IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.     

Decolourisation of Remazol Brilliant Blue R via a novel Bjerkandera sp. strain

A novel strain of Bjerkandera sp. (B33/3), with particularly high decolourisation activities upon Poly R-478 and Remazol Brilliant Blue R (RBBR) dyes, was isolated. The role of the ligninolytic extracellular enzymes produced by this strain on decolourisation of RBBR was studied in some depth. The basis of decolourisation is an enzyme-mediated process, in which the main enzyme responsible is a recently described peroxidase with capacity for oxidation of manganese, as well as veratryl alcohol and 2,6-dimethoxyphenol in a manganese-independent reaction.     

Modeling retrovirus production for gene therapy. 2. Integrated optimization of bioreaction and downstream processing

In this work a model envisaging the integrated optimization of bioreaction and downstream processing is presented. This model extends the work presented in part 1 of this pair of papers by adding ultrafiltration to process optimization. The new operational parameters include ultrafiltration time, pressure, and stirring rate. For global optimization, the model uses as constraints the final product titer and quality to be achieved after downstream processing. This extended model was validated with the same system used in part 1, i.e., PA317 cells producing a recombinant retrovirus containing the LacZ gene as a marker in stirred tanks using porous supports. Optimization of the extended model led to the conclusion that bioreaction should have two steps, batch and perfusion, similar to what was found in part 1. Ultrafiltration in a stirred cell should be performed at low pressures and stirring rates to reduce the losses of infective retroviruses. Sensitivity analysis performed on the results of the integrated optimization showed that under optimal conditions the productivity is less sensitive to the parameters related to ultrafiltration than to those associated with bioreaction. These results were interpreted as reflecting the high yield of ultrafiltration (90%). The relevance of the model extension to perform integrated optimization was also demonstrated since a restriction in the specific ultrafiltration area in downstream processing conditioned perfusion duration and perfusion rate in bioreaction. This clearly indicates that overall process optimization cannot be achieved without integrated optimization.     

Acyloxymethyl as a drug protecting group. Part 6: N-acyloxymethyl- and N-[(aminocarbonyloxy)methyl]sulfonamides as prodrugs of agents containing a secondary sulfonamide group

Tertiary N-acyloxymethyl- and N-[(aminocarbonyloxy)methyl]sulfonamides were synthesised and evaluated as novel classes of potential prodrugs of agents containing a secondary sulfonamide group. The chemical and plasma hydrolyses of the title compounds were studied by HPLC. Tertiary N-acyloxymethylsulfonamides are slowly and quantitatively hydrolysed to the parent sulfonamide in pH 7.4 phosphate buffer, with half-lives ranging from 20 h, for 7d, to 30 days, for 7g. Quantitative formation of the parent sulfonamide also occurs in human plasma, the half-lives being within 0.2-2.0 min for some substrates. The rapid rate of hydrolysis can be ascribed to plasma cholinesterase, as indicated by the complete inhibition observed at [eserine] = 0.10 mM. These results suggest that tertiary N-acyloxymethylsulfonamides are potentially useful prodrugs for agents containing a secondary sulfonamide group, especially with pKa < 8, combining a high stability in aqueous media with a high rate of plasma activation. In contrast, N-[(aminocarbonyloxy)methyl]sulfonamides 7h-j do not liberate the parent sulfonamide either in aqueous buffers or in human plasma and thus appear to be unsuitable for development as sulfonamide prodrugs.     

Morphological Differentiation and Possible Origin of B Chromosomes in Natural Brazilian Population of Astyanax Scabripinnis (PISCES, CHARACIDAE)

Specimens of Astyanax scabripinnisfrom three different altitudes (1920, 1800 and 700?m) along the Ribeirão Grande stream in the Campos do Jordão region (São Paulo State, Brazil) were investigated. The same diploid number, 2n?=?50, was detected in the three populations, with the following karyotypic constitution: 6M, 22SM, 10ST and 12A. The populations located at 1920 and 1800?m altitude presented a high incidence of B chromosomes varying in number (0–2), shape (meta- and submetacentrics), size (large and small) and sex-related frequency (they were more frequent among females). The two morphologically variant B chromosomes probably evolved from a metacentric macrochromosome, which is the most commonly observed B chromosome in several A. scabripinnispopulations.     

Metabolically optimised BHK cell fed-batch cultures

The aim of this work was the optimisation of a fed-batch culture by metabolic confinement of BHK21 cells producing an antibody/cytokine fusion protein with potential application in tumour-targeted therapy. Previous results showed that at very low nutrient concentrations, a metabolic shift towards more efficient metabolic pathways occurs. The application of those results in the optimisation of a fed-batch culture resulted in higher cell growth (0.020 vs. 0.016 h(-1)) and cell viability, higher maximum cell concentration (2.5 vs. 1.1x10(6) cell ml(-1)), longer culture span (17 versus nine days) and higher product titre (60% increase), in relation to batch culture. This was achieved by maintaining glucose at 0.3 mM and glutamine at 0.2 mM through the addition of a concentrated solution based on the estimations of future nutrient consumption and growth rates through off line measurements. The production of toxic metabolites such as lactate and ammonia was reduced, especially the lactate production, which was markedly decreased due to the metabolic confinement of the cells. In conclusion, it was possible to increase the final titre of the recombinant antibody/cytokine fusion protein by confining the metabolism of the cells to an energetically more efficient state.     

Evaluation of timed insemination during summer heat stress in lactating dairy cattle

We wished to compare the effect of summer heat stress on pregnancy rate in cows that were inseminated at a set interval associated with a synchronized ovulation vs those inseminated upon routine estrus detection. The study was carried out on a commercial dairy farm in Florida from May to September 1995. Lactating dairy cows were given PGF2 alpha (25 mg i.m.) at 30 + 3 d postpartum and randomly assigned to be inseminated at a set time (Timed group) or when estrus was detected (Control group). Cows in the Timed group were synchronized by sequential administration of Buserelin (8 micrograms i.m.) on Day 0 at 1600 h, PGF2 alpha (25 mg i.m.) on Day 7 at 1600 h and Buserelin (8 micrograms i.m.) on Day 9 at 1600 h. They were inseminated on Day 10 between 0800 and 0900 h (Day 9 + 16 h). Cows in the Control group were given PGF2 alpha at 57 + 3 d postpartum and inseminated when detected in estrus. Estrus detection or insemination rate for control insemination cows was 18.1 +/- 2.5% versus 100% for time inseminated cows (P < 0.01). Mean interval from PGF2 alpha to insemination was shorter for time inseminated cows (3 +/- 2.1 d < 35.5 +/- 1.9 d; P < 0.01). Pregnancy rate was greater for time inseminated cows (13.9 +/- 2.6 > 4.8 +/- 2.5%; P < 0.01) as was overall pregnancy rate by 120 d postpartum (27.0 +/- 3.6 > 16.5 +/- 3.5%; P < 0.05). Number of days open for cows conceiving by 120 d postpartum was less for time inseminated cows (77.6 +/- 3.8 < 90.0 +/- 4.2 d; P < 0.05), as was interval to first service (58.7 +/- 2.1 < 91.0 +/- 1.9 d; P < 0.01). Services per conception were greater for time inseminated cows (1.63 +/- 0.10 > 1.27 +/- 0.11; P < 0.05). The timed insemination program did improve group reproductive performance. However, the timed insemination program will not protect the embryo from temperature-induced embryonic mortality, but management limitations induced by heat stress on estrus detection are eliminated. An economical evaluation of the timed insemination program indicates an increase in net revenue per cow with implementation of timed insemination for first service during the summer months.     

Circadian Rhythmicity of Grooming Behavior During Pregnancy in a Marmoset (Callithrix jacchus) Family Group in Captivity

One family group of common marmoset ( Callithrix jacchus ) was observed in captivity under natural environmental conditions to describe the circadian pattern of grooming behavior during the pregnancy of the reproductive female. The animals were observed by focal sampling, from the beginning to the end of animal's active phase, during two non-consecutive days per week, from August to November 1996. The duration of self-grooming and social grooming behavior of each animal were totalled each hour. The time series obtained were analyzed by Cluster Analysis and by Sin gle Cosinor Analysis. The daily patterns of grooming behavior during pregnancy were divided by Cluster Analysis into two stages, one corresponding to the second half of the third month of pregnancy, and the other from the last half of the third month to the last week before parturition. The confidence intervals of acrophases overlapped in all stages. The acrophases values of the first stage occurred between 10:09 h and 11:00 h, and on the second stage between 8:51 h and 11:13 h. The daily pattern of grooming behavior of adult male and female showed a trend to increase in the first hours of the active phase. This trend to advance grooming behavior during the second stage of pregnancy may be due to an effect of the coming parturition itself or to seasonal influences.     

Characterization of the Circadian System in a Brazilian Species of Monkey (Callithrix jacchus): Immunohistochemical Analysis and Retinal Projections

The hypothalamic suprachiasmatic nucleus (SCN) and the thalamic pregeniculate nucleus (PGN), which appears to include the intergeniculate leaflet (IGL), comprise circadian related centers in the primate brain. In this study, these centers were analysed in respect to their cytoarchitecture, retinal afferents and chemical of major cells and axon terminals with tract tracers and immunohistochemical techniques to define cytoarchitecture and connections, in the common marmoset. The SCN was shown to be a triangularly shaped cluster of compact cells just dorsal to the optic chiasm and lateral to the third ventricle. It is innervated in its ventral portion by terminals from the retina, and NPY-ergic fibers. Serotonergic and SP-staining processes are distributed throughout. VIP-neurons form a dorsolateral group of cells and CB-immunoreactive neurons fill much of the nucleus. The PGN was shown to be a wedge-shaped cluster of cells located dorsomedially to the dorsal lateral geniculate nucleus. It appears to comprise a ventral portion which receives a bilateral retinal projection and contains NPY-neurons, suggesting that this portion may correspond to IGL. The PGN also contains CB-neurons, PV-neurons and fibers, and SP- and 5-HT-fibers. These results in marmoset show that, beside a common plan revealed for most mammals, there are significant interspecific variations in the circadian timing system. Future studies are needed in order to elucidate the circadian organization in this primate species.