Methods to identify the substrates of thiol-disulfide oxidoreductases.

The formation of a disulfide bond is a critical step in the folding of numerous secretory and membrane proteins and catalyzed in vivo. A variety of mechanisms and protein structures have evolved to catalyze oxidative protein folding. Those enzymes that directly interact with a folding protein to accelerate its oxidative folding are mostly thiol-disulfide oxidoreductases that belong to the thioredoxin superfamily. The enzymes of this class often use a CXXC active-site motif embedded in their thioredoxin-like fold to promote formation, isomerization, and reduction of a disulfide bond in their target proteins. Over the past decade or so, an increasing number of substrates of the thiol-disulfide oxidoreductases that are present in the ER of mammalian cells have been discovered, revealing that the enzymes play unexpectedly diverse physiological functions. However, functions of some of these enzymes still remain unclear due to the lack of information on their substrates. Here, we review the methods used by researchers to identify the substrates of these enzymes and provide data that show the importance of using trichloroacetic acid in sample preparation for the substrate identification, hoping to aid future studies. We particularly focus on successful studies that have uncovered physiological substrates and functions of the enzymes in the periplasm of Gram-negative bacteria and the endoplasmic reticulum of mammalian cells. Similar approaches should be applicable to enzymes in other cellular compartments or in other organisms.

Identification of the physiological substrates of PDIp, a pancreas-specific protein-disulfide isomerase family member.

About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells. They are thought to catalyze thiol-disulfide exchange reactions within secretory or membrane proteins to assist in their folding or to regulate their functions. PDIp is a PDI family member highly expressed in the pancreas and known to bind estrogen in vivo and in vitro However, the physiological functions of PDIp remained unclear. In this study, we set out to identify its physiological substrates. By combining acid quenching and thiol alkylation, we stabilized and purified the complexes formed between endogenous PDIp and its target proteins from the mouse pancreas. MS analysis of these complexes helped identify the disulfide-linked PDIp targets in vivo, revealing that PDIp interacts directly with a number of pancreatic digestive enzymes. Interestingly, when pancreatic elastase, one of the identified proteins, was expressed alone in cultured cells, its proenzyme formed disulfide-linked aggregates within cells. However, when pancreatic elastase was co-expressed with PDIp, the latter prevented the formation of these aggregates and enhanced the production and secretion of proelastase in a form that could be converted to an active enzyme upon trypsin treatment. These findings indicate that the main targets of PDIp are digestive enzymes and that PDIp plays an important role in the biosynthesis of a digestive enzyme by assisting with the proper folding of the proenzyme within cells.

Treatable Bedridden Elderly -Recovery from Flexion Contracture after Cortisol Replacement in a Patient with Isolated Adrenocorticotropic Hormone Deficiency.

Isolated adrenocorticotropic hormone deficiency (IAD) is a rare disorder with diverse clinical presentations. A 79-year-old man was bedridden for six months due to flexion contractures of the bilateral hips and knees, along with hyponatremia. He was diagnosed with IAD based on the results of endocrine tests. After one month of corticosteroid replacement, he recovered and was able to stand up by himself. Although flexion contracture is a rare symptom of IAD, steroid replacement therapy may be effective, even for seemingly irreversibly bedridden elderly patients. In bedridden elderly patients with flexion contractures, we should consider and look for any signs of adrenal insufficiency.

Successful Fecal Microbiota Transplantation as an Initial Therapy for Clostridium difficile Infection on an Outpatient Basis.

A 64-year-old woman developed diarrhea after taking clindamycin for a dental infection. We diagnosed her with Clostridium difficile infection (CDI) and performed fecal microbiota transplantation (FMT) as the initial therapy using colonoscopy on an outpatient basis. The frequency of her bowel movements decreased from 10 times per day to two times per day three days after the procedure. The key component of FMT is to restructure the protective microbiome of the natural intestinal flora. We consider that FMT could be used as an effective first-line therapy for CDI if the efficacy and safety of this procedure is established in the future.

[The characteristics of urinary tract infection with urosepsis].

BACKGROUND AND PURPOSE: Urinary tract infections (UTIs) are frequently complicated with bacteremia. Many cases of this infection are diagnosed and treated by general practitioners. We retrospectively exam- ined the characteristics of UTIs presenting with urosepsis. METHODS: We assigned 57 UTI patients into two groups according to the results of the blood cultures. Patients were admitted to the Department of General Practice at Sakai City Hospital from January 1, 2011 to December 31, 2011. We investigated the medical records retrospectively. RESULTS: 22 patients presented with urosepsis and 35 did not. Urosepsis in the patients was significantly associated with diabetes mellitus and ureteral stones (diabetes: 32 vs. 3%, p = 0.004; ureteral stone: 23 vs. 3%, p = 0.03). Nausea or vomiting and hydronephrosis were seen in about one half of the patients with urosepsis and were significantly more frequent (nausea or vomiting: 45 vs. 17%, p = 0.03; hydronephrosis: 36 vs. 11%, p = 0.04). Leukocytosis (white blood cell (WBC) count > 12,000/gL) or leukopenia (WBC count < 4,000 /µL) were significantly more frequent (68% vs. 29%) but no significant association was found between urosepsis and body temperature or C-reactive proteins. CONCLUSIONS: Nausea or vomiting, ureteral stones or hydronephrosis, diabetes mellitus and leukocytosis or leukopenia had significantly higher rates in the patients with urosepsis.