed associated with flocculent material in the perivitelline space and with the vitelline membrane. These results indicate that the bovine oviduct-specific secretory glycoproteins become associated with oviductal embryos. This association may be biologically important to the developing embryo.

Induction of multiple follicular development and superovulation in the olive baboon, Papio anubis.

The objective of this study was to induce multiple follicular development and superovulation in the olive baboon. Beginning at menses, adult female baboons were treated with hMG for 10 days followed by hCG on day 11. Multiple follicular development was seen in all 19 animals; superovulation occurred in 11 of these. Serum E2 and P levels were consistent with multiple follicular and corpora lutea development, respectively. Ovulated ova were able to be fertilized. These results indicate that olive baboons can be superovulated using a regimen of hMG and hCG, however, development of antibodies against the human hormones precludes restimulation.

Identification and characterization of bovine oviductal glycoproteins synthesized at estrus.

Published reports indicate that in several mammalian species the oviduct synthesizes and secretes specific glycoproteins which are components of the luminal fluids at the time of ovulation and fertilization. The present study characterized the secretory glycoproteins synthesized by the bovine oviduct at estrus. Oviducts obtained from four crossbred cows in estrus were flushed with saline, and segments of the ampullary and isthmic regions were fixed for immunocytochemical analyses. The remainder of the tissue was subjected to explant culture for 24 h in medium containing either 3H-leucine or 3H-glucosamine. Analysis of culture media by one- and two-dimensional SDS-PAGE followed by fluorography indicated that both the ampullary and isthmic regions synthesized a major class of Mr 97,000 glycoproteins with isoelectric points ranging from 5.5 to 8.1. A polyclonal antibody was generated to the glycoproteins after their isolation by gel filtration followed by electrophoretic separation. Western blot analysis of oviductal culture media indicated that the antisera cross-reacted with a doublet at Mr 97,000 and to a lesser extent with two additional bands at Mr greater than 200,000. Immunoreactive antigens were not identified in serum or in culture media of ovary, uterus, and nonreproductive tract tissues. The Mr 97,000 glycoproteins were present in oviductal flushings obtained from cows in estrus. They were also detected to a lesser degree in oviductal flushings obtained from cows at Days 5, 10, 15, and 18 of the estrous cycle, with the least amount of immunoreactivity being observed in Day 10 samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of oviductal glycoproteins within the zona pellucida and perivitelline space of ovulated ova and early embryos in baboons (Papio anubis).

The estrogen-dominated baboon oviductal epithelium synthesizes and secretes a family of oviduct-specific glycoproteins. The objective of this study was to determine if these glycoproteins become associated with ova and early embryos. Ovarian and oviductal eggs obtained from superovulated baboons 72 h post-hCG were subjected to an indirect immunofluorescent assay that used a polyclonal antibody prepared toward the baboon oviduct-specific glycoproteins. Oviductal ova as well as 2-cell and 4-cell embryos showed intense, specific fluorescence within their zonae pellucidae. Ovarian ova did not exhibit fluorescence. Oviductal eggs were also fixed and processed for peroxidase-antiperoxidase immunocytochemistry and colloidal gold immunoelectron microscopy to confirm the immunofluorescent data and to determine the subcellular distribution of the antigens. Oviductal ova as well as 2-cell and 3-cell embryos exhibited immunolabeling localized within the zona. Gold particles were distributed uniformly throughout the width of the zona. Occasional groupings of gold particles were observed within the zona. Also, in most eggs, immunoreactivity was observed associated with flocculent material in the perivitelline space as well as the vitelline membrane. Furthermore, immunogold labeling above background level was noted in the cytoplasm of the eggs, particularly in the blastomeres of 3-cell embryos. Collectively, these results indicate that baboon estrogen-dependent oviductal secretory glycoproteins become intimately associated with oviductal ova and with embryos.

Rabbit sperm function after co-culture with uterine epithelial cells.

The effects of spermatozoa:uterine epithelial cell interactions in vitro on various sperm functions were studied using monolayers of uterine epithelial cells, endometrial stromal cells and fetal fibroblasts. Epithelial and stromal cells were isolated from uteri of rabbits in estrus, while fibroblasts were derived from 12-d-old rabbit fetuses. Twenty-nine to 31 h after culture initiation, washed, ejaculated rabbit spermatozoa were incubated with epithelial cells, uterine stromal or fetal fibroblastic cells, medium or conditioned medium. Sperm viability and loss of acrosome were measured after 10 to 20 h of incubation. Progressive sperm motility and fertilizing ability, which was assessed by an in vivo fertilization assay, were determined after 12 h co-culture. Sperm viability decreased throughout the culture period and was not affected by treatment. Sperm co-cultured with epithelial cells or incubated in medium had fewer acrosomes after 20 h than after 10 h. Fewer sperm co-cultured with stromal or fibroblastic cells lost their acrosomes. Progressive motility was positively affected by sperm-epithelium interaction as 39% of the co-cultured sperm were motile compared with 18% of the sperm incubated in media. In vivo fertilization experiments suggested that sperm incubated with epithelial cells or in medium had similar fertilizing ability. The co-culture of sperm with uterine cells provides an in vitro model to evaluate the effect of gamete-genital tract interaction on sperm function.

Oviductal epithelium of the baboon: hormonal control and the immuno-gold localization of oviduct-specific glycoproteins.

Oviducts were obtained from a series of cycling and ovariectomized steroid-treated baboons. The lining epithelium of the ampulla and isthmus was analyzed by light and electron microscopy. Both morphological and cytomorphometric analyses revealed that the morphological and functional state of the oviductal epithelium in the baboon is controlled by the ovarian steroids. Additionally, a clear cephalocaudal steroid-responsive gradient was observed when the data from the ampulla and isthmus of the same animal were compared. Within the ampulla, estradiol induced hypertrophy, hyperplasia, ciliogenesis, and secretory activity, whereas adding progesterone to the treatment regimen (+/- estradiol) resulted in atrophy, deciliation, apoptosis, and loss of the secretory activity. These cyclic processes were less evident in the isthmus. We also used an indirect electron microscopic immunogold technique and a previously characterized polyclonal antibody to determine the localization of oviduct-specific glycoproteins. These glycoproteins were present in every secretory granule observed, regardless of oviduct region, electron density, or size of the secretory granule. In summary, our data show that 1) estradiol induces and maintains the mature epithelium of the baboon oviduct, 2) steroid withdrawal or the administration of progesterone causes regression of the epithelium, and 3) the previously identified estradiol-dependent oviduct-specific glycoproteins are synthesized within and released from the secretory epithelial cells.

Immunological characterization and immunocytochemical localization of oviduct-specific glycoproteins in the baboon (Papio anubis).

Oviducts obtained from estradiol-treated ovariectomized baboons synthesize and release a family of high mol wt (100,000-130,000) glycoproteins during short term explant culture. The objective of this study was to make a polyclonal antibody to these glycoproteins and then use the antibody to determine the presence of the glycoproteins in oviduct flushings, tissue culture media, and tissues obtained from cycling and steroid-treated baboons. Oviduct culture medium proteins from estradiol-treated baboons were separated on one-dimensional polyacrylamide gels and transferred to nitrocellulose membranes. The region containing the glycoproteins was cut out, solubilized in dimethylsulfoxide, mixed with Freund's adjuvant, and injected at 2-week intervals into a male rabbit. The anti-serum used in this study was obtained 6 weeks after the initial injection and cross-reacted with antigens on Western blots of oviduct flushings and oviduct culture media obtained from follicular stage and estradiol-treated baboons. The antigens were absent in oviduct flushings obtained from luteal stage, ovariectomized and estradiol-primed baboons treated with estradiol and progesterone or progesterone alone. The antigens were not detected on Western blots of other reproductive and nonreproductive tract culture media or in serum obtained from follicular stage or estradiol-treated baboons. Immunoperoxidase staining was limited to discrete granules in the apical cytoplasm of secretory cells in oviducts obtained from follicular stage and estradiol-treated baboons. Thus, the secretory cells of the baboon oviduct synthesize and secrete a family of estradiol-dependent oviduct-specific glycoproteins that may have potential functional significance during fertilization and embryo development.

Spontaneous heteroploidy in one-cell mouse embryos.

First cleavage divisions were analyzed, after chromosome banding, in 321 zygotes recovered from superovulated, outbred ICR mice 33-35 h after injection of human chorionic gonadotropin. With 72.1% of all zygotes at metaphase and late prophase analyzed, there was 87% diploidy, 3.6% trisomy, 2.5% monosomy, 0.4% structural rearrangement, and 0.93% triploidy. Aneuploid zygotes in which there were 37 or 38 chromosomes accounted for 6.5% of the population with both haploid complements analyzable. In 45 diploid zygotes in which only one complement was analyzable, there was 93.4% haploidy, 2.2% disomy, and 4.4% hypoploidy. The chromosome most often involved in aneuploidy was the 19. The parental origin of the chromosome anomalies was determined on the basis of differential condensation of the chromosomes. The source of trisomy was shown to be the male in three and the female in five of the eight cases determined. This indicates a paternal contribution to trisomy and a female nondisjunction frequency of 1.5% (five of 321). For monosomy where the origin was identified in five of the cases, the male complement was implicated in three zygotes. The single break was seen in a sperm-derived chromosome. Two of the three triploids were dispermic and one was digynic. The outbred Swiss mouse might be a useful model for studying factors responsible for the induction of cytogenetic anomalies in early development of mammals.

Normal development following in vitro fertilization in the cow.

A repeatable procedure for fertilization of bovine ova in vitro is described. Oocytes were recovered from ovarian follicles or from oviducts near the time of ovulation following treatment of donors with pregnant mare's serum gonadotropin (PMSG) and prostaglandin F2 alpha (PGF2 alpha). For in vitro capacitation semen was incubated, then high ionic strength treated and subsequently incubated in defined medium prior to insemination of oocytes. In one experiment frozen bull semen was successfully used. In experiments with 4 bulls (B, C, D, F), 34 (43.6%) of 78 ova and 13 (19.7%) of 66 follicular oocytes were fertilized in vitro. In the last series (spermatozoa from Bull F) the fertilization of 22 (62.9%) of 35 tubal ova was achieved. In vitro development proceeded to the 8-cell stage. No fertilization in vitro followed use of one male (Bull E), even though his spermatozoa could penetrate zona-free hamster ova in vitro, and higher than usual bacterial contamination of his semen was implicated as the probable cause. Findings suggested vigorous progressive sperm motility and acrosome integrity to be important features of good sperm samples. In one experiment a 4-cell stage embryo was transferred with the result that the recipient gave birth to a normal bull calf on June 9, 1981. The first calf resulting from in vitro fertilization has been found to be completely normal.