Effects of early contact between non-littermate piglets and of the complexity of farrowing conditions on social behaviour and weight gain

In this study we tested if contact possibilities between non-littermate piglets and complexity of farrowing conditions affect the pre- and post-weaning behaviour, weight gain and skin lesions of piglets. Suckling sows were either kept in a group housing system (GH), in a single pen loose housing system (LH), or in conventional farrowing crates (FC). In the single pen systems a piglet door to the adjacent pen was opened on d 10 after farrowing in half of the pens so that piglets were able to enter the neighbouring pen (LH+ and FC+). For control, in the other half of single pens no piglet doors were opened (LH− and FC−). In the group housing system piglets also were allowed to freely move within the whole system on d 10 after farrowing. After weaning on d 28 piglets were kept in littered rearing pens in an open stable holding 20 piglets each. Piglets from contact pens were mixed with those they previously had contact to whereas piglets from control pens were mixed with unfamiliar litters. Data were obtained from 230 litters (113 sows with 1935 farrowed piglets). All piglets were scored for skin lesions immediately before and 4 days after opening the piglet doors, as well as immediately before and 4 days after moving into rearing pens. Behaviour (biting, fighting, drinking and laying) of piglets was recorded in the rearing pens in a 48-h period after weaning for 2 × 4 h. Treatments did not affect the level of skin lesions in the rearing period (H = 8.72, df 4, ns) nor daily weight gain until weaning (F_4,216 = 1.21, ns). In the 48 h after moving to rearing pens, less intensive agonistic behaviour (fighting and biting) was observed in contact piglets (H = 53.36, df 4, P < 0.0001). Four days after weaning control piglets showed significantly higher numbers and more severe skin lesions than contact piglets and, in addition, lesion scores of piglets from the larger single farrowing pens with straw bedding were significantly lower compared to the single farrowing crate (H = 33.86, df 4, P < 0.0001). The latency for lying in the new rearing pen was decreasing (F_4,93 = 25.76, P < 0.001) and the latency for drinking (F_4,81 = 3.43, P = 0.01) was increasing with decreasing complexity and space allotment of the housing system but were not related to whether the piglets have had contact to other litters before weaning. Five weeks after weaning weight gain (F_4,204 = 7.01, P < 0.0001) and BW (F_4,207 = 5.34, P < 0.001) were higher in treatments offering contact. Our results show that familiarising piglets from different litters 10 day post partum by establishing contact possibilities through a piglet door reduces social stress at weaning and increases weight gain after weaning. Farrowing pens with straw bedding and enlarged space as offered in the farrowing pens and the group housing system can further decrease the level of harmful agonistic interactions after mixing unacquainted litters at weaning and can improve the adaptation of piglets towards the new environment of the rearing pen.     

Fast rearrangement of the neuronal growth cone’s actin cytoskeleton following VEGF stimulation

The neuronal growth cone plays a crucial role in the development of the nervous system. This highly motile structure leads the axon to its final destination by translating guidance cues into cytoskeletal rearrangements. Recently, vascular endothelial growth factor (VEGF), which is essential for angiogenesis and vascular sprouting, has been found to exert a trophic activity also on neurons, leading to an increased axonal outgrowth, similar to the well-known nerve growth factor (NGF). The neurotrophic properties of VEGF are likely to be promoted via the VEGF receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1). In the long term, VEGF attracts and influences the growth cone velocity and leads to growth cone enlargement. The present study focuses on immediate VEGF effects using RFP-actin and GFP-NF-M microinjected chicken dorsal root ganglia for live cell imaging of the neuronal growth cone. We analyzed actin and neurofilament dynamics following VEGF and NGF treatment and compared the effects. Furthermore, key signaling pathways of VEGF were investigated by specific blocking of VEGFR-2 or NRP-1. With the aid of confocal laser scanning microscopy and stimulated emission depletion microscopy, we show for the first time that VEGF has a quick effect on the actin-cytoskeleton, since actin rearrangements were identifiable within a few minutes, leading to a dramatically increased motion. Moreover, these effects were strongly enhanced by adding both VEGF and NGF. Most notably, the effects were inhibited by blocking VEGFR-2, therefore we propose that the immediate effects of VEGF on the actin-cytoskeleton are mediated through VEGFR-2.     

Disruption of the Podosome Adaptor Protein TKS4 (SH3PXD2B) Causes the Skeletal Dysplasia, Eye, and Cardiac Abnormalities of Frank-Ter Haar Syndrome

Frank-Ter Haar syndrome (FTHS), also known as Ter Haar syndrome, is an autosomal-recessive disorder characterized by skeletal, cardiovascular, and eye abnormalities, such as increased intraocular pressure, prominent eyes, and hypertelorism. We have conducted homozygosity mapping on patients representing 12 FTHS families. A locus on chromosome 5q35.1 was identified for which patients from nine families shared homozygosity. For one family, a homozygous deletion mapped exactly to the smallest region of overlapping homozygosity, which contains a single gene, SH3PXD2B. This gene encodes the TKS4 protein, a phox homology (PX) and Src homology 3 (SH3) domain-containing adaptor protein and Src substrate. This protein was recently shown to be involved in the formation of actin-rich membrane protrusions called podosomes or invadopodia, which coordinate pericellular proteolysis with cell migration. Mice lacking Tks4 also showed pronounced skeletal, eye, and cardiac abnormalities and phenocopied the majority of the defects associated with FTHS. These findings establish a role for TKS4 in FTHS and embryonic development. Mutation analysis revealed five different homozygous mutations in SH3PXD2B in seven FTHS families. No SH3PXD2B mutations were detected in six other FTHS families, demonstrating the genetic heterogeneity of this condition. Interestingly however, dermal fibroblasts from one of the individuals without an SH3PXD2B mutation nevertheless expressed lower levels of the TKS4 protein, suggesting a common mechanism underlying disease causation.     

Methodology and evaluation of a highly sensitive algae toxicity test based on multiwell chlorophyll fluorescence imaging

A new phytotoxicity bioassay based on chlorophyll fluorescence imaging of algae suspensions in multiwell plates is introduced. Phytotoxicity is quantified via inhibition of photosystem II quantum yield, Y(II), assessed with the saturation pulse method. The basics of this approach as well as the factors enhancing and limiting its performance are outlined. Compared to other established techniques the new system allows exceptionally rapid and accurate measurements of phytotoxicity using pulse-amplitude-modulation (PAM) fluorometry. While instrument related errors are negligibly small, optimal performance depends on appropriate choice of algae and illumination conditions. Illustrative examples for the response of Phaeodactylum tricornutum to diuron are presented. The standard deviation involved in the Y(II) determination of a single well amounts to the equivalent of 44 ng/L diuron. A decisive role is played by the light (measuring light, saturation pulses, actinic light) to which samples are exposed during the bioassay: (1) the inhibitor response is enhanced at high measuring light intensity. (2) Saturation pulses may be considered non-invasive only, if applied at low frequency and as long as physiologically healthy algae cultures are used. (3) Continuous actinic light may be problematic, as it induces complex physiological reactions that limit the performance of the approach; it is not required for assessment of diuron-type inhibitors at high measuring light intensity.     

Stress-induced expression of cyclophilins in proembryonic masses of Digitalis lanata does not protect against freezing/thawing stress

Using proembryonic masses (PEMs) of Digitalis lanata Erh., it was demonstrated that cold, hormonal or osmotic stress, which increased freezing tolerance during cryopreservation, induced an increasing level of two peptidyl-prolyl-cis/trans-isomerases (PPIases). The difference in pI (9.2 ± 0.2 and 9.5 ± 0.2, ±SD; n = 3) allowed the separation of the two enzymes by free-flow isoelectrophoresis. Both were inhibited by cyclosporin A and thus belong to the cyclophilin family of PPIases. The enzymes differed slightly in their substrate specificity and their relative molecular masses of 18038 ± 4 Da (D. lanataCyp18.0) and 18132 ± 3 Da (D. lanataCyp18.1). Both cyclophilins were blocked N-terminally. Partial internal amino acid sequences from the two cyclophilins, with a length of 34 amino acids, displayed 82% sequence identity to each other. Pretreatment of PEMs with abscisic acid, sorbitol or a combination of both substances led to a 270 ± 30% elevation of the total cytosolic cyclophilin concentration determined with a cyclophylin affinity sensor. During the first 4 d of pretreatment, the total PPIase activity was enhanced up to 230 ± SD% compared with the control culture. The lag phase between maximal PPIase concentration after 4 d of pretreatment and maximal effect of freezing tolerance after 10 d of pretreatment indicated that increasing levels of cytosolic PPIases may be necessary to overcome the stress induced by hormones and osmotica during pretreatment but not to protect against freezing/thawing stress. Received: 17 September 1998 / Accepted: 29 January 1999     

Assembly of infectious Kaposi’s sarcoma-associated herpesvirus progeny requires formation of a pORF19 pentamer

Herpesviruses cause severe diseases particularly in immunocompromised patients. Both genome packaging and release from the capsid require a unique portal channel occupying one of the 12 capsid vertices. Here, we report the 2.6 Å crystal structure of the pentameric pORF19 of the γ-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) resembling the portal cap that seals this portal channel. We also present the structure of its β-herpesviral ortholog, revealing a striking structural similarity to its α- and γ-herpesviral counterparts despite apparent differences in capsid association. We demonstrate pORF19 pentamer formation in solution and provide insights into how pentamerization is triggered in infected cells. Mutagenesis in its lateral interfaces blocked pORF19 pentamerization and severely affected KSHV capsid assembly and production of infectious progeny. Our results pave the way to better understand the role of pORF19 in capsid assembly and identify a potential novel drug target for the treatment of herpesvirus-induced diseases.

In herpesviruses, genome packaging and release from the capsid require a unique portal channel. Here, the authors have resolved the crystal structure of a pentameric KSHV pORF19 assembly and find that it resembles the herpesviral portal cap and provides insights how the viral genome is retained within the capsid.