Significance of serum early pregnancy factor concentrations during pregnancy and embryonic development in Sminthopsis macroura (Spencer) (Marsupialia : Dasyuridae)

Marsupial pregnancy differs from that in eutherians in duration, placentation and hormonal profile so much so that maternal recognition of pregnancy may not occur in polyovular marsupials. However, a comparison of gravid and non-gravid uteri reveals differences indicative of histological and physiological adaptations to pregnancy. In the present study, the hypothesis that embryo-maternal signalling occurs in polyovular marsupials was tested by examining serum from non-pregnant and pregnant Sminthopsis macroura for the presence of early pregnancy factor (EPF), a serum protein secreted by the ovary in response to the presence of a newly fertilized egg in the oviduct. EPF is detectable in the serum of pregnant, but not in non-pregnant, females in all eutherians studied to date. In the present study, EPF was detected in S. macroura serum by the rosette inhibition test during the first 9 days of the 10.7 day gestation period in this marsupial. However, EPF was not detected on day 10, just before parturition, or in non-pregnant or preovulatory animals. Immunohistochemical analysis of ovaries from gravid and non-gravid animals demonstrates that EPF is found in the capillaries, interstitial spaces and secretory cells of the corpus luteum. It is concluded that the spatiotemporal pattern of EPF activity described strongly indicates that maternal recognition of pregnancy in marsupials is mediated, at least in part, by EPF. Because the endocrinological milieu is the same in pregnant and non-pregnant marsupials, the possibility of using marsupials as an experimental system for studying EPF function unconfounded by hormonal effects is presented.

Chronic hypoxia induced down-regulation of angiotensinogen expression in rat epididymis

The presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis has been previously established by showing the expression of several key RAS components, and in particular angiotensinogen, the indispensable element for the intracellular generation of angiotensin II. In this study, the possible involvement of this local epididymal RAS in the testicular effects of chronic hypoxia was investigated. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and by in situ hybridization histochemistry of the rat epididymis were used to show changes in localization and expression of angiotensinogen. Results from RT-PCR analysis demonstrated that chronic hypoxia caused a marked decrease (60%) in the expression of angiotensinogen mRNA, when compared with that in the normoxic epididymis. Western blot analysis demonstrated a less decrease (35%) in the expression of angiotensinogen protein. In situ hybridization histochemistry showed that the reduced angiotensinogen mRNA in chronic hypoxia was specifically localized to the epididymal epithelium from the cauda, corpus and caput regions of the epididymis; a distribution similar to that of normoxic rats. It was concluded that chronic hypoxia decreases the transcriptional and translational expression of angiotensinogen, and thus local formation of angiotensin II, in the rat epididymis. (C) 2001 Elsevier Science B.V. All rights reserved.

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

dEndocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wildtype Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and enclocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension

To test the hypothesis that Vegf-B contributes to the pulmonary vascular remodelling, and the associated pulmonary hypertension, induced by exposure of mice to chronic hypoxia. Methods: Right ventricular systolic pressure, the ratio of right ventricle/[left ventricle+septum] (RV/[LV+S]) and the thickness of the media (relative to vessel diameter) of intralobar pulmonary arteries (o.d. 50-150 and 151-420 mum) were determined in Vegfb knockout mice (Vegfb(-/-); n=17) and corresponding wild-type mice (Vegfb(+/+); n=17) exposed to chronic hypoxia (10% oxygen) or housed in room air (normoxia) for 4 weeks. Results: In Vegfb(+/+) mice hypoxia caused (i) pulmonary hypertension (a 70% increase in right ventricular systolic pressure compared with normoxic Vegfb(+/+) mice; P

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

Crown ether appended cyclam receptors for cationic guests

A series of crown ether appended macrocyclic amines has been prepared comprising benzo-12-crown-4, benzo-15-crown-5, or benzo-18-crown-6 attached to a diamino-substituted cyclam. The Co-III complexes of these three receptors have been prepared and characterized spectroscopically and structurally. Crystal structures of each receptor in complex with an alkali metal ion and structures of the benzo-12-crown-4 and benzo-15-crown-5-receptors without guest ions are reported. 2D NMR and molecular mechanics modeling have been used to examine conformational variations upon guest ion complexation. Addition of cations to these receptors results in an appreciable anodic shift in the Co-III:II 11 redox potential, even in aqueous solution, but little cation selectivity is observed. Evidence for complex formation has been corroborated by Na-23 and Li-7 NMR spectroscopy and electrospray mass spectrometry.

Factors influencing paternity success in Antechinus agilis: last-male sperm precedence, timing of mating and genetic compatibility

We describe the patterns of paternity success from laboratory mating experiments conducted in Antechinus agilis, a small size dimorphic carnivorous marsupial (males are larger than females). A previous study found last-male sperm precedence in this species, but they were unable to sample complete Utters, and did not take male size and relatedness into account. We tested whether last-male sperm precedence regardless of male size still holds for complete litters. We explored the relationship between male mating order, male size, timing of mating and relatedness on paternity success. Females were mated with two males of different size with either the large or the small male first, with 1 day rest between the matings. Matings continued for 6 h. in these controlled conditions male size did not have a strong effect on paternity success, but mating order did. Males mating second sired 69.5% of the offspring. Within first mated males, males that mated closer to ovulation sired more offspring, To a lesser degree, variation appeared also to be caused by differences in genetic compatibility of the female and the male, where high levels of allele-sharing resulted in lower paternity success.

Mating aggregations and mating success in the flower thrips, Frankliniella schultzei (Thysanoptera: Thripidae), and a possible role for pheromones

Aggregations of Frankliniella schultzei males were observed on the corollas of Hibiscus rosasinensis and Gossypium hirsutum flowers in southeast Queensland. Aggregations were seen only on the upper surfaces of corollas but may have occurred on other flower parts, which were hidden from view. Conspecific females entered aggregations and a small proportion of them mated [18% (n = 163), H. rosasinensis; 30% (n = 181), G. hirsutum]. Most females (87 and 72%, respectively) that did not mate in aggregations walked to other flower parts. Behavior was difficult to observe on these parts, but mating was sometimes observed there. The number of females that landed within aggregations on the upper surfaces of both H. rosasinensis and G. hirsutum corollas was highly correlated with the number of males (r = 0.88, r = 0.93, respectively; P < 0.001). Significantly more mating pairs were observed in high-density aggregations (mean +/- SE, 1.10 +/- 0.22 and 4.44 +/- 0.48, respectively) than in low-density aggregations (0.37 +/- 0.11 and 1.67 +/- 0.29, respectively) (P < 0.05) on flowers of both species. More F. schultzei females were attracted to sticky traps baited with live conspecific males set among flowering Ipomoea indica (mean +/- SE, 8.83 +/- 0.32) and G. hirsutum (10.90 +/- 0.79) plants than to control traps (0.10 +/- 0.05 and 0.70 +/- 0.25, respectively)( P < 0.05), presumably in response to male-produced pheromones. Significantly more females were attracted to traps with high male densities than to traps with low densities. We found no statistical evidence that aggregation size influenced mating success (proportion males that mated). Mating success, however, should be evaluated with respect to mating on all flower parts and not just the upper surfaces of corollas. The results of this study constitute the first behavioral evidence for an attractant sex pheromone in thrips.

Mating behavior of Trissolcus basalis (Wollaston) (Hymenoptera : Scelionidae): Potential for outbreeding in a predominantly inbreeding species

The mating behavior of the quasi-gregarious egg parasitoid Trissolcus basalis (Wollaston) was investigated under field conditions. Trissolcus basalis has female-biased sex ratios and is a protandrous species, with males emerging 1-2 days before females. Males competed aggressively for control of the egg mass, with one male assuming dominance and control of the egg mass, although changes in dominance occurred at least once on each egg mass observed. Typical mating behavior involved the dominant male mating his sisters immediately upon their emergence from the egg mass. These behaviors are characteristic of an inbreeding species that manifests local mate competition. However, several aspects of the mating behavior of T. basalis are inconsistent with that of an inbreeding species. Over 18% of emerging females were not mated by the dominant male upon emergence, 13% of females were not observed to be mated at all and may have left their natal site as virgins, 25% of females were mated multiple times and sometimes by multiple males, females remained near the natal site for up to several hours after emergence before emigrating, and males dispersed away from the natal site during female emergence. Trissolcus basalis may be a predominantly inbreeding species but its emergence and mating behavior suggest that low-frequency outbreeding is also likely to occur.

Rational combinatorial chemistry-based selection, synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca2+-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (∼1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability. © 2002 Elsevier Science B.V. All rights reserved.

Effect of periparturient diseases accompanied by excessive vulval discharge and weaning to mating interval on sow reproductive performance

Objective To evaluate the effect of periparturient disease accompanied by vulval discharge, and weaning-to-mating intervals, on sow fertility and litter size. Design Reproductive data were collected and analysed from 19 Hungarian swine herds over a 4 year period. Conception rates, farrowing rates and litter sizes of sows with periparturient disease accompanied by vulval discharge were used to evaluate the relationship between duration of vulval discharge and subsequent fertility and litter size. The possibility of interactions between weaning-to-mating intervals and duration of vulval discharges was investigated to determine if there was any effect on subsequent fertility and litter size. Results and conclusions Both parity 1 and parity 2 to 8 sows having had periparturient disease accompanied by vulval discharge in excess of 6 days duration had significantly (P < 0.001) lower subsequent fertility (conception, farrowing and adjusted farrowing rates) compared with sows of similar parity where the duration of vulval discharge was < 4 or 4 to 6 days. There was no difference in fertility rates between sows, in both parity categories, with vulval discharge for < 4 days compared with 4 to 6 days. A duration of vulval discharge in excess of 6 days in parity 1 sows significantly reduced litter size (total born and live-born) in subsequent farrowings, but not in parity 2 to 8 sows. There was no interaction between the duration of vulval discharge and post-weaning to mating intervals. However sows with weaning to mating intervals between 7 and 10 days had smaller (P < 0.001) subsequent litter sizes compared with 3 to 6 or 11 to 14 day intervals. It was concluded that the duration of vulval discharge in excess of 6 days was an indication of a severe persistent endometritis adversely affecting fertility of sows.