RNA polymerase II transcription complex assembly in nuclear extracts

Variation in superovulatory responses in cattle may be related to the stage of follicular growth at the time of gonadotropin treatment. Waves of follicle growth are regulated by both follicle-stimulating hormone (FSH) and oestradiol. The objective of experiment 1 was to determine the dynamics of follicle wave emergence and the relationship with FSH and oestradiol concentrations, after treatment of heifers with oestradiol benzoate (ODB) in the presence of an intravaginal progesterone-releasing device (CIDR-B). Experiment 2 examined the superovulatory response, embryo yield and quality following treatment with porcine follicle-stimulating hormone (pFSH) at different times relative to ODB injection. In experiment 1, 28 beef heifers were treated with a CIDR for 9 days and allocated at random to one of four groups to receive either: (I) CIDR only, or 5 mg ODB given as a single intramuscular injection at (II) day 0 (d0); (III) day 1.5 (d1.5); or (IV) day 3 (d3) post CIDR insertion. Ovaries were examined using daily ultrasound and blood samples were collected twice daily for 11 days. In experiment 2, 96 heifers were treated with a CIDR and 5 mg ODB as in experiment 1, and were allocated using a 4 x 3 factorial design plan to a superovulation programme using three doses (400 IU; 600 IU; 800 IU) of pFSH. FSH was given for 4 days at 12-h intervals beginning 6.5 days after CIDR insertion. Heifers received prostaglandin analogue 12 h before CIDR removal and were inseminated (AI) at 48 and 60 h post CIDR withdrawal and embryos were recovered 7 days after AI. In experiment 1, the interval from CIDR insertion to follicle wave emergence (FWE) was longer (P < 0.05) in heifers treated with ODB at d1.5 (5.4 +/- 0.4 days) and d3 (5.1 +/- 0.6 days) compared to heifers treated with CIDR only (2.4 +/- 0.4 days). On the basis of time to proposed injection of pFSH heifers would have had follicle emergence 4.4, 2.3, 1.5 and 1.4 days prior to pFSH for groups I, II, III and IV, respectively. In experiment 2, heifers treated with ODB at d1.5 had a higher (P < 0.05) superovulatory response (18.2 +/- 1.7) than heifers treated at d3 (12.8 +/- 1.7), but superovulatory response in both groups did not differ (P > 0.05) from heifers treated at d0 (14.4 +/- 2.0) or with CIDR only (15.0 +/- 1.8). There were fewer (P < 0.05) freezable-grade embryos recovered from heifers treated with ODB at d0 (1.5 +/- 0.7) and d3 (2.1 +/- 0.5) compared to heifers treated at d1.5 (3.0 +/- 0.6) or in heifers treated with CIDR only (3.4 +/- 0.7). Increasing the dose of pFSH caused a linear increase in the superovulatory response (11.7 +/- 1.0, 15.8 +/- 1.4 and 18.0 +/- 1.9) and in the number of embryos recovered (5.8 +/- 0.9, 7.0 +/- 0.8 and 9.1 +/- 1.0) for 400 IU, 600 IU and 800 IU, respectively. In conclusion, heifers treated with ODB had wide variation in time to follicle wave emergence and there was not a consistent beneficial effect of pretreatment with ODB on embryo yield and quality following superovulation.     

Five days of oral topotecan (Hycamtin), a phase I and pharmacological study in adult patients with solid tumours

Topotecan is a specific inhibitor to topoisomerase I. An oral formulation of topotecan is available with a bioavailability of 32-44% in humans. A phase I and pharmacological study of the oral formulation of topotecan administered daily for 5 days every 21 days was performed in adult patients with solid tumours to determine the maximum tolerated dose (MTD). Adult patients with a WHO performance status < or = 2 adequate haematological, hepatic and renal functions, with malignant solid tumours refractory to standard forms were entered into the study. Pharmacokinetics were performed on days 1 and 4 of the first course using a validated high performance liquid chromatographic assay. 29 patients entered the study, all patients were evaluable for toxicity and response. The doses studied in the 29 patients were 1.2, 1.8, 2.3, 2.7 mg/m2/day and a fixed dose of 4 mg/day without surface area adjustment. A total of 109 courses were given. Dose limiting toxicity (DLT) was reached at a dose of 2.7 mg/m2/day and consisted of CTC (NCI-Common Toxicity Criteria) grade IV granulocytopenia. The regimen was well tolerated. Non-haematological toxicities were mild, including fatigue, anorexia, nausea, vomiting and diarrhoea. A significant correlation was observed between the percentage decrease in white blood cells versus the area under the curve (AUC(t)) of topotecan lactone (R = 0.76 P < 0.01) which was modelled by a sigmoidal Emax function. The correlation coefficient between the absolute topotecan dose administered and the AUC(t) was R = 0.52 (P = 0.04). Pharmacokinetics of the fixed dose of 4 mg/day were comparable to the 2.3 mg/m2/day dose. DLT in this phase I study of five daily doses of oral topotecan every 21 days was granulocytopenia. The recommended dose for phase II studies is 2.3 mg/m2/day or alternatively, a fixed dose of 4 mg/day.     

Spontaneous and ligand-induced trafficking of CXC-chemokine receptor 4

A chimeric protein consisting of CXC-chemokine receptor 4 (CXCR4) and the green fluorescent protein (GFP) was used for studying receptor localization and trafficking in real time in stably transduced HeLa, U-937, CEM, and NIH/3T3 cells. CXCR4-GFP was fully active as a co-receptor in mediating human immunodeficiency virus (HIV) entry. Both CXCR4 and CXCR4-GFP were found to undergo significant spontaneous endocytosis. Only 51.5 +/- 7.8% of receptor molecules were found on the plasma membrane in CD4-positive cells, 43.9 +/- 8.5% were found in CD4-negative HeLa cells, 75.6 +/- 9.7% were found in U-937 cells, 72.5 +/- 7.9 were found in CEM cells, and almost none were found in in NIH/3T3 cells. Stromal cell-derived factor-1alpha induced rapid endocytosis of cell surface receptor molecules. A significant part of CXCR4 was targeted to lysosomes upon binding of the ligands, and recycling of internalized CXCR4 was not efficient. Only about 30% of receptor molecules recycled back to the cell surface in HeLa cells, 5% recycled in U937, and 10% recycled in CEM cells, suggesting that the protective effect of chemokines against HIV infection can be attributed not only to competition for binding but also to depletion of the co-receptor molecules from the cell surface. Envelope glycoprotein gp120 of syncytia-inducing/lymphocyte tropic HIV-1 strains induced rapid internalization of CXCR4 in both CD4-negative and CD4-positive cells, suggesting that gp120 is a high affinity ligand of CXCR4.     

Functional nonequivalence of Drosophila actin isoforms

We show that different Drosophila actin isoforms are not interchangeable. We sequenced the six genes that encode conventional Drosophila actins and found that they specify amino acid replacements in 27 of 376 positions. To test the significance of these changes we used directed mutagenesis to introduce 10 such conversions, independently, into the Act88F flight muscle-specific actin gene. We challenged these variant actins to replace the native protein by transforming germline chromosomes of a Drosophila strain lacking flight muscle actin. Only one of the 10 reproducibly perturbed myofibrillar function, demonstrating that most isoform-specific amino acid replacements are of minor significance. In order to establish the consequences of multiple amino acid replacements, we substituted portions of the Drosophila Act88F actin gene with corresponding regions of genes encoding other isoforms. Only one of five constructs tested engendered normally functioning flight muscles, and the severity of myofibrillar defects correlated with the number of replacements within the chimeric genes. Finally, we completely converted the flight muscle actin-encoding gene to one specifying a nonmuscle isoform, a change entailing a total of 18 amino acid replacements. Transformation of flies with this construct resulted in disruption of flight muscle structure and function. We conclude that actin isoform sequences are not equivalent and that effects of the amino acid replacements, while minor individually, collectively confer unique properties.     

Time-dependent differential changes of immune function in rats exposed to chronic intermittent noise

Noise is a highly relevant environmental and clinical stressor. Compared to most other experimental stressors, noise is a modest activator of neuroendocrine pathways that mimic the situation in human health where neuroendocrine activation by environmental stressors is often absent or difficult to establish. Little is known about the effects of noise exposure on the immune system. In the present work, the effects of a low-intensity chronic intermittent unpredictable noise regimen on various parameters of immune function was studied. Male wistar rats were exposed to a randomized noise protocol (white noise, 85 dB, 2-20 kHz) for 10 h per day, 15 min per h over a total period of 3 weeks. Control animals were exposed to ambient sound only. Immune function was monitored after 24 h, 7 days, and 21 days of noise exposure. Noise induced several significant changes in immune function in a time-dependent differential pattern involving both immunosuppression and immunoenhancement. After 24 h, serum IgM levels were increased and peripheral phagocytic activity was decreased. Splenic lymphocytic proliferation to mitogens was significantly decreased after 7 days, but slightly elevated after 3 weeks. The activity of splenic NK cells was increased significantly after 24 h and 7 days, but suppressed after 3 weeks. These results show that various parameters of immune function are affected differentially over time in a period of chronic mild noise stress, possibly due to sequential activation of different physiological mechanisms.