Perk-dependent repression of miR-106b-25 cluster is required for ER stress-induced apoptosis.

Activation of the unfolded protein response sensor PKR-like endoplasmic reticulum kinase (Perk) attenuates endoplasmic reticulum (ER) stress levels. Conversantly, if the damage is too severe and ER function cannot be restored, this signaling branch triggers apoptosis. Bcl-2 homology 3-only family member Bim is essential for ER stress-induced apoptosis. However, the regulatory mechanisms controlling Bim activation under ER stress conditions are not well understood. Here, we show that downregulation of the miR-106b-25 cluster contributes to ER stress-induced apoptosis and the upregulation of Bim. Hypericin-mediated photo-oxidative ER damage induced Perk-dependent cell death and led to a significant decrease in the levels of miRNAs belonging to miR-106b-25 cluster in wild-type (WT) but not in Perk⁻/⁻ MEFs. Further, we show that expression of miR-106b-25 and Mcm-7 (host gene of miR-106b-25) is co-regulated through the transcription factors Atf4 (activating transcription factor 4) and Nrf2 (nuclear factor-erythroid-2-related factor 2). ER stress increased the activity of WT Bim 3'UTR (untranslated region) construct but not the miR-106b-25 recognition site-mutated Bim 3'UTR construct. Overexpression of miR-106b-25 cluster inhibits ER stress-induced cell death in WT but did not confer any further protection in Bim-knockdown cells. Further, we show downregulation in the levels of miR-106b-25 cluster in the symptomatic SOD1(G86R) transgenic mice. Our results suggest a molecular mechanism whereby repression of miR-106b-25 cluster has an important role in ER stress-mediated increase in Bim and apoptosis.

Differential diagnosis of hereditary hemochromatosis from other liver disorders by genetic analysis: gene mutation analysis of patients previously diagnosed with hemochromatosis by liver biopsy.

BACKGROUND: Hereditary hemochromatosis, a common autosomal recessive trait caused by mutations in the HLA-H gene, is often diagnosed by the pathologist at the time of histologic examination. Unfortunately, histologic parameters alone do not differentiate between hereditary hemochromatosis and other causes of iron overload. We performed a retrospective study to determine the frequency of familial hemochromatosis in patients diagnosed with he mochromatosis by abnormal liver histology. METHODS AND RESULTS: DNA was isolated from paraffin-embedded tissue sections from 15 patients and used in a polymerase chain reaction-based assay in which we tested for the C282Y and H63D mutations. We found that in this group of patients, 5 (33%) were homozygous for the common C282Y genetic mutation, 3 (20%) were heterozygous, and 7 (47%) were normal. CONCLUSIONS: Our study shows that the molecular assay is the gold standard for the diagnosis of hereditary hemochromatosis. The case study also illustrates that a definitive diagnosis of familial hemochromatosis has significant counseling implications allowing for accurate family studies.

Localization of ectopic corticotropin-producing carcinoid tumor with use of indium-111 pentetreotide scintigraphy.

OBJECTIVE: To report on the diagnosis of ectopic corticotropin (adrenocorticotropic hormone [ACTH])-producing bronchial carcinoid tumor by indium-111 pentetreotide (octreotide scan) scintigraphy. METHODS: We present a case of ectopic ACTH syndrome caused by an occult bronchial carcinoid tumor arising in a lymph node and review the pertinent literature. RESULTS: Biochemical diagnosis of ACTH syndrome can be difficult, and conventional imaging modalities often do not demonstrate these small carcinoid tumors. After biochemical proof of the presence of ectopic ACTH syndrome in our patient, conventional radiographic studies did not demonstrate any lesions. An octreotide scan showed a lesion in the lung, which was confirmed surgically. ACTH values returned to normal after resection of the lesion, and octreotide scans confirmed the completeness of surgical resection. The carcinoid tumor originated in a lymph node outside the bronchus. The differential diagnosis of ACTH syndrome, the localization of ectopic ACTH-producing tumors, the bronchial carcinoids, and the uniqueness of the carcinoid tumor arising in a lymph node are briefly discussed. CONCLUSION: Octreotide scintigraphy is useful in localizing occult carcinoid tumors and can be used in the follow-up of patients after successful removal of these tumors.

Global capitation rates. A survival primer for capitated environment--Part 2.

This article is the second of a two-part series in Michigan Medicine designed to provide Michigan physicians with a framework to approaching capitation. Capitation in healthcare financing consists of risk transfers from insurers to providers for cost and utilization of services via fixed prepayment. Unfortunately, a fairly large number of physician practices currently are searching for a starting point on capitation processes in these rapidly evolving times. The focus of the first article (September 1996 Michigan Medicine) was on primary care and specialty subcapitation; this article addresses global capitation arrangements.

Capitation rates. A survival primer for capitated environment--Part 1.

This article is the first of a two-part series designed to provide Michigan physicians with a conceptual framework to approaching capitation. Capitation in financing consists of prepaid risk transfers from insurers to providers for cost and utilization of services. Unfortunately, a number of physician practices are searching for a starting point on capitation processes in these evolving times. The focus of this first article is on primary and specialty subcapitation. Global capitation will be covered in a second article.

Characterization of the gene for rat phosphorylase kinase catalytic subunit.

Phosphorylase kinase, a key enzyme in glycogen metabolism, has a subunit composition of (alpha beta gamma delta)4, in which the alpha and beta subunits are regulatory, delta is calmodulin, and the gamma subunit is catalytic. As one segment of our studies on the regulation of the expression of phosphorylase kinase subunits, we present in this report the structure of the gene for the catalytic gamma subunit. The gene extends over 16 kilobase pairs (kb) of DNA, and contains eight introns within the coding region plus one 3.3-kb intron upstream in the 5'-untranslated region. Within this first intron, and also upstream of the transcription start site, are sequences homologous to defined regulatory elements, including some found in other muscle-specific genes. The positions of intron splice junctions for this gene have been compared with similar data for other protein kinase genes. A somewhat unexpected finding for the gamma subunit is that two of the splice junctions fall in the midst of highly conserved strings of amino acids, both of which have been nominally defined as functional domains for the protein kinases and appear to make key contributions to substrate binding and phosphotransferase catalysis.

Coordinated expression of phosphorylase kinase subunits in regenerating skeletal muscle.

The developmental expression of the alpha, beta, and gamma subunits of skeletal muscle phosphorylase kinase has been examined in regenerating muscle. Rat extensor digitorum longus (EDL) muscles, treated with bupivacaine, promptly undergo a rapid degeneration of the muscle, followed by regeneration and recovery of essentially normal morphology and physiology by 3-4 weeks post-treatment (Hall-Craggs, E. C. B., and Seyan, H. S. (1975) Exp. Neurol. 46, 345-354). Phosphorylase kinase activity dropped to approximately 10% of control within 3 days of bupivacaine treatment and remained at this low level for several days but had attained at least 60% of normal levels by day 21. The pH 6.8/8.2 activity ratio was unusually high during the period of low activity, suggesting that the catalytic activity was not under normal regulation at this time. The subunit mRNAs were readily detected in control EDL but were undetectable at day 3 post-bupivacaine treatment. Very small amounts of message for all three subunits were evident by day 6 and began to approach normal levels by day 12-15. The mRNA for both the alpha and alpha' subunits of phosphorylase kinase exhibited a similar pattern of recovery, as did also the mRNA for phosphorylase. In contrast to both phosphorylase kinase and phosphorylase, actin mRNA exhibited a quite a different pattern, with a nearly full recovery of message levels by day 6 post-bupivacaine. These data indicate that synthesis of phosphorylase and the alpha, beta, and gamma subunits of phosphorylase kinase appears to be coordinately regulated at the level of message accumulation and that the expression of phosphorylase kinase activity is likely to be also regulated post-transcriptionally.

Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.

Normal anatomic variant: scintigraphy of the ischiopubic synchondrosis.

Radionuclide bone imaging in pediatric patients occasionally shows a focus of distinct localized increase of radiotracer uptake at the ischiopubic synchondrosis. Correlation of radionuclide bone images and conventional radiographs of this area in a group of pediatric patients demonstrates the positive bone scans to correlate with the period of beginning but incomplete fusion of the synchondrosis. This represents a normal phase of skeletal development that radiographically and scintigraphically may mimic disease and should not be confused with a focus of pathologic activity.