Genomic variants within chromosome 14q32.32 regulate bone mass through MARK3 signaling in osteoblasts

Bone mineral density (BMD) is a highly heritable predictor of osteoporotic fracture. GWAS have identified hundreds of loci influencing BMD, but few have been functionally analyzed. In this study, we show that SNPs within a BMD locus on chromosome 14q32.32 alter splicing and expression of PAR-1a/microtubule affinity regulating kinase 3 (MARK3), a conserved serine/threonine kinase known to regulate bioenergetics, cell division, and polarity. Mice lacking Mark3 either globally or selectively in osteoblasts have increased bone mass at maturity. RNA profiling from Mark3-deficient osteoblasts suggested changes in the expression of components of the Notch signaling pathway. Mark3-deficient osteoblasts exhibited greater matrix mineralization compared with controls that was accompanied by reduced Jag1/Hes1 expression and diminished downstream JNK signaling. Overexpression of Jag1 in Mark3-deficient osteoblasts both in vitro and in vivo normalized mineralization capacity and bone mass, respectively. Together, these findings reveal a mechanism whereby genetically regulated alterations in Mark3 expression perturb cell signaling in osteoblasts to influence bone mass.     

Educational interventions to reduce use of unsafe abbreviations.

PURPOSE: Educational interventions to reduce the use of abbreviations and dosage designations that were deemed unsafe at a level 1 trauma center are described. SUMMARY: Strategies to reduce the use of unsafe abbreviations at Detroit Receiving Hospital were studied. Six abbreviations and dosage designations were deemed as unsafe by the site's medication-use and patient medical safety committees: (1) U for units, (2) microg for microgram, (3) TIW for three times a week, (4) the degree symbol for hour, (5) trailing zeros after a decimal point, and (6) the lack of leading zeros before a decimal point. Data on abbreviation use was collected starting in September 2003 by examining copies of patients' order sheets, which are sent from nursing units to the pharmacy for processing. Data were collected during three 24-hour periods each month, with 7-10 days between each period. A data collection sheet was developed to assist in documenting the number of opportunities for each unsafe abbreviation and the actual incidence of each. Educational strategies were developed and implemented starting in October 2003 to decrease the use of the unsafe abbreviations. These strategies included inservice education programs for the medical, pharmacy, and nursing staffs; laminated pocket cards; patient chart dividers; stickers; and interventions by pharmacists and nurses during medication prescribing. During the eight-month evaluation period, 20,160 orders were reviewed, representing 27,663 opportunities to use a designated unsafe abbreviation. Educational interventions successfully reduced the overall incidence of unsafe abbreviations from 19.69% to 3.31%. CONCLUSION: Educational interventions markedly reduced the use of unsafe abbreviations in medication orders over an eight-month evaluation period.     

Protective activity of different hepatic cytosolic glutathione S-transferases against DNA-binding metabolites of aflatoxin B1

To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations.     

Circulating Pancreatic Polypeptide in Patients with Adenocarcinoma of the Bile Duct

Using radioimmunoassay methods, the blood of patients with pancreatic tumors was screened for circulating polypeptide hormones. This screening discovered pancreatic polypeptide in abnormally high concentration in the serum of six of seven patients with adenocarcinomas of the bile duct. the assay appears to be very sensitive finding excessive residual pancreatic polypeptide production after palliative resections. Serum pancreatic polypeptide assays warrant evaluation as an aid in the diagnosis and management of patients with bile duct tumors.     

Collateral blood flow following acute coronary artery occlusion: comparison of a new intracellular calcium antagonist (KT-362) and diltiazem

The effects of a new intracellular calcium antagonist, KT-362 (150 and 300 micrograms/kg per min), on hemodynamics and collateral function (retrograde pressure and flow, radioactive microspheres) distal to an acute coronary artery occlusion were studied in anesthetized dogs and compared with the effects of the structurally related classical calcium channel blocker, diltiazem (15 and 30 micrograms/kg per min), and a saline-treated control group. In the saline series, there were no changes in systemic hemodynamics or coronary collateral blood flow over the 90-min ischemic period. KT-362 reduced mean aortic pressure, heart rate, and dP/dt whereas diltiazem only decreased aortic blood pressure. When blood pressure was controlled by a distal aortic cuff, heart rate was significantly reduced in both groups and dP/dt was reduced in the KT-362 series and increased in diltiazem-treated dogs. In both drug-treated groups, retrograde pressure and flow were significantly increased only when aortic pressure was controlled. Regional myocardial tissue blood flow in the nonischemic or ischemic region did not change significantly after KT-362 treatment despite its hypotensive actions, and in the presence of a constant aortic pressure, transmural collateral blood flow and the ischemic/nonischemic blood flow ratio tended to increase. In contrast, diltiazem treatment resulted in a significant decrease in the ischemic/nonischemic blood flow ratio in the absence of blood pressure control. In the presence of constant aortic pressure, blood flow to the nonischemic area was markedly increased by diltiazem whereas subendocardial blood flow was significantly increased in the ischemic area.(ABSTRACT TRUNCATED AT 250 WORDS)     

The prevalence of airways hyperresponsiveness in members of an exercise training facility.

Athletes have a high prevalence (11-50%) of exercise-induced asthma, which may be caused by the hyperventilation accompanying repetitive bouts of strenuous exercise. We hypothesized that recreational exercisers would display a similar trend. Eucapnic voluntary hyperventilation (EVH) bronchoprovocation (breathing 21% O2, 5% CO2, and 74% N2 at 60% of MVV for 5 minutes) was performed to determine the prevalence of airways hyperresponsiveness (AHR) in adults (n=212, 146 males, mean +/- standard deviation, age 32 +/- 10 years) who exercised regularly (10 +/- 10 years, 31 +/- 28% of their lives): none had a previous diagnosis of asthma. AHR was defined by at least a 10%, 20%, or 25% decline in FEV1, FEF(25-75), or PEFR, respectively, by spirometry at 1, 5, 10, and 15 minutes post-EVH. Forty-one of 212 (19%) tested positive for AHR: 20 of 41 (49%) were positive by FEV1, 28 of 41 (68%) by FEF(25-75), and 27 of 41 (66%) by PEFR. Comparing responders with nonresponders: pre-EVH lung function was equivalent, except for FEV1, which was reduced (p<0.05) in responders (96 +/- 13 vs. 102 +/- 12% predicted). Mean maximal negative deflections for responders were: for FEV1, -17 +/- 7%; FEF(25-75), -31 +/- 10%; PEFR, -38 +/- 11%. Ranges of decline for responders were: FEV1, -10 to -33%; FEF(25-75), -20 to -59%; PEFR, -25- to -70%. We conclude that in these regular exercisers, the prevalence of AHR is high and comparable with some athletic populations.     

Autophagic degradation of protein generates a pool of ferric iron required for the killing of cultured hepatocytes by an oxidative stress

Pretreatment of cultured hepatocytes with the ferric iron chelator deferoxamine prevents the killing of the cells by tert-butyl hydroperoxide (TBHP). Incubation of the deferoxamine-pretreated hepatocytes in a serum-free medium containing only 0.25 nM iron restored the sensitivity of the cells to TBHP within 4 to 6 hr. An amino acid-free medium accelerated the restoration of sensitivity in parallel with an enhanced rate of degradation of 14C-prelabeled protein. By contrast, inhibitors of the autophagic degradation of protein, including chymostatin, 3-methyladenine, benzyl alcohol, colchicine, oligomycin, and methylamine, inhibited the restoration of sensitivity of deferoxamine-treated hepatocytes to TBHP in parallel with their inhibition of protein degradation. With chymostatin, 3-methyladenine, benzyl alcohol, and colchicine, there was a parallel dose dependency of both the inhibition of protein turnover and the inhibition of the restoration of sensitivity to TBHP. Ascorbic acid, known to specifically retard the autophagic degradation of ferritin, inhibited the restoration of sensitivity to TBHP without effect on the general rate of protein turnover. None of the agents studied had any protective effect on the toxicity of TBHP for hepatocytes that were not pretreated with deferoxamine. These data indicate that the autophagic degradation of protein generates a pool of ferric iron required for the killing of cultured hepatocytes by TBHP.     

Mosaicism for an FMR1 gene deletion in a fragile X female

Most cases of fragile X syndrome result from expansion of CGG repeats in the FMR1 gene; deletions and point mutations of FMR1 are much less common. Mosaicism for an FMR1 full mutation with a deletion or with a normal allele has been reported in fragile X males. Here we report on a fragile X female who is mosaic for an FMR1 full mutation and an intragenic deletion. The patient is a 4-year-old girl with developmental delay, autistic-like behaviors, and significant speech and language abnormalities. Southern blotting demonstrated the presence of a methylated full mutation, a normal allele in methylated and unmethylated forms, and an additional fragment smaller than the normal methylated allele. This result indicates that the patient is mosaic for a full mutation and a deletion, in the presence of a normal allele. By DNA sequence analysis, we mapped the 5' breakpoint 63/65 bp upstream from the CGG repeat region and the 3' breakpoint 86/88 bp downstream of the CGG repeats within the FMR1 gene. The deletion removed 210 bp, including the entire CGG repeat region. The full mutation was inherited from a premutation in the patient's mother. The deletion, which remained methylated at the Eag I and Nru I sites, was probably derived from the full mutation allele. Mosaicism of this type is rare in females with a fragile X mutation but should be kept in mind in the interpretation of Southern blots.     

Studies on Acute Methionine Toxicity: I. Nucleolar Disaggregation in Guinea Pig Hepatic Cells with Methionine or Ethionine and Its Reversal with Adenine

The effects of methionine and ethionine on the fine structure of hepatic cell nucleoli of guinea pigs and rats were investigated. A single intraperitoneal injection of methionine into guinea pigs results in the disruption of nucleolonema as early as 2 hours after the injection. By 4 hours, nucleoli show complete fragmentation consisting of many small fragments and small remnants of nucleoli. Large aggregates of interchromatinic granules and condensation of chromatin appear in the nucleoplasm. These changes are remarkably similar to the lesions induced by ethionine in the liver of the rat or the guinea pig. The methionine-induced nuclear and nucleolar lesions persist up to 10 hours after the injection. The administration of adenine 4 hours after the methionine injection reverses the nucleolar lesions by 8 hours. The appearance of incompletely reconstructed nucleoli with twisted ropelike structures suggests a pattern of recovery very similar to the adenine-induced nucleolar reformation in ethionine-treated rats. Injecting methionine into rats induced no nucleolar abnormalities. It is suggested that the mechanism of nucleolar fragmentation induced by methionine or ethionine is related to the accumulation of S-adenosyl compounds with concomitant ATP deficiency in the liver.