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In vitro progesterone-induced maturation of Xenope ovocyte was studied as a function of progesterone uptake and extracellular progesterone concentration. Maturation depends on the initial total amount of progesterone in incubation medium and does not depend on the extracellular hormonal concentration. This result strongly argues for an intracellular receptor site for progesterone.
FTY720, given i.v. or orally at 0.03 mg/kg or more, significantly prolonged skin allograft survival in a dose-dependent manner and showed more potent immunosuppressive activity than cyclosporin A (CsA) or tacrolimus (FK506) in MHC-incompatible rat strains of WKAH donors and F344 recipients. However, unlike CsA or FK506, FTY720 up to 1000 nM did not affect IL-2 production in allogeneic MLC. Within 3 to 24 h after a single oral administration of FTY720 at 0.1 to 1 mg/kg, the number of lymphocytes in the rats was markedly decreased in the peripheral blood and thoracic duct lymph and partially in spleen. By contrast, the number of lymphocytes in peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP) was significantly increased at the same time. Intravenous transfusion of calcein-labeled rat lymphocytes into rats revealed that FTY720 significantly accelerated lymphocyte homing to PLN, MLN, and PP, dose dependently. Since FTY720-induced lymphocyte homing was completely blocked by simultaneous treatment of the calcein-labeled lymphocytes with mAbs against CD62L, CD49d, and CD11a before the transfusion, the acceleration of lymphocyte homing by FTY720 appears to be mediated by lymphocyte-homing receptors. These findings indicate that FTY720 sequesters circulating mature lymphocytes into PLN, MLN, and PP by acceleration of lymphocyte homing and thereby decreases the number of lymphocytes in peripheral blood, thoracic duct lymph, and spleen. Based on these observations, sequestration of circulating mature-lymphocytes is presumed to be a main mechanism of the immunosuppressive activity of FTY720.
In starfish oocytes, activity of the maturation-promoting factor (MPF) and that of a major cAMP-independent protein kinase dropped at the time of meiotic cleavage, and rose again after the first but not the second meiotic cleavage. Protein synthesis was required before the first meiotic cleavage for both MPF and protein kinase activity to rise again after the first meiotic cleavage. Microinjection of either leupeptin or soybean trypsin inhibitor early enough prior to first polar body emission suppressed both the meiotic cleavage and the associated drop of MPF activity. Microinjection of leupeptin or soybean trypsin inhibitor during the 10-min period before the first meiotic cleavage also suppressed cytokinesis but did not prevent a decrease in MPF activity at the normal time of cytokinesis. The lysosomotropic inhibitor ammonia neither suppressed cytokinesis nor the drop of MPF activity at the time of first meiotic cleavage. Activity of neutral proteases sensitive to leupeptin and soybean trypsin inhibitor was demonstrated in oocyte homogenates prepared at the time of first meiotic cleavage. It is proposed that such proteases might be involved in degradation of protein kinase(s) and in the drop of MPF activity at the time of first meiotic cleavage.
OBJECTIVE: To assess the usefulness of the recently introduced TDx-FLM assay in managing pregnant women with diabetes. METHODS: Participating institutions were recruited from the 1993 and 1994 Society of Perinatal Obstetricians Diabetes Special Interest Group meetings. Study patients consisted of insulin-dependent diabetic women who had undergone transabdominal amniocentesis with assay of the fluid by the TDx-FLM method. Pertinent data were requested concerning pregnancy and respiratory outcomes of the corresponding neonates. RESULTS: Data from 261 pregnancies at 13 institutions were collected. Eight of the 182 infants born within 4 days of amniocentesis developed respiratory distress syndrome (RDS); five of the eight infants with RDS required intubation, and all five had TDx-FLM values less than 70 mg of surfactant per gram of albumin. Three of the eight infants with RDS required hood oxygen only; two of these infants had TDx-FLM values at least 70 mg/g. Thirteen of 144 (9%) subjects who delivered within 4 days of amniocentesis and for whom a TDx-FLM assay and phosphatidylglycerol level were both reported had a TDx-FLM level of at least 70 mg/g and a negative phosphatidylglycerol result. No infant with this combination of results developed RDS. Fifteen of the 40 patients who delivered more than 4 days after amniocentesis, with both tests available, had TDx-FLM values at least 70 mg/g and were phosphatidylglycerol negative. CONCLUSION: In infants of diabetic mothers, TDx-FLM values at least 70 mg/g were not associated with RDS requiring intubation. The TDx-FLM assay may be useful in determining the best time of delivery for pregnant patients with diabetes, especially in a situation in which the TDx-FLM assay is mature and the phosphatidylglycerol result is immature.
We evaluated a modified TDx-fetal lung maturity (FLM) test for estimating surfactant in small volume tracheal aspirates in 140 infants requiring mechanical ventilation. Respiratory distress syndrome was present in 75 infants, and 68 had TDx FLM less than 60 mg/dL (sensitivity, 90.6%). Respiratory distress was absent in 65 infants, 56 had TDx FLM more than 60 mg/dL (specificity, 86.1%). The modified TDx-FLM assay is highly automated, has a fast turn-around time (less than 30 minutes), has high reproducibility, and is less expensive than other available methods. This method has potential routine application in the diagnosis of surfactant deficiency, and the assessment of surfactant replacement and mechanical ventilation therapies.
The artificial induction of sexual maturation of European eel males was carried out by using weekly hCG administrations. Histological pictures showed that the testis tissues developed and regressed naturally and no pathological changes took place under the conditions of artificial rearing in freshwater. According to light and electron microscopic investigations the morphology and motility of the spermatozoa of males kept in freshwater proved to be similar to those in seawater. The authors suppose that freshwater rearing of males is not a barrier factor in the artificial propagation of European eels.
The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 +/- 12% of the oocytes were at the germinal vesicle stage compared to 97 +/- 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 +/- 18% were at MII stage, compared with 93 +/- 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 +/- 8% vs 89 +/- 7%; P < 0.01), oocyte activation higher (11 +/- 5% vs 2 +/- 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 +/- 7% vs 4 +/- 4%; P > 0.05), compared with the control group. Cleavage was lower (61 +/- 13% vs 81 +/- 6%; P < 0.001), as well as day 8 blastocyst formation (17 +/- 7% vs 36 +/- 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development.
Yeast protoplasts were pulse labelled with [5-3H] uridine and the labelling kinetics of the low molecular weight rRNA species were determined in order to gain more insight into the position of those small rRNAs in the process of ribosome maturation. 7-S RNA, the immediate precursor of 5.8-S rRNA, is found to be present only in the nucleus, indicating that the conversion of 7-S to 5.8-S RNA is a nuclear event. 5.8-S rRNA is observed in the cytoplasm almost immediately after its formation. This as well as the presence of only a small amount of 5.8-S RNA in the nucleus, shows that the ribosomal precursor particles of the large ribosomal subunit are very rapidly transported into the cytoplasm once 5.8-S rRNA is formed. Most of the newly synthesized 5-S RNA is found in the nucleus. This nuclear 5-S rRNA is mainly present in the ribosomal precursor particles. However, a small pool of free 5-S rRNA is probably also present.
A gradation of small rostral to larger caudal neurosecretory cells is found in the preoptic area of fish. Regional variations in ultrastructural features were assessed in this region of fish ranging widely in size (1.7-283.1 g), some of which had received intraperitoneal injections of horseradish peroxidase in order to label neurons projecting beyond the blood-brain barrier (i.e. neurosecretory cells). In small (less than or equal to 6.6 g) fish, neurosecretory cells of the caudal preoptic area were 10-15 microns in diameter and contained dense core vesicles. More rostral smaller cells had few or no dense core vesicles, but were labeled following intraperitoneal horseradish peroxidase injections. Cells in both areas were progressively larger, with larger nuclei and more granular reticulum in larger fish. In large fish, neurons that contained dense core vesicles ranged from 10 microns in diameter rostrally to 70 microns in diameter caudally. Some intermediate and large cells were extensively vacuolated and contained extremely convoluted and possibly multiple nuclei. Degenerating material was seen in apparently normal large fish. A few neurosecretory cells were extensively surrounded by perineuronal electron-dense glial cells. Most neurosecretory cells were involved in extensive soma-somatic apposition, which was especially pronounced in fish which were removed from cold water in late fall. Gap junctions were also clearly present on the somata of preoptic neurosecretory cells of these fall fish. These data imply that increased capacity for hormone secretion in large fish may be accomplished in part by neuronal hypertrophy. Maturational and seasonal variation in structure suggest that physiological characteristics are also variable in this nucleus.
Glomerular filtration rate (GFR) was measured in 39 healthy infants (gestation 27-40 weeks, birthweight 0.68-3.71 kg) by prolonged inulin infusion between 2 and 63 days of age. Absolute GFR showed a logarithmic rise with conceptional age (gestational plus postnatal age) which was independent of postnatal age. GFR per kilogram showed a slow rise with gestational age, and a rapid rise with increasing postnatal age which was shown to be due to a temporary cessation of growth rather than a true acceleration in renal maturation. GFR per unit surface area showed similar, but steeper rises. Formulae were constructed to predict GFR in the first month of life from postnatal age (PA), weight and birthweight (BW); (GFR = (0.24 BW + 0.18 PA + 0.45) X weight), or from conceptional age (CA); (GFR = 100.0618 CA-1.859). 95% of the predictions fell within 66 and 151%, and 58 and 172% of the measured values, respectively. Data from 14 studies were expressed in the same format where possible. The agreement between the reported data and this study was close. Apparent contradictions between these studies had been largely due to their different forms of presentation.
Mechanically isolated early preantral mouse follicles were cultured singly for 16 d and fully grown oocytes were obtained from these follicles. We then compared in vitro and in vivo follicle growth by trypsinising the follicles and counting their cell numbers in a Neubauer-counting chamber and recording the diameter and meiotic status of oocytes under an inverted microscope. As long as the granulosa cells were within the basal membrane, proliferation was slow. From Day 6, when granulosa cells had broken through the basal membrane, the proliferation rate progressed up to Day 10 and decreased thereafter to approximately 12,000 cells per culture droplet. Incorporation of BrdU revealed that proliferating cells were evenly distributed throughout the follicle until antrum formation. As granulosa cell differentiation progressed, proliferation of mural-granulosa cells ceased, while cells around the oocytes continued dividing. Oocyte diameter increased discontinuously in relation to follicle remodelling. During the first growth phase, diameters increased from 56.5 (+/- 4.4 microns) to 67 (+/- 4.1 microns) until the onset of antral-like cavity formation. The last growth phase started after Day 10, and by Day 14 oocyte diameters were not significantly different from those of 26-d-old in vivo control oocytes. The potential to resume meiosis after mechanical removal of granulosa cells was first reached on Day 8; thereafter, removal of the corona showed that all oocytes cultured with FSH remained arrested at the GV stage up to Day 16. After Day 8, approximately 70% of all oocytes underwent GVBD as a result of granulosa-cell removal, but only 23% of these reached MII after 24 h. The in vivo controls reached a comparable GVBD rate (66%) when the granulosa was removed, but most of the oocytes (82%) underwent first polar body extrusion 24 h later. These results suggest that although oocyte diameters after IVM are not different from those of the controls, culture conditions are not yet adequate to support complete meiotic maturation.
Despite intensive molecular biology investigations over the past 10 years, and an important breakthrough on how PML-RARalpha, the fusion protein resulting from t(15;17), can alter RARalpha and PML functions, no definitive views on how leukemia is generated and by what mechanism(s) the normal phenotype is restored, are yet available. 'Resistances' to pharmacological levels of all-trans-retinoic acid (ATRA) have been observed in experimental in vivo and in vitro models. In this review, we emphasize the key role played by signal cross-talk for both normal and neoplastic hemopoiesis. After an overview of reported experimental data on APL-cell maturation and apoptosis, we apply our current knowledge on signaling pathways to underline those which might generate signal cross-talks. The design of biological models suitable to decipher the integration of signal cross-talks at the transcriptional level should be our first priority today, to generate some realistic therapeutic approaches After 'Ten Years of Molecular APL', we still know very little about how the disease develops and how effective medicines work.
beta-Defensins are microbicidal peptides implicated in host defense functions of phagocytic leukocytes and certain surface epithelial cells. Here we investigated the genetic structures and cellular expression of BNBD-4, -12, and -13, three prototypic bovine neutrophil beta-defensins. Characterization of the corresponding cDNAs indicated that BNBD-4 (41 residues) derives from a 63-amino acid prepropeptide and that BNBD-12 (38 residues) and BNBD-13 (42 residues) derive from a common 60-amino acid precursor (BNBD-12/13). The peptides were found to be encoded by two-exon genes that are closely related to bovine epithelial beta-defensin genes. BNBD-4 and BNBD-12/13 mRNAs were most abundant in bone marrow, but were expressed differentially in certain non-myeloid tissues. In situ hybridization and immunohistochemical studies demonstrated that BNBD-4 synthesis is completed early in myelopoiesis. BNBD-12 was localized exclusively to the novel dense granules, organelles that also contain precursors of cathelicidins, antimicrobial peptides that undergo proteolytic processing during phagocytosis. In contrast to cathelicidins, Western blot analyses revealed that mature beta-defensins are the predominant organellar form in myeloid cells. Stimulation of neutrophils with phorbol myristate acetate induced secretion of BNBD-12, indicating that it is co-secreted with pro-cathelicidins. The exocytosis of BNBD-12 by activated neutrophils reveals different mobilization pathways for myeloid alpha- and beta-defensins.
The correlation between the dental maturity and the different measures of sexual development, i.e. the age at menarche, the ages at breast and pubic hair development, and the age of peak height velocity, was found to be low in healthy girls. Multiple regression analyses between the predictors height and growth rate at the chronological age of a certain stage of the dental development and each of the predictands age at menarche, breast development, and age of peak height velocity improved the residual standard deviation significantly in comparison with the standard deviation of the distribution of these variables.
Dendritic cells (DCs) can be matured by CD40 stimulation to upregulate their MHC class II/peptide complexes and costimulatory molecule surface expression to become adept at presenting antigen to and activating naive T lymphocytes. The use of anti-CD40 antibodies as adjuvants for DC-based therapy has been advanced. Little is known as to how DC biology in response to CD40 ligation differs between in vitro versus in vivo ligation. Therefore, the authors analyzed the expression kinetics of MHC class II (I-Ak)/HEL peptide "complex," total MHC class II, CD80, and CD86 on in vitro or in vivo CD40-stimulated DCs over a period of 5 days. MHC class II, "complex," and costimulatory molecule expression was elevated at 1 day in vitro and stayed high for the culture period, whereas in vivo expression of the cohort of molecules peaked earlier and then declined. When purified DCs were co-cultured in vitro with antigen-specific T cell hybridomas, the DCs had lower expression of total MHC class II and "complex," but did not reduce their CD80 and CD86 expression. The lower expression was dependent on cognate interaction as a non-antigen-specific T cell hybridoma was without effect. Blocking antigen-specific MHC class II/peptide-T cell receptor (TcR) complex interaction with antibody inhibited the reduction of MHC class II expression on CD40-stimulated DCs in vitro. Overall, their studies suggest distinct response of DCs to typical conditions that feature anti-CD40 monoclonal antibody (mAb)-activated DCs in vitro or in vivo.
Maturation of bacteriophage SPPI is imprecise. Although terminally redundant and circularly permuted molecules were always formed, individual molecules varied by more than 200 base pairs from each other.
Low-dense mature thymocytes (MT) and high-dense immature thymocytes (IMT) were investigated. Immature thymocyte-potentiating factor (IMPF) was found in the supernatant from MT stimulated by Con A. On the other hand, suppressing activity was present in the supernatant from the unstimulated IMT. IMPF was different from Con A itself. Sephadex G-100 gel chromatography of IMPF revealed that the activity was recovered at the position between bovine serum albumin and ovalbumin.
In Heteropneustes fossilis, in vitro incubation of postvitellogenic follicles with 2-hydroxyestradiol-17beta (2-OHE2, 5 micromol l(-1)) decreased significantly the total cAMP level, concomitant with germinal vesicle breakdown (GVBD). The incubation of the follicles with cAMP or cAMP-elevating drugs [phosphodiesterase (PDE) inhibitors], such as IBMX (3-isobutyl-1-methyl-xanthine), theophylline and caffeine, inhibited the 2-OHE2-induced GVBD in a concentration-dependent manner. The magnitude of the response varied: both cAMP and IBMX were effective at all concentrations (0.1-2.0 mmol l(-1)), followed by theophylline (0.5-2.0 mmol l(-1)) and caffeine (1-2.0 mmol l(-1)). The protein kinase A (PKA) inhibitor H89 stimulated oocyte maturation in a concentration-dependent manner. However, when co-incubated with 2-OHE2 for 24 h it produced a biphasic effect: low concentrations (0.1 and 1.0 micromol l(-1)) did not alter the 2-OHE2-induced GVBD, but high concentrations (5 and 10 micromol l(-1)) inhibited it. The incubation of the follicles with H89 lowered the inhibitory effect of IBMX on the 2-OHE2-induced GVBD. The incubation of the follicles with okadaic acid (OA), a protein phosphatase 1 and 2A inhibitor did not affect GVBD but when co-incubated with 2-OHE2, it enhanced the GVBD response. OA reversed the inhibitory effect of IBMX. The results suggest that OA may overcome the inhibition of 2-OHE2-induced GVBD by IBMX at a step distal to the cAMP-PKA pathway.