Fos protein expression in the circadian clock is not associated with phase shifts induced by a nonphotic stimulus, triazolam

A mutant human lysozyme, designated as C77A-a, in which glutathione is bound to Cys95, has been shown to mimic an intermediate in the formation of a disulfide bond during folding of human (h)-lysozyme. Protein disulfide isomerase (PDI), which is believed to catalyze disulfide bond formation and associated protein folding in the endoplasmic reticulum, attacked the glutathionylated h-lysozyme C77A-a to dissociate the glutathione molecule. Structural analyses showed that the protein is folded and that the structure around the disulfide bond, buried in a hydrophobic core, between the protein and the bound glutathione is fairly rigid. Thioredoxin, which has higher reducing activity of protein disulfides than PDI, catalyzed the reduction with lower efficiency. These results strongly suggest that PDI can catalyze the disulfide formation in intermediates with compact structure like the native states in the later step of in vivo protein folding.     

Neutrophil Fc gamma and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fc gamma receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fc gamma and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.     

Isolation and characterization of a DnaJ-like protein in rats: the C-terminal 10-kDa domain of hsc70 is not essential for stimulating the ATP-hydrolytic activity of hsc70 by a DnaJ-like protein

A DnaJ-like protein, RDJ1, was isolated from a rat brain cDNA library. The protein is predicted to have 397 amino acid residues and shares 99% identity to that of HDJ2, a human DnaJ-like protein. RDJ1 was also shown to rescue the temperature-sensitive lethality of a strain containing a mutated cytosolic DnaJ in yeast, ydj1-151. Fragments containing the J-domain of RDJ1 either with or without the G/F motif were expressed in Escherichia coli. The purified proteins stimulated the ATPase activity of hsc70 and of the 60-kDa N-terminal fragment of hsc70. These results imply that RDJ1 can interact with the N-terminal 60-kDa fragment of hsc70 to activate ATP hydrolysis by hsc70.     

Chronic active hepatitis induced by Helicobacter hepaticus in the A/JCr mouse is associated with a Th1 cell-mediated immune response

Helicobacter hepaticus infection in A/JCr mice results in chronic active hepatitis characterized by perivascular, periportal, and parenchymal infiltrates of mononuclear and polymorphonuclear cells. This study examined the development of hepatitis and the immune response of A/JCr mice to H. hepaticus infection. The humoral and cell-mediated T helper immune response was profiled by measuring the postinfection (p.i.) antibody response in serum, feces, and bile and by the production of cytokines and proliferative responses by splenic mononuclear cells to H. hepaticus antigens. Secretory immunoglobulin A (IgA) and systemic IgG2a antibody developed by 4 weeks p.i. and persisted through 12 months. Splenocytes from infected mice proliferated and produced more gamma interferon (IFN-gamma) than interleukin-4 (IL-4) or IL-5 when cultured with H. hepaticus outer membrane proteins. The predominantly IgG2a antibody response in serum and the in vitro production of IFN-gamma in excess of IL-4 or IL-5 are consistent with a Th1 immune response reported in humans and mice infected with Helicobacter pylori and Helicobacter felis, respectively. Mice infected with H. hepaticus developed progressively severe perivascular, periportal, and hepatic parenchymal lesions consisting of lymphohistiocytic and plasmacytic cellular infiltrates. In addition, transmural typhlitis was observed at 12 months p.i. The characterization of a cell-mediated Th1 immune response to H. hepaticus infection in the A/JCr mouse should prove valuable as a model for experimental regimens which manipulate the host response to Helicobacter.     

The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.     

Overexpression of the PC3/TIS21/BTG2 mRNA is part of the stress response induced by acute pancreatitis in rats

We have previously shown that the acute phase reaction of the pancreas is a powerful emergency mechanism which protects the organism against further pancreatic aggression. In an attempt to understand the mechanisms involved in this protective effect we tried to characterize at the molecular level the phenotypic changes of the pancreatic cell during acute stress. Using a systematic approach, we identified the PC3/TIS21/BTG2 mRNA as strongly overexpressed in pancreas during the acute phase of pancreatitis. PC3/TIS21/BTG2 mRNA is also overexpressed in liver and kidney during acute pancreatitis but not in the other tissues analyzed. In addition, PC3/TIS21/BTG2 mRNA is overexpressed in kidney after a 30-min ischemia. Since acute pancreatitis and kidney ischemia-reperfusion-induced injury were associated with apoptosis, and PC3/TIS21/BTG2 has an antiapoptotic activity, we speculate that this protein may play a role in the control of apoptosis progression in these tissues.     

Lack of a specific immune response against a recombinant capsid protein of Jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis

Enzootic nasal tumour (ENT) and sheep pulmonary adenomatosis (SPA) are two contagious adenocarcinomas of the respiratory tract of sheep and goats. Both diseases are associated with related, but distinct, type-D-retroviruses (ENTV and JSRV respectively). No evidence of circulating antibodies has been described in animals affected by either ENT or SPA using antigens from natural sources. We evaluated the usefulness of a recombinant JSRV capsid protein (JSRV-CA) as antigen to study the antibody responses of animals naturally affected by ENT or SPA, using immunoblotting. Positive reactions were detected in the sera of both affected and unaffected sheep and goats. The reactivity was abolished completely by absorption with the GST fusion partner but not by JSRV-CA, suggesting that it was not specific. The results support prior observations indicating that sheep and goats infected by JSRV and ENTV do not develop specific humoral responses to these retroviruses.