Welfare Issues on Israeli Dairy Farms: Attitudes and Awareness of Farm Workers and Veterinary Practitioners.

Attitudes toward practical dairy cow welfare issues were evaluated based on a questionnaire answered by 500 dairy farm workers and 27 veterinary practitioners. Primarily, the effect of demographic characteristics on attitudes toward cattle welfare was tested. Professionally, five themes were identified: effect of welfare awareness on productivity, knowledge of cattle's senses and social structure, effects of man-animal interactions on milk yield, pain perception and prevention, and knowledge transfer from veterinary practitioners to farm workers. Farms with a higher welfare awareness score also had higher annual milk yield, with an annual mean difference of 1000 L of milk per cow between farms with higher and lower awareness scores. Veterinary practitioners showed high awareness of cows' social structure, senses, and pain perception. Farm workers were aware of the influence of man-animal interactions during milking and stress effects on milk yield, and the possible effect of man's behavior on heifers and cows. Practitioners and farm workers had different views regarding pain perception, mostly involving mutilation procedures. All veterinary practitioners advocated the use of pain alleviation in painful procedures, but only some of them instructed the farm workers to administer it. The survey results emphasize the variation in welfare knowledge and practical applications across farms, and the interest of both the animals and their managers to improve applied knowledge of best practice.

Targeting of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: cross-talk between E3s in the endoplasmic reticulum.

There are an increasing number of ubiquitin ligases (E3s) implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) in mammals. The two for which the greatest amount of information exists are the RING finger proteins gp78 and Hrd1, which are the structural orthologs of the yeast ERAD E3 Hrd1p. We now report that Hrd1, also known as synoviolin, targets gp78 for proteasomal degradation independent of the ubiquitin ligase activity of gp78, without evidence of a reciprocal effect. This degradation is observed in mouse embryonic fibroblasts lacking Hrd1, as well as with acute manipulation of Hrd1. The significance of this is underscored by the diminished level of a gp78-specific substrate, Insig-1, when Hrd1 expression is decreased and gp78 levels are consequently increased. These finding demonstrate a previously unappreciated level of complexity of the ubiquitin system in ERAD and have potentially important ramifications for processes where gp78 is implicated including regulation of lipid metabolism, metastasis, cystic fibrosis and neurodegenerative disorders.

Computed tomographic analysis of calvarial hyperostosis in captive lions.

Osseous malformations in the skull and cervical vertebrae of lions in captivity are believed to be caused by hypovitaminosis A. These often lead to severe neurologic abnormalities and may result in death. We describe the characterization of these abnormalities based on computed tomography (CT). CT images of two affected and three healthy lions were compared with define the normal anatomy of the skull and cervical vertebrae and provide information regarding the aforementioned osseous malformations. Because bone structure is influenced by various factors other than the aforementioned disease, all values were divided by the skull width that was not affected. The calculated ratios were compared and the most pronounced abnormalities in the affected lions were, narrowing of the foramen magnum, thickening of the tentorium osseus cerebelli and thickening of the dorsal arch of the atlas. CT is useful for detection of the calvarial abnormalities in lions and may be useful in further defining this syndrome.

Mdm2: p53's lifesaver?

In a recent issue of Molecular Cell, Taira et al. (2007) and Rinaldo et al. (2007) provide insight into the involvement of the DYRK2 kinase and a surprising role of MDM2 in regulation of DNA damage-induced apoptosis via p53 phosphorylation.

A positive feedback loop between the p53 and Lats2 tumor suppressors prevents tetraploidization.

Damage to the mitotic spindle and centrosome dysfunction can lead to cancer. To prevent this, cells trigger a succession of checkpoint responses, where an initial mitotic delay is followed by slippage without cytokinesis, spawning tetraploid G1 cells that undergo a p53-dependent G1/S arrest. We describe the importance of Lats2 (Large Tumor Suppressor 2) in this checkpoint response. Lats2 binds Mdm2, inhibits its E3 ligase activity, and activates p53. Nocodazole, a microtubule poison that provokes centrosome/mitotic apparatus dysfunction, induces Lats2 translocation from centrosomes to the nucleus and p53 accumulation. In turn, p53 rapidly and selectively up-regulates Lats2 expression in G2/M cells, thereby defining a positive feedback loop. Abrogation of Lats2 promotes accumulation of polyploid cells upon exposure to nocodazole, which can be prevented by direct activation of p53. The Lats2-Mdm2-p53 axis thus constitutes a novel checkpoint pathway critical for the maintenance of proper chromosome number.

E4F1 is an atypical ubiquitin ligase that modulates p53 effector functions independently of degradation.

p53 is regulated by multiple posttranslational modifications, including Hdm2-mediated ubiquitylation that drives its proteasomal degradation. Here, we identify the p53-associated factor E4F1, a ubiquitously expressed zinc-finger protein first identified as a cellular target of the viral oncoprotein E1A, as an atypical ubiquitin E3 ligase for p53 that modulates its effector functions without promoting proteolysis. E4F1 stimulates oligo-ubiquitylation in the hinge region of p53 on lysine residues distinct from those targeted by Hdm2 and previously described to be acetylated by the acetyltransferase PCAF. E4F1 and PCAF mediate mutually exclusive posttranslational modifications of p53. E4F1-dependent Ub-p53 conjugates are associated with chromatin, and their stimulation coincides with the induction of a p53-dependent transcriptional program specifically involved in cell cycle arrest, and not apoptosis. Collectively, our data reveal that E4F1 is a key posttranslational regulator of p53, which modulates its effector functions involved in alternative cell fates: growth arrest or apoptosis.

Life, death, and ubiquitin: taming the mule.

Ubiquitin-mediated protein degradation is an efficient way for the cell to get rid of unwanted proteins. A key player in this process is the E3 ubiquitin ligase. In this issue of Cell, and describe a new E3 ligase, ARF-BP1/Mule, which targets two very different substrates, p53 and Mcl-1, with completely different cellular outcomes.

Regulation of p53 by Mdm2: fate is in the numbers.

The p53 tumor suppressor protein is normally restrained by the Mdm2 oncoprotein, which promotes p53 ubiquitination. In a recent issue of Science, report that p53 may face two alternative fates, depending on Mdm2 levels: high Mdm2 drives p53 polyubiquitination and degradation within the cell nucleus, whereas low Mdm2 promotes p53 monoubiquitination and nuclear exclusion.

A novel mammalian endoplasmic reticulum ubiquitin ligase homologous to the yeast Hrd1.

The yeast hHrd1 is a ubiquitin-protein ligase (E3) involved in ER-associated degradation. It was originally identified by genetic methods as an E3 of the yeast cholesterol biosynthetic enzyme HMG-CoA reductase (HMGR). We report the identification and cloning of a human homologue of Hrd1 (hHrd1). Immunofluorescence imaging confirms that the endogenous hHrd1 resides in the ER and in vitro assay demonstrates that it has a ubiquitin-ligase activity. However, the homology between the human and yeast Hrd1 is limited to the N-terminal domain of the proteins, and hHrd1 does not appear to be involved in the degradation of mammalian HMGR.

An essential role for tripeptidyl peptidase in the generation of an MHC class I epitope.

Most of the peptides presented by major histocompatibility complex (MHC) class I molecules require processing by proteasomes. Tripeptidyl peptidase II (TPPII), an aminopeptidase with endoproteolytic activity, may also have a role in antigen processing. Here, we analyzed the processing and presentation of the immunodominant human immunodeficiency virus epitope HIV-Nef(73-82) in human dendritic cells. We found that inhibition of proteasome activity did not impair Nef(73-82) epitope presentation. In contrast, specific inhibition of TPPII led to a reduction of Nef(73-82) epitope presentation. We propose that TPPII can act in combination with or independent of the proteasome system and can generate epitopes that evade generation by the proteasome-system.