Zmpste24 is an integral membrane metalloproteinase of the endoplasmic reticulum. Biochemical studies of tissues from Zmpste24-deficient mice (Zmpste24 ؊/؊ ) have indicated a role for Zmpste24 in the processing of CAAX-type prenylated proteins. Here, we report the pathologic consequences of Zmpste24 deficiency in mice. Zmpste24 ؊/؊ mice gain weight slowly, appear malnourished, and exhibit progressive hair loss. The most striking pathologic phenotype is multiple spontaneous bone fractures-akin to those occurring in mouse models of osteogenesis imperfecta. Cortical and trabecular bone volumes are significantly reduced in Zmpste24 ؊/؊ mice. Zmpste24 ؊/؊ mice also manifested muscle weakness in the lower and upper extremities, resembling mice lacking the farnesylated CAAX protein prelamin A. Prelamin A processing was defective both in fibroblasts lacking Zmpste24 and in fibroblasts lacking the CAAX carboxyl methyltransferase Icmt but was normal in fibroblasts lacking the CAAX endoprotease Rce1. Muscle weakness in Zmpste24 ؊/؊ mice can be reasonably ascribed to defective processing of prelamin A, but the brittle bone phenotype suggests a broader role for Zmpste24 in mammalian biology.metalloproteinase ͉ knockout mice ͉ brittle bones ͉ CAAX motif T he mammalian zinc metalloproteinase Zmpste24 has attracted attention because it shares a high degree of sequence identity with Ste24p, a Saccharomyces cerevisiae enzyme required for the maturation of the farnesylated mating pheromone a-factor (1-3). Ste24p plays two distinct roles in a-factor biogenesis (2, 4). First, it acts as a CAAX endoprotease, clipping off the C-terminal three amino acids from the protein (i.e., the ϪAAX of the CAAX motif) (3). Release of the ϪAAX from a-factor can also be mediated by Rce1p, the CAAX endoprotease involved in Ras processing (3). The removal of the ϪAAX exposes a carboxyl-terminal farnesylcysteine, which is methylated by Ste14p (5). Second, Ste24p clips the amino-terminal extension of a-factor, rendering it susceptible to a final endoproteolytic cleavage by Axl1p or Ste23p (6). Aside from a-factor, no other substrates for Ste24p have been identified, but other substrates likely exist because genetic screens in yeast have demonstrated that STE24 mutations can reverse the topological orientation of membrane proteins (7) and can affect the viability of yeast with mutations in genes encoding actin cytoskeleton proteins (8).Zmpste24 faithfully carries out both of Ste24p's processing steps in a-factor biogenesis and thus is a bona fide Ste24p ortholog (2, 9). Although it would be tempting to speculate that Zmpste24 processes an ''a-factor-like'' peptide in mammals, no a-factor ortholog has yet been identified. We have previously speculated that prelamin A (a precursor to lamin A, a component of the nuclear lamina) might be a Zmpste24 substrate (2, 6) because prelamin A (like yeast a-factor) is a farnesylated CAAX protein that undergoes more than one proteolytic processing step (10). After the removal of the C-terminal ϪAAX, an additional 15 res...

One key adaptation that Mycobacterium tuberculosis established to survive long term in vivo is a reliance on lipids as an energy source. M. tuberculosis H37Rv has 36 fadD genes annotated as putative fatty acyl-CoA synthetase genes, which encode enzymes that activate fatty acids for metabolism. One such gene, fadD5 (Rv0166), is located within the mce1 operon, a cluster of genes associated with M. tuberculosis persistence. We disrupted the putative fatty acid binding site of fadD5 in H37Rv M. tuberculosis. No significant differences were found in the growth of the mutant and wild-type strains in vitro in nutrient-rich broth or in activated RAW264.7 cells. However, the fadD5 mutant was diminished in growth in minimal medium containing mycolic acid, but not other long-chain fatty acids. C57BL/6 mice infected with the fadD5 mutant survived significantly longer than those infected with wild-type, and the mutant never attained the plateau phase of infection in the mouse lungs. The steady-state infection phase was maintained for up to 168 days at a level one to two logs less than that shown by wild-type. These observations raise a rather intriguing possibility that FadD5 may serve to recycle mycolic acids for the long-term survival of the tubercle bacilli.

The use of effective regimens for mitigating pain remain underutilized in research rodents despite the general acceptance of both the ethical imperative and regulatory requirements intended to maximize animal welfare. Factors contributing to this gap between the need for and the actual use of analgesia include lack of sufficient evidence-based data on effective regimens, under-dosing due to labor required to dose analgesics at appropriate intervals, concerns that the use of analgesics may impact study outcomes, and beliefs that rodents recover quickly from invasive procedures and as such do not need analgesics. Fundamentally, any discussion of clinical management of pain in rodents must recognize that nociceptive pathways and pain signaling mechanisms are highly conserved across mammalian species, and that central processing of pain is largely equivalent in rodents and other larger research species such as dogs, cats, or primates. Other obstacles to effective pain management in rodents have been the lack of objective, science-driven data on pain assessment, and the availability of appropriate pharmacological tools for pain mitigation. To address this deficit, we have reviewed and summarized the available publications on pain management in rats, mice and guinea pigs. Different drug classes and specific pharmacokinetic profiles, recommended dosages, and routes of administration are discussed, and updated recommendations are provided. Nonpharmacologic tools for increasing the comfort and wellbeing of research animals are also discussed. The potential adverse effects of analgesics are also reviewed. While gaps still exist in our understanding of clinical pain management in rodents, effective pharmacologic and nonpharmacologic strategies are available that can and should be used to provide analgesia while minimizing adverse effects. The key to effective clinical management of pain is thoughtful planning that incorporates study needs and veterinary guidance, knowledge of the pharmacokinetics and mechanisms of action of drugs being considered, careful attention to individual differences, and establishing an institutional culture that commits to pain management for all species as a central component of animal welfare.

SummaryMycobacterium tuberculosis causes a variety of clinical outcomes determined by host as well as bacterial factors. M. tuberculosis disrupted in the mce1 operon causes increased mortality in immunocompetent mice. This operon is negatively regulated by mce1R (Rv0165c). We studied the role of mce1R in infection outcome in mice. At 5 ¥ 10 4 tail vein infectious dose, the median survival time (MST) of mice infected with the mce1R mutant M. tuberculosis H37Rv was 293 days, while mice infected with the wild-type H37Rv survived more than 350 days (P < 0.0001). At a higher dose (5 ¥ 10 6 ), the MST of mutant-infected mice was 32 days, compared with 127 days for wild type-infected mice (P < 0.0001). With either tail vein or aerosol infection, mutantinfected mice developed larger granulomatous lesions in their lungs than mice infected with the wild type. Mutant-infected mice were unable to control the bacterial burden in the first 4 weeks of infection, but even after achieving control later, these mice succumbed to granulomatous pneumonia. These observations suggest that the early deregulated expression of the mce1 operon products determines later granulomatous tissue response. mce1 operon may homeostatically regulate the cell wall architecture in vivo that elicits a steady-state granuloma tissue response permitting M. tuberculosis to establish a long-term infection.