Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo.

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2's capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

Utility of hair shafts from study skins for mitochondrial DNA analysis.

The condition of mtDNA in hair shafts preserved in a museum was examined using 30 study skins of masked palm civets, Paguma larvata (Viverridae), collected between 1924 and 2011. Comparisons of extracts from fresh and burnt alum-fixed hair shafts showed that burnt alum, which is commonly used in taxidermy, had no harmful effect on the amount of total DNA and lengths of the mtDNA fragments. Burnt alum-fixed hair shafts had a tendency to develop a small degree of melanin hindering PCR amplification compared with fresh hair shafts, although that observation was not supported statistically (Wilcoxon signed-rank test, Z = -0.183, P = 0.855). However, the amount of total DNA decreased after preparation of specimen in an exponential relation (regression of log DNA amount with year, regression analysis, F = 7.065, P = 0.013). Nevertheless, the oldest specimen, collected in 1924, yielded 1341.5 ng of DNA per 100 hair shafts, which was sufficient for PCR amplification. In addition, the mtDNA fragment length and amount of melanin in the hair shaft were not significantly correlated with the passage of time after preparation (F = 0.244, P = 0.625 for mtDNA fragment length; F = 0.039, P = 0.845 for the amount of melanin). Therefore, hair shafts prepared and preserved by chemical treatment in museums are good sources of mtDNA and useful for genetic analysis.

Stress on a postpartum mother inhibits the secretion of growth hormone in the offspring and causes persistent growth impairment.

Children exposed to environmental stress in the early neonatal period often develop psychiatric or somatic diseases in adulthood. In the present study in mice, we examined how postpartum stress on the mother influences their pups and thus tried to provide new insight into the management of idiopathic short stature. The dams were exposed to daily 3-h immobilization stress (IS) only for 3 weeks from the day after delivery. When compared to the pups of nonstressed dams (control pups), those of the IS dams (IS pups) showed lower body weight and height, which persisted even into adulthood. Their nutritional status was normal. The IS pups also showed low serum concentrations of insulin-like growth factor I (IGF-I) and poor responses to growth hormone-releasing hormone (GHRH) stimulation on day 22 and were behaviorally hyperactive at 8 weeks. Immunohistochemical analysis demonstrated that the number of pituitary GH-positive cells in response to treatment with GHRH was markedly decreased in the IS pups compared to the control pups. The IS dams did not show apparent behavioral abnormalities except downregulation of glucocorticoid receptor (GR) gene expression in the hippocampus. These results suggest that the perturbation of GH secretion in the pituitary glands is involved in the lifelong growth impairment of the IS pups.

Detection of venous emboli using Doppler ultrasound.

OBJECTIVES: To detect emboli in the venous system using a Doppler ultrasound device with embolism detecting software. METHODS: Pulmonary embolism (PE) was induced by injecting thrombus through the iliac vein of castrated swine under general anaesthesia. Data recorded from the Doppler system were analysed for high intensity transient signals (HITS) using receiver operating characteristic curves. Four different thrombi (5 or 10mm long and 3 or 5mm in diameter) were then injected to assess the quantitative analysis. RESULTS: Thrombus could be detected in the venous system by the Doppler ultrasound device with an embolism detecting function. Appropriate confidence level was 60%. If thrombus were assumed to travel at the maximum flow rate (30 cm/s) in the inferior vena cava, the estimated embolism size was 10.4 S.D. 2.8mm for 3mm and 10.8 S.D. 4.9 mm for 5mm, both of which were close to 10mm. CONCLUSION: Thrombi could be detected as high intensity transient signals in the venous system. The appropriate confidence level was 60%. The size of emboli can be estimated if they are more than 3mm in diameter when the venous flow rate is 30 cm/s or less. Our results suggest that it may be possible to detect emboli in the subclavian vein, axillary vein or inferior vena cava in clinical cases.

Oral bacteria are a possible risk factor for valvular incompetence in primary varicose veins.

OBJECTIVES: To investigate a possible link between valvular incompetence in primary varicose veins and chronic infection of periodontal disease by assessing the presence of oral bacteria in the great saphenous vein from patients with varicose veins and control subjects. MATERIAL AND METHODS: Forty-four primary varicose vein patients were enrolled in the study. 12 control saphenous veins were obtained from patients undergoing peripheral arterial bypass without clinical evidence of venous reflux. In total, 56 saphenous vein specimen (44 varicose veins and 12 control veins) were examined for 7 periodontal bacteria using a polymerase chain reaction (PCR) method. RESULTS: Of the 44 primary varicose vein patients, 31 patients were women and mean age was 59 years (range, 39-79 years). PCR examination of the diseased vein specimens showed that 48% were positive for at least one of 7 periodontal bacterial DNA. No bacteria were detected in the control specimens. CONCLUSION: Bacterial colonisation or infection of varicose veins is a frequent event although we were not able to establish whether this is a cause or consequence of the development of varices but this could be considered a risk factor for the development of varices.

Detection and localization of periodontopathic bacteria in abdominal aortic aneurysms.

OBJECTIVES: We examined a possible link between periodontal disease and abdominal aortic aneurysm (AAA) by studying resected aneurysmal specimens from AAA patients for the presence of periodontopathic bacteria. DESIGN: Prospective case control study. MATERIAL AND METHODS: Thirty-two AAA patients were enrolled in the study. Periodontitis was classified according to the probing depth of periodontal pocket. Thirty-two aneurysmal walls, 16 mural thrombi, 5 atherosclerotic occlusive aorta and 5 control arterial tissue, were examined for 7 periodontal bacteria using polymerase chain reaction (PCR) method. The localization of the bacteria in the aneurysmal/atherosclerotic wall was determined by thromboendarterectomy. RESULTS: All patients had periodontal disease, and most cases were severe. PCR examination of the aneurysmal specimens showed that 86% were positive for periodontal bacterial DNA. No bacteria were detected in the control specimens. The bacteria were found in both the intimal/medial layer and the adventitial layer of the aneurysmal wall but only in intimal/medial layer of the atherosclerotic occlusive aorta. CONCLUSION: Periodontopathic bacteria were present in a high percentage of specimens of diseased arteries from AAA patients and were found throughout the whole aneurysmal wall. These bacteria may play a role in the development of AAAs and/or contribute to weakening the aneurysmal wall.

[Spinal cord ischemia complicated with acute aortic dissection and intramural hematoma; report of two cases].

Spinal cord ischemic injury is one of te most serious complications in patients with aortic dissection. We experienced 2 cases with severe upper back pain and paraplegia of the lower extremities as initial manifestations of acute DeBakey type IIIb aortic dissection (case 1) and aortic intramural hematoma (IMH) from ascending aorta to abdominal aorta (case 2). Paraplegia was permanent and spinal cord atrophy was proved at Th 9-10 by the magnetic resonance imaging (MRI) in case 1. The aortic IMH regressed rapidly and paraplegia was transient in case 2. The MRI obtained 18 days after the onset showed scattered small lesions within the spinal cord at Th 4-7.

Muscle sarcoidosis following malignant lymphoma: diagnosis by MR imaging.

We report on a case of intramuscular sarcoidosis in a patient with gastric malignant lymphoma that had been operated on before, and discuss the role of magnetic resonance (MR) imaging in characterizing sarcoid nodules in muscle and distinguishing them from lymphoma.

The proximal form of mural thrombus in aortoiliac occlusive disease using computed tomography.

BACKGROUND: It is important to know the nature of proximal thrombus in patients with aortoiliac occlusive disease (AIOD) when juxtarenal aortic clamping is scheduled. This study was aimed to evaluate the shape and nature of thrombi at several sites of the abdominal aorta in patients with AIOD using enhanced computed tomography (CT). Final judgment was made according to the operative findings. METHODS: Between the years 1999 and 2001, 22 patients, who underwent aortobifemoral bypass, were enrolled. The shape and nature of their thrombi were examined at 4 points (superior mesenteric, suprarenal, juxtarenal and infrarenal arteries at the level of the 2 cm before the renal artery) and 88 slices of CT were examined retrospectively. RESULTS: There was mural thrombus in 31 slices, which could be classified into 4 shapes (crescent-shaped: 10 cases; magatama: 2; wavy: 12; circular: 6). The wavy and circular shaped thrombi were found to be atheromatous. Nine cases (40.9%) on operative findings were atheroma (wavy: 4; circular shaped: 5). The crescent shape might correspond to fibrin thrombus. Atheromatous thrombus clamping near the renal artery was thought to cause microthromboembolism to surrounding organs. CONCLUSIONS: It is recommended that the more proximal aorta or splanchnic arteries should be temporarily clamped during proximal procedures in patients with wavy or circular shaped thrombi at the juxtarenal aorta to prevent kidney or bowel infarction.

Colonic epithelial cell proliferation decreases with increasing levels of serum 25-hydroxy vitamin D.

Epidemiological evidence suggests a potential role for vitamin D in colon cancer prevention. Vitamin D, absorbed from the intestine or derived from solar ultraviolet light, is metabolized in the liver to 25-hydroxyvitamin D (25-OH D(3)). Previous studies examining effects of vitamin D upon carcinogenesis have focused upon the active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)(2) D(3)], which interacts with nuclear vitamin D receptors in several organs. Until recently, the metabolism of 25-OH D(3) to 1,25-(OH)(2) D(3) was believed to occur only in the kidney, but more recent studies have shown that 25-OH D(3) conversion to 1,25-(OH)(2) D(3) can occur in other tissues. We examined the association between fasting levels of 25-OH D(3), 1,25-(OH)(2) D(3), and BsmI polymorphism of the vitamin D receptor (VDR) gene with indices of colonic epithelial cell proliferation and differentiation in a chemoprevention study, after giving vitamin D or calcium and taking rectal biopsies that were incubated with bromodeoxyuridine. Vitamin D receptor polymorphism was determined by genotyping of the 3' BsmI polymorphism in intron eight of the VDR gene. No significant changes in cell proliferation or in differentiation were found in subjects between study start and end. However, fasting serum levels of 25-OH D(3) showed a highly significant decrease with whole crypt labeling index and the size of the proliferative compartment (phi h). There was no correlation between serum levels of 1,25-(OH)(2) D(3) and the proliferative parameters. Calcium supplementation induced a significant effect upon the relationship between serum 25-OH D(3) and rectal epithelial cell labeling index and phi h when studied by covariance analysis without a relationship with 1,25-(OH)(2) D(3) levels. VDR genotype did not influence the effects of serum 25-OH D(3) or serum 1,25-(OH)(2) D(3) levels upon proliferation. These data suggest that there might be a local effect of 25-OH D(3) on colonic epithelial cells through conversion of 25-OH D(3) to 1,25-(OH)(2) D(3). Subsequent studies have demonstrated the presence of 1alpha-hydroxylase mRNA in normal colorectal epithelium and in colorectal cancer. Thus, vitamin D may have an important role in determining the effects of calcium on colorectal epithelial proliferation and may explain some of the discrepancies found previously in studies that examine the direct role of calcium on the colorectal epithelium.

Decreased expression of DNA-dependent protein kinase, a DNA repair protein, during human colon carcinogenesis.

DNA-dependent protein kinase (DNA-PK), consisting of a catalytic subunit (DNA-PKcs) and the Ku70 and Ku86 proteins, participates in the repair of DNA double-strand breaks (DSBs). We assessed its expression immunohistochemically in normal human colon tissue, colon adenomas, colon carcinomas, and normal tissue distant from carcinomas. Normal colonocytes expressed all DNA-PK proteins. Compared with the expression in normal tissue [176.62 +/- 18.56 (the intensity of expression x the percentage of cells expressing this protein), mean + SE], the expression of Ku70 was significantly reduced in adenomas (36.62 +/- 11.09; P < 0.001) and carcinomas (85.68 +/- 15.76; P < 0.01), as was the expression of Ku86 [(113.10 +/- 10.22 versus 41.66 +/- 14.71 in adenomas (P < 0.01) or versus 85.68 +/- 15.76 in carcinomas (P < 0.05)]. The expression of DNA-PKcs was not significantly changed. The marked underexpression of Ku70 and Ku86 starting at the adenoma stage may be crucial to the development of colon cancer.

Paget's disease of bone: a disease of the osteoclast.

Paget's disease is a chronic focal disease of the skeleton that affects up to 2-3% of the population over the age of 60 years. There is a genetic predisposition for Paget's disease, with one predisposition locus identified on chromosome 18q-21-22. Osteoclasts and osteoclast precursors from Paget's patients are abnormal and appear hyperresponsive to 1,25(OH)2D3 and RANK ligand and contain paramyxoviral transcripts (Fig. 1). The basis for the abnormalities detected in Paget's disease and the role that the paramyxoviruses may play in this disease are still unclear.

Osteoclast inhibitory peptide 2 inhibits osteoclast formation via its C-terminal fragment.

Osteoclast inhibitory peptide 2 (OIP-2) is a novel autocrine/paracrine factor produced by osteoclasts (OCLs) that inhibits bone resorption and OCL formation in vitro and in vivo. It is identical to the asparaginyl endopeptidase legumain. During maturation of OIP-2, a signal peptide and a 17-kDa C-terminal fragment (CTF) are cleaved to produce the mature enzyme. To determine if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His-tagged complementary DNA (cDNA) constructs for the CTF of OIP-2, the proform of OIP-2, and the "mature enzyme" form of OIP-2. The proform or the CTF portion of OIP-2 inhibited OCL formation in a dose-dependent manner in murine bone marrow cultures stimulated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The mature form of OIP-2, which was enzymatically active, did not inhibit OCL formation. In addition, OIP-2 inhibited OCL formation in cultures of highly purified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/ml of receptor activator of NF-kappaB (RANK) ligand. Binding studies with His-tagged OIP-2 showed expression of a putative OIP-2 receptor on RAW264.7 cells treated with RANK ligand for 4 days and human marrow cultures treated with 1,25(OH)2D3 for 3 weeks. These data show that the CTF of OIP-2, rather than the mature enzyme, mediates the inhibitory effects of OIP-2 through a putative receptor on OCL precursors.