Degradation of HMG-CoA reductase in rat liver is cholesterol and ubiquitin independent

In contrast with the accelerated degradation observed in tumor cells in response to sterols, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase turnover in whole animals was not increased by dietary cholesterol. Furthermore, treating rats with lovastatin to lower hepatic cholesterol levels did not decrease the rate of degradation. The half-life remained in the 6 h range. Co-immunoprecipitation studies revealed that the amount of ubiquitin associated with the reductase was entirely dependent upon the amount of microsomal protein subjected to immunoprecipitation. The results indicate that in liver, neither the rate of reductase protein degradation nor the ubiquitin-proteasome system appear to play roles in mediating changes in HMG-CoA reductase protein levels in response to dietary cholesterol.     

Multiple-mutation at a potential ligand-binding region decreased allergenicity of a mite allergen Der f 2 without disrupting global structure

We assessed the effect of multiple-mutations within one IgE-binding area on allergenicity of Der f 2. The triple-mutant of Der f 2, P34/95/99A, exhibited the most significant reduction of allergenicity and circular dichroism analysis showed that the global structure of Der f 2 was maintained in P34/95/99A. These results indicate that such a strategy is effective when designing allergen-vaccines, which achieve less allergenicity for a broad population of patients without disrupting the global structure. Structurally, Der f 2 is a member of the MD-2 related lipid-recognition proteins. The sites for the triple-mutation located on the characteristically charged entrance of a cavity and corresponded to the regions critical to ligand-binding in the Niemann-Pick type 2 disease protein and MD-2.     

Analysis of mGluR1a constitutive internalization using a pulse-chase enzyme-linked immuno-sorbant assay (ELISA)

The surface expression of G protein-coupled receptors is regulated by internalization. For many receptors, a constitutive level of internalization in the absence of agonist has been reported. The constitutive internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse-chase ELISA method that allows the investigation of mGluR1a constitutive internalization. When investigated by pulse-chase ELISA, the constitutive internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse-chase labelling procedure. The application of the pulse-chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive internalization. Interestingly, only DMII underwent significant constitutive internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse-chase ELISA appears to be an efficient tool to analyze the constitutive internalization of different mGluR1 splice variants.     

"Distant spin trapping": a method for expanding the availability of spin trapping measurements

The technique of spin trapping is used to study a wide range of free radicals in various systems, including those generated in vitro and in vivo. But unfortunately, EPR spectrometers are not always immediately accessible at the site of experimentation, and therefore it is important to find a method that can preserve a radical adduct over longer periods of time. We describe here an alternative method in which the samples can be frozen and transported for EPR measurements at another site. Various spin adducts of DEPMPO were frozen and measured at 0 degrees C at various intervals after freezing to determine their stability in the frozen state. The radical adducts were generated by established methods and stored at two different temperatures; -196 degrees C (liquid nitrogen) and -80 degrees C (dry ice). The experiments were carried out in an aqueous solution with and without a model of reducing environment (2 mM ascorbate). The results indicate that it is feasible to store and transport spin adducts for subsequent analysis. We conclude that this approach, which we term "distant spin trapping", makes it feasible to transport samples to another site for EPR measurements. This should significantly expand the ability to use spin trapping in biology and medicine.     

An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and l-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of l-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP]². When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of l-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in l-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.     

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.     

Schistosoma japonicum: localization of calpain in the penetration glands and secretions of cercariae

A monoclonal antibody was generated against the large subunit of Schistosoma japonicum calpain to study the localization and possible function of the molecule in vivo. Mice were immunized with recombinant S. japonicum calpain and polyclonal antisera and a monoclonal antibody specific to schistosome calpain was obtained. In immunohistochemistry, a monoclonal antibody against S. japonicum calpain, KG-2E11, bound weakly to calpain expressed at the surface of adult worm tegument, however, it bound strongly to the cercarial secretions ("footprints") of S. japonicum, emitted from the penetration glands. The present study indicates that calpain is multifunctional as it is expressed at various locations in different developmental stages. Calpain-based vaccines could thus possibly induce protective immunity against cercariae and the following early developing stages.     

Boophilus microplus: the pattern of bovine immunoglobulin isotype responses to high and low tick infestations

Cattle present variable levels of resistance to ticks and the immune correlates of these heritable phenotypes must be known in order to develop effective vaccines. The antibody responses to tick salivary antigens were examined in cattle of tick-susceptible (Holstein) and tick-resistant (Nelore) breeds. After heavy infestations, levels of IgG1 and IgG2 antibodies decreased in Holsteins and remained the same in Nelores. Conversely, levels of IgE antibodies increased in Holsteins. Different sizes of tick burdens modulated the IgG1 antibody response in a susceptible breed (Aberdeen): levels were higher than in controls in heavily infested animals, but not in those undergoing intermediary or minimal infestations. The three experimental groups presented similar levels of IgG2 antibodies. Levels of IgE antibodies were higher only in animals undergoing intermediate infestations. These results indicate that tick infestations suppress the IgG antibody response in susceptible breeds, that IgE antibodies are not protective, and that the dose of tick saliva modulates the isotype of host antibody responses.     

ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul-Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB-containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5-Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5-Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5-Rbx2. Furthermore, all three tested ASB complexes containing Cul5-Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5-Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination-mediated pathway.