Biomedical subjects
Publications and source records attributed to F Stewart.
We report two unrelated children with a new syndrome consisting of polydactyly and tibial aplasia in the lower limbs. severe brachyphalangy in the hands, malformed ears and a micropenis.
Large quantities of an unusual 19 kDa protein (p19) are secreted into the lumen of the uterus of the mare (Equus caballus) during the oestrous cycle and early pregnancy. p19 associates strongly with the acellular capsule that surrounds the young horse conceptus and is believed to be important in maintaining pregnancy. Here we report the complete cDNA sequence encoding p19, its expression patterns in horse tissues and a Southern blot analysis of the gene in horse DNA. The predicted amino acid sequence of the p19 cDNA demonstrated a signal peptide of 18 residues and a mature protein of 162 residues, giving a predicted molecular mass of 18.8 kDa for the secreted protein. Analysis of the predicted amino acid sequence showed that p19 belongs to the lipocalin family of proteins, which are classified on the basis of three conserved amino acid sequence motifs and an eight-stranded anti-parallel beta-barrel conformation. Overall, p19 was most similar to the mouse major urinary protein (MUP) lipocalins (30-34% identity). Secondary structural predictions and structural modelling using the MUP 1 crystal structure coordinates gave convincing evidence that the three-dimensional structure of p19 closely resembles that of the lipocalins. However, p19 has several unusual substitutions in one of the three conserved lipocalin motifs and therefore seems to be a novel member of the family. Lipocalins perform many different functions but most bind small hydrophobic molecules and the majority act as transport proteins. The function of p19 might therefore be as a carrier of a maternal factor needed to sustain the developing embryo during pregnancy, or it could be incorporated into the embryonic capsule and perform some other function. Northern blot analysis demonstrated that expression of p19 is confined to the endometrial lining of the mare's uterus, and hybridization studies in situ showed that the mRNA for p19 is localized to the glandular and luminal epithelia of the endometrium. A Southern blot analysis of horse DNA indicated a single gene for p19 that seems to be at least 4.5 kb in size.
OBJECTIVE: To evaluate the efficacy and safety of high-dose therapy with the human immunodeficiency virus (HIV) protease inhibitor saquinavir and to establish the duration of the effect of this therapy. DESIGN: Open-label study. SETTING: Clinical research referral center. PATIENTS: 40 adults with human immunodeficiency virus type 1 (HIV-1) infection and CD4+ T-cell counts of 200 to 500 cells/mm3. INTERVENTION: Monotherapy with 3600 mg or 7200 mg of saquinavir per day, in six divided doses, for 24 weeks. MEASUREMENTS: Patients were monitored for adverse events and were evaluated monthly for CD4+ T-cell count, HIV-1 viral load (as measured by reverse transcriptase polymerase chain reaction [PCR] for plasma HIV RNA levels), immune-complex-disassociated p24 antigen levels, peripheral blood mononuclear cell viral DNA levels (as measured by PCR), and resistance mutations to saquinavir. Quantitative peripheral blood mononuclear cell cultures were also done every 2 months. RESULTS: The low-dose saquinavir regimen (3600 mg/d) resulted in a maximal mean decrease in plasma HIV RNA levels of 1.06 log RNA copies/mL of plasma and a mean maximal increase in CD4 counts of 72 cells/mm3. At week 24, the plasma HIV RNA level remained 0.48 log RNA copies/mL of plasma lower than baseline (P < 0.001) and the CD4 count remained 31 cells/mm3 higher than baseline (P = 0.165). The high-dose saquinavir regimen (7200 mg/d) produced a mean maximal decrease in the plasma HIV RNA level of 1.34 log RNA copies/mL of plasma and a mean maximal increase in CD4 count of 121 cells/mm3. At week 24, the plasma HIV RNA level remained 0.85 log RNA copies/mL of plasma lower than baseline (P < 0.001) and the CD4 count remained 82 cells/mm3 higher than baseline (P = 0.002). The high-dose regimen produced a greater reduction in plasma HIV RNA level (P = 0.08), a greater reduction in peripheral blood mononuclear cell cultures (P = 0.008), and a greater increase in CD4 count (P = 0.002) than did the low-dose regimen. Higher plasma drug concentrations in individual patients correlated with greater reductions in plasma HIV RNA levels over the two doses. Nine patients receiving the low-dose regimen and four patients receiving the high-dose regimen developed key saquinavir resistance mutations. Adverse reactions, most commonly gastrointestinal problems and elevated serum aminotransferase levels, were more common in patients receiving the high-dose regimen, but most adverse events were mild and all were reversible. CONCLUSION: Saquinavir is a potent antiviral agent that has a favorable toxicity profile at high doses. Higher doses produce a greater and more durable suppression of viral load and elevation in CD4+ T-cell counts and may delay the development of resistance mutations. Therapy with high-dose saquinavir alone or in combination with other antiretroviral agents should be investigated further.
To create a strategy for inducible gene targeting we developed a Cre-lox recombination system which responds to the synthetic steroid RU 486. Several fusions between Cre recombinase and the hormone binding domain (HBD) of a mutated human progesterone receptor, which binds RU 486 but not progesterone, were constructed. When tested in transient expression assays recombination activities of all fusion proteins were responsive to RU 486, but not to the endogenous steroid progesterone. However, the observed induction of recombination activity by the synthetic steroid varied between the different fusion proteins. The fusion with the highest activity in the presence of RU 486 combined with low background activity in the absence of the steroid was tested after stable expression in fibroblast and embryonal stem (ES) cells. We could demonstrate that its recombination activity was highly dependent on RU 486. Since the RU 486 doses required to activate recombination were considerably lower than doses displaying anti-progesterone effects in mice, this system could be used as a valuable tool for inducible gene targeting.
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The major components of the mammalian placental membranes are an epithelial surface layer, the trophoblast, and a heavily vascularized mesenchyme, the allantoic mesenchyme. The trophoblast layer makes the most intimate contact with maternal tissues and it displays a wide range of unusual, often invasive, phenotypes. However, one common feature of trophoblast development in all species is a strong correlation between the proliferation and differentiation of this epithelial layer and its physical contact with developing allantoic mesenchyme. This suggests an epithelial-mesenchymal interaction involving paracrine signals from allantoic mesenchyme acting on adjacent trophoblast. The expression patterns of several growth factors and their receptors, including hepatocyte growth factor-scatter factor (HGF-SF) and its receptor, c-met, support the hypothesis. Furthermore, HGF-SF and c-met gene knockout studies in mice indicate that HGF-SF and c-met are both essential for placental development. HGF-SF, in addition to being a potent mitogen, causes scattering and morphogenic changes in cultured cells and is believed to be an important mediator of the induction of epithelial differentiation during embryogenesis. This review evaluates the importance of mesenchymal induction of trophoblast growth and differentiation in placental development and argues that HGF-SF is a crucial component of the mesenchymal stimulus.
Reverse transcription-PCR was used to clone the coding region of the donkey (Equus asinus) glycoprotein hormone alpha-subunit transcript from pituitary gland RNA. The donkey alpha-subunit sequence demonstrated considerable identity with the horse (97% at the nucleotide level), confirming the very close evolutionary linkage between these two species. The predicted amino acid sequence revealed that the donkey alpha-subunit has the same unusual C-terminus as the horse alpha-subunit, when compared with all other mammalian alpha-subunits, including a Tyr-His transposition between positions 87 and 93 and Ile instead of Ser as the C-terminal residue. Since recent evidence indicates important involvement of this region of the alpha-subunit in receptor binding, these findings provide a possible partial explanation for the unique biological properties of the equine gonadotrophins.
Coeliac disease is an important cause of malabsorption, particularly of folic acid, in adults. We investigated the possibility that it might be a maternal risk factor for neural tube defect (NTD)-associated pregnancy by screening affected mothers using serum endomysial antibody (EmA) which has high sensitivity and specificity for coeliac disease. One (1.6%) of 60 patients was EmA positive and had a diagnosis of coeliac disease confirmed by the finding of villous atrophy on jejunal biopsy. In conclusion, the majority of NTD-associated pregnancies are not associated with maternal coeliac disease and our study is additional evidence that abnormalities of folic acid metabolism rather than absorption are the most important risk factors for NTD. Further studies are needed to determine whether the coeliac disease prevalence among women with NTD-affected pregnancy is higher than that of the general population.
The mature preinvasive chorionic girdles of horse, mule, donkey and extraspecies donkey-in-horse conceptuses, and the very young endometrial cups on d 37 of gestation in mares carrying horse, mule and transferred donkey-in-horse conceptuses, were compared histologically and ultrastructurally to determine possible mechanisms underlying failure of endometrial cup development in the donkey-in-horse model of equine abortion. The progenitor chorionic girdle from the failing donkey-in-house pregnancy was similar in size to the normal donkey chorionic girdle but the trophoblast cells within the former were smaller, less organised and showed definite signs of degeneration and pyknosis. In the 37 d endometrial cups both in the horse and mule pregnancies, the recently invaded, differentiated and enlarging endometrial cup cells had penetrated deeply into the endometrial stroma and were becoming tightly packed between the persisting endometrial glands. In the donkey-in-horse pregnancy, on the other hand, relatively few donkey chorionic girdle cells had begun the invasion process and the majority of these, having penetrated and dislodged the horse luminal epithelium, did not penetrate the basement membrane beneath. Very few cells had reached the endometrial stroma and these had already attracted considerable numbers of lymphocytes to the area. It is concluded that unknown factors in the horse uterus affect adversely all phases of the development, attachment and invasion of donkey chorionic girdle cells, thereby leading to very little or no endometrial cup development and equine chorionic gonadotropin secretion in the extraspecific donkey-in-horse pregnancy created by embryo transfer.
The coding regions of the horse (Equus caballus) and donkey (E. asinus) luteinizing hormone (LH) beta-subunit transcripts were cloned from pituitary gland RNA, in order to investigate their relationships to the corresponding equine chorionic gonadotropin (CG) beta-subunits and to further understand the unusual receptor-binding properties of equine LH and CG. The horse and donkey LH beta-subunit sequences were very similar (97% identity at the nucleotide (nt) level; 93% at the amino acid (aa) level), confirming their very close evolutionary linkage and also indicating that the C-terminal extension in both subunits occurred prior to the divergence of horse and donkeys. Furthermore, sequence comparisons and Southern blot analysis confirmed that in donkeys, as in horses, the pituitary (LH) and placental (CG) beta-subunits are almost certainly derived from a single gene. This contrasts with primates which have a single LH beta gene expressed in the pituitary and a family of separate CG beta genes that are expressed in the placenta. The deduced aa sequences also revealed several differences between the horse and donkey LH/CG beta-subunits that could explain their differences in biological activity. In addition, the study confirmed that the donkey subunit contains an additional Cys residue that is not present in other gonadotropin beta-subunits.
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In situ hybridization, Northern blotting, and immunohistochemical techniques were used to study the expression of transforming growth factor beta 1 (TGF beta 1) in the endometrium of the mare during the first 150 days of pregnancy (term = 330-340 days). In situ hybridization using an oligonucleotide (45mer) probe, based on a homologous region within all known mammalian TGF beta 1 DNA sequences, demonstrated TGF beta 1 mRNA accumulation in the glandular and lumenal epithelial cells of the endometrium from day 33 onwards which corresponds to the time of implantation (day 33-45). Expression in the endometrium remained at a high level to the end of the sampling period (day 150). There was also marked expression of TGF beta 1 in the mononuclear cells accumulated around the periphery of the specialized trophoblast cells of the endometrial cup within the endometrium and in the mononuclear cells accumulated in the endometrial stroma of mares carrying failing donkey-in-horse pregnancies created by embryo transfer. The sense (control) oligonucleotide probe exhibited no hybridization to any tissue at any stage. Northern blot analysis demonstrated that the oligonucleotide probe and a porcine TGF beta 1 cDNA clone hybridized to a single 2.5 kb transcript in horse endometrial and lymphocyte RNA, thus validating the oligonucleotide probe for detection of horse TGF beta 1 mRNA. Furthermore, both probes demonstrated an increased signal in the pregnant endometrium from day 33 onwards, thereby confirming the in situ hybridization results. Immunostaining with a specific anti-bovine TGF beta 1 serum also showed increasing TGF beta 1 accumulation in endometrial epithelia during pregnancy, and localization of the protein in endometrial stroma and in the trophoblast layer of the placenta after day 60 of pregnancy. These results show that TGF beta 1 expression increases in the maternal endometrium of the mare at the time of implantation and that it may play a role in regulating endometrial and/or trophoblast growth and differentiation during placentation in this species. It may also influence fetal development (via placental transfer) at a later stage of gestation.
Placentation in equids involves two types of trophoblast: a minor invasive component, the chorionic girdle, that gives rise to transient endocrine structures known as endometrial cups, and a major non-invasive component, the allantochorion, that forms the diffuse, microcotyledonary placenta. Growth factors are likely to be important in controlling these complex events at implantation and this study describes the use of in situ hybridization and northern blotting techniques to monitor expression of insulin-like growth factor II (IGF-II) in the fetus and placenta of the horse (Equus caballus), using 12 conceptuses recovered between 14 and 150 days of gestation (term is about 340 days). The anti-sense, but not the sense, ovine IGF-II oligonucleotide probe (45 mer) hybridized to a total of seven IGF-II mRNA transcripts (6.2-1.3 kb) in RNA extracted from horse fetal liver, demonstrating the specificity and validity of the probe for equine IGF-II mRNA. In situ hybridization demonstrated that the IGF-II gene was expressed intensely in the fetus at all stages examined, predominantly in tissues of mesodermal origin, but also in the endoderm-derived liver and epithelia of the gut and lung bronchioles, and the ectoderm-derived facial mesenchyme and choroid plexus. High concentrations of IGF-II mRNA were also detected in the extraembryonic mesoderm, invasive chorionic girdle and mature endometrial cup tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Endometrial tissue from the gravid uterine horn of pregnant mares was examined by northern analysis and in situ hybridization for mRNA that hybridized to cDNA and RNA probes generated from a mouse TGF-beta 2 1.2 kb cDNA clone. The mouse cDNA probe hybridized to characteristic TGF-beta 2 mRNA transcripts on a northern blot of total RNA isolated from horse endometrium collected at day 45 of gestation. Two major 4.0 and 3.5 kb transcripts and possibly a minor 1.6 kb transcript were observed, consistent with specific hybridization to equine TGF-beta 2 mRNA. By in situ hybridization, riboprobes transcribed from the same fragment used in northern analysis hybridized to clusters of cells scattered between endometrial glands at days 38, 40, 42, 43, 78 and 81 of gestation. Positive cells were absent before day 38. From day 38 to day 43 there was marked hybridization over maternal leucocytes in the region of the developing endometrial cups, and at later stages (day 78 and day 81) clusters of cells positive for mRNA encoding TGF-beta 2 were localized within the dense band of leucocytes at the periphery of the degenerating endometrial cups. There was no hybridization to invasive or non-invasive trophoblast or to fully differentiated endometrial cup cells. Approximately 90% of the TGF-beta 2 positive cells detected in the sections taken at day 78 also stained with chromotrope 2R used to detect eosinophils and the morphology of approximately 50% of these cells was characteristic of eosinophils.(ABSTRACT TRUNCATED AT 250 WORDS)
An apparently unique protein produced in large quantities by the endometrium of the mare which adheres to, or is incorporated into, the acellular capsule that surrounds the equine conceptus in early pregnancy, has been characterized and partially sequenced. It has a molecular mass of approximately 18 kDa on SDS-PAGE gels and is nonglycosylated as assessed by a sensitive carbohydrate detection kit. Comparison of its first 24 amino-terminal amino acids with all entries in the databases failed to show any significant identity with any other protein sequence. Secretion of the protein appears to be progesterone dependent, as its presence in uterine flushings correlates with peripheral serum progesterone profiles during the oestrous cycle and its secretion can be induced in anoestrous mares by administration of a synthetic progestagen. However, in pregnant mares, the protein disappears from the uterus after about day 20 (term = 320-340 days), despite the persistence of high serum concentrations of progesterone, indicating that additional mechanisms control its synthesis and secretion. The strong association of the protein with the glycoprotein capsule that surrounds the equine blastocyst suggests that it may be incorporated into the capsule as the capsule expands from day 11 after ovulation. Alternatively, or additionally, it may be involved in the transport of nutrients or other substances through the capsule, and may therefore play an important role in the maintenance of pregnancy.
OBJECTIVES: The purpose of the study was to determine the clinical and economic impact of alternative contraceptive methods. METHODS: Direct medical costs (method use, side effects, and unintended pregnancies) associated with 15 contraceptive methods were modeled from the perspectives of a private payer and a publicly funded program. Cost data were drawn from a national claims database and MediCal. The main outcome measures included 1-year and 5-year costs and number of pregnancies avoided compared with use of no contraceptive method. RESULTS: All 15 contraceptives were more effective and less costly than no method. Over 5 years, the copper-T IUD, vasectomy, the contraceptive implant, and the injectable contraceptive were the most cost-effective, saving $14,122, $13,899, $13,813, and $13,373, respectively, and preventing approximately the same number of pregnancies (4.2) per person. Because of their high failure rates, barrier methods, spermicides, withdrawal, and periodic abstinence were costly but still saved from $8933 to $12,239 over 5 years. Oral contraceptives fell between these groups, costing $1784 over 5 years, saving $12,879, and preventing 4.1 pregnancies. CONCLUSIONS: Contraceptives save health care resources by preventing unintended pregnancies. Up-front acquisition costs are inaccurate predictors of the total economic costs of competing contraceptive methods.
The PCR technique and highly degenerate oligonucleotide primers were used to amplify a 282 bp fragment of the horse (Equus caballus) epidermal growth factor (EGF) cDNA. The clone corresponded to 94 amino acids of the EGF precursor molecule. The deduced amino acid sequence of the 53 residue EGF mitogenic peptide within the precursor sequence showed 60-70% identity with five other published EGF sequences. The PCR cDNA fragment hybridized to a 4.9 kb transcript in horse kidney and endometrial RNA which was of a similar size to the mature EGF transcript found in other mammalian species. The horse cDNA clone was used in Northern blots to monitor EGF expression in the endometrium of pregnant mares up to day 83 of gestation (term = 330-340 days). The level of expression increased from day 33 and showed a further dramatic increase between days 35 and 45, which coincides with the onset of implantation and placentation in this species. Levels remained elevated up to day 83. The horse DNA sequence was used to design sense and antisense oligonucleotide probes (45-mers) for in situ hybridization studies. The antisense probe showed specific hybridization to the glandular, but not lumenal, epithelial cells of the endometrium and there was no signal in fetal membranes. The in situ hybridization signal increased between days 35 and 45 to a similar degree to that observed in the Northern blot analysis. This dramatic increase in EGF expression in the glandular epithelium of the mare's endometrium during pregnancy may provide a mitogenic stimulus to the endometrium and/or trophoblast to facilitate placental differentiation and attachment. Alternatively, the precursor could be involved in the endometrial gland secretory process which is necessary to produce uterine milk for fetal sustenance. The PCR cloning methods used in this study should be generally applicable to the cloning of EGF cDNAs from other species.
Stereological analysis has been performed to investigate the late changes in the normal mouse bladder one year after radiation alone (10-25 Gy) or radiation (10-15 Gy) combined with cyclophosphamide (CTX), 100 mg/kg, or cis-diamminedichloroplatinum (II) (cis-DDP), 6 mg/kg. A significant increase in total volume of urothelium was found in the group treated with CTX combined with radiation compared with the control. The total volume of connective tissue in the muscular layer in the groups which received X-rays only showed a significant increase compared to the control. There also seemed to be an increase in the amount of connective tissue in muscular tissue, although this was not statistically significant, in the groups treated with CTX and irradiation compared with CTX alone. This indicates the importance of radiation in the development of late damage. Results from bladders treated with cis-DDP alone or combined with radiation were statistically inconclusive due to the small number of animals in each group. The group treated with CTX and radiation (15 Gy) showed a significant decrease in the total volume of lamina propria, but no difference was found in the other treatment groups when comparing connective tissue, the muscular layer or the amount of vessels and capillaries with their corresponding control groups. Stereological analysis seems a feasible method to demonstrate late bladder tissue changes encountered after irradiation alone or combined with chemotherapy.