Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli.

To improve our understanding of the mechanism that couples nucleotide-excision repair to transcription in expressed genes, we have examined the effects of mutations in several different DNA repair genes on the removal of cyclobutane pyrimidine dimers from the individual strands of the induced lactose operon in UV-irradiated Escherichia coli. As expected, we found little repair in either strand of the lactose operon in strains with mutations in established nucleotide excision-repair genes (uvrA, uvrB, uvrC, or uvrD). In contrast, we found that mutations in either of two genes required for DNA-mismatch correction (mutS and mutL) selectively abolish rapid repair in the transcribed strand and render the cells moderately sensitive to UV irradiation. Similar results were found in a strain with a mutation in the mfd gene, the product of which has been previously shown to be required for transcription-coupled repair in vitro. Our results demonstrate an association between mismatch-correction and nucleotide-excision repair and implicate components of DNA-mismatch repair in transcription-coupled repair. In addition, they may have important consequences for human disease and may enhance our understanding of the etiology of certain cancers which have been associated with defects in mismatch correction.

Myelin-associated neurite growth-inhibitory proteins and suppression of regeneration of immature mammalian spinal cord in culture.

Neurite outgrowth across spinal cord lesions in vitro is rapid in preparations isolated from the neonatal opossum Monodelphis domestica up to the age of 12 days. At this age oligodendrocytes, myelin, and astrocytes develop and regeneration ceases to occur. The role of myelin-associated neurite growth-inhibitory proteins, which increase in concentration at 10-13 days, was investigated in culture by applying the antibody IN-1, which blocks their effects. In the presence of IN-1, 22 out of 39 preparations from animals aged 13-17 days showed clear outgrowth of processes into crushes. When 34 preparations from 13-day-old animals were crushed and cultured without antibody, no axons grew into the lesion. The success rate with IN-1 was comparable to that seen in younger animals but the outgrowth was less profuse. IN-1 was shown by immunocytochemistry to penetrate the spinal cord. Other antibodies which penetrated the 13-day cord failed to promote fiber outgrowth. To distinguish between regeneration by cut neurites and outgrowth by developing uncut neurites, fibers in the ventral fasciculus were prelabeled with carbocyanine dyes and subsequently injured. The presence of labeled fibers in the lesion indicated that IN-1 promoted regeneration. These results show that the development of myelin-associated growth-inhibitory proteins contributes to the loss of regeneration as the mammalian central nervous system matures. The definition of a critical period for regeneration, coupled with the ability to apply trophic as well as inhibitory molecules to the culture, can permit quantitative assessment of molecular interactions that promote spinal cord regeneration.

Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

Function of von Willebrand factor after crossed bone marrow transplantation between normal and von Willebrand disease pigs: effect on arterial thrombosis in chimeras.

von Willebrand factor (vWF) is essential for the induction of occlusive thrombosis in stenosed and injured pig arteries and for normal hemostasis. To separate the relative contribution of plasma and platelet vWF to arterial thrombosis, we produced chimeric normal and von Willebrand disease pigs by crossed bone marrow transplantation; von Willebrand disease (vWD) pigs were engrafted with normal pig bone marrow and normal pigs were engrafted with vWD bone marrow. Thrombosis developed in the chimeric normal pigs that showed normal levels of plasma vWF and an absence of platelet vWF; but no thrombosis occurred in the chimeric vWD pigs that demonstrated normal platelet vWF and an absence of plasma vWF. The ear bleeding times of the chimeric pigs were partially corrected by endogenous plasma vWF but not by platelet vWF. Our animal model demonstrated that vWF in the plasma compartment is essential for the development of arterial thrombosis and that it also contributes to the maintenance of bleeding time and hemostasis.

Body composition and bone mineral density in male adults with hyperthyroidism. Which association?

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Vitamin D in patients with atherosclerosis : a link between atherosclerosis and bone mass ?

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Alcoholic neurobiology: Changes in dependence and recovery

This article presents the proceedings of a symposium held at the meeting of the International Society for Biomedical Research on Alcoholism (ISBRA) in Mannheim, Germany, in October, 2004. Chronic alcoholism follows a fluctuating course, which provides a naturalistic experiment in vulnerability, resilience, and recovery of human neural systems in response to presence, absence, and history of the neurotoxic effects of alcoholism. Alcohol dependence is a progressive chronic disease that is associated with changes in neuroanatomy, neurophysiology, neural gene expression, psychology, and behavior. Specifically, alcohol dependence is characterized by a neuropsychological profile of mild to moderate impairment in executive functions, visuospatial abilities, and postural stability, together with relative sparing of declarative memory, language skills, and primary motor and perceptual abilities. Recovery from alcoholism is associated with a partial reversal of CNS deficits that occur in alcoholism. The reversal of deficits during recovery from alcoholism indicates that brain structure is capable of repair and restructuring in response to insult in adulthood. Indirect support of this repair model derives from studies of selective neuropsychological processes, structural and functional neuroimaging studies, and preclinical studies on degeneration and regeneration during the development of alcohol dependence and recovery from dependence. Genetics and brain regional specificity contribute to unique changes in neuropsychology and neuroanatomy in alcoholism and recovery. This symposium includes state-of-the-art presentations on changes that occur during active alcoholism as well as those that may occur during recovery-abstinence from alcohol dependence. Included are human neuroimaging and neuropsychological assessments, changes in human brain gene expression, allelic combinations of genes associated with alcohol dependence and preclinical studies investigating mechanisms of alcohol induced neurotoxicity, and neuroprogenetor cell expansion during recovery from alcohol dependence.

Hearing sensitivity and bone mineral density in older adults: the Health, Aging and Body Composition Study

Bone mineral density (BMD) may be associated with hearing loss in older adults. Demineralization of the cochlear capsule has been associated with hearing loss in those with Paget's disease of the bone and otosclerosis. Osteoporosis may also result in cochlear capsule demineralization. We hypothesized that lower hip BMD and lower heel ultrasound measurements would be associated with hearing loss in a population-based sample of 2,089 older black and white men and women. Bone parameters and hearing function were measured at the fourth clinical follow-up visit. Audiometric threshold testing was used to measure air- and bone-conduction hearing sensitivity. BMD of the hip and its subregions was measured using dual-energy X-ray absorptiometry. Calcaneal bone measurements [broadband ultrasound attenuation (BUA), speed of sound (SOS) and the quantitative ultrasound index (QUI)] were obtained using heel ultrasound. After adjusting for known hearing loss risk factors, no association was found between hearing and any of the bone measurements in whites and black women. In black men, however, lower hip BMD was associated with higher odds of hearing loss; for each standard deviation decrease in total hip BMD, the odds of hearing loss were 1.41 (95% confidence interval 1.08, 1.83), 1.39 (95% CI 1.07, 1.82) for femoral neck BMD and 1.65 (95% CI 1.26, 2.16) for trochanter BMD. Conductive hearing loss was associated with lower heel ultrasound measurements, though only among white men. The results of this study are mixed and inconclusive. Lower BMD of the hip and its subregions was associated with hearing loss among black men, but not among whites or black women. Lower measurements on heel ultrasound were associated with conductive hearing loss, though only among white men. These results suggest that axial and appendicular bone parameters may be modestly associated with hearing loss in older men, but not in women.

Renal structural and functional repair in a mouse model of reversal of ureteral obstruction

The end point of immune and nonimmune renal injury typically involves glomerular and tubulointerstitial fibrosis. Although numerous studies have focused on the events that lead to renal fibrosis, less is known about the mechanisms that promote cellular repair and tissue remodeling. Described is a model of renal injury and repair after the reversal of unilateral ureteral obstruction (UUO) in male C57b1/6J mice. Male mice (20 to 25 g) underwent 10 d of UUO with or without 1, 2, 4, or 6 wk of reversal of UUO (R-UUO). UUO resulted in cortical tubular cell atrophy and tubular dilation in conjunction with an almost complete ablation of the outer medulla. This was associated with interstitial macrophage infiltration; increased hydroxyproline content; and upregulated type I, III, IV, and V collagen expression. The volume density of kidney occupied by renal tubules that exhibited a brush border was measured as an assessment of the degree of repair after R-UUO. After 6 wk of R-UUO, there was an increase in the area of kidney occupied by repaired tubules (83.7 +/- 5.9%), compared with 10 d UUO kidneys (32.6 +/- 7.3%). This coincided with reduced macrophage numbers, decreased hydroxyproline content, and reduced collagen accumulation and interstitial matrix expansion, compared with obstructed kidneys from UUO mice. GFR in the 6-wk R-UUO kidneys was restored to 43 to 88% of the GFR in the contralateral unobstructed kidneys. This study describes the regenerative potential of the kidney after the established interstitial matrix expansion and medullary ablation associated with UUO in the adult mouse.

Hypothalamic control of bone formation: Distinct actions of leptin and Y2 receptor pathways

Leptin and Y2 receptors on hypothalamic NPY neurons mediate leptin effects on energy homeostasis; however, their interaction in modulating osteoblast activity is not established. Here, direct testing of this possibility indicates distinct mechanisms of action for leptin anti-osteogenic and Y2(-/-) anabolic pathways in modulating bone formation. Introduction: Central enhancement of bone formation by hypothalamic neurons is observed in leptin-deficient oblob and Y2 receptor null mice. Similar elevation in central neuropeptide Y (NPY) expression and effects on osteoblast activity in these two models suggest a shared pathway between leptin and Y2 receptors in the central control of bone physiology. The aim of this study was to test whether the leptin and Y2 receptor pathways regulate bone by the same or distinct mechanisms. Materials and Methods: The interaction of concomitant leptin and Y2 receptor deficiency in controlling bone was examined in Y2(-/-) oblob double mutant mice, to determine whether leptin and Y2 receptor deficiency have additive effects. Interaction between leptin excess and Y2 receptor deletion was examined using recombinant adeno-associated viral vector overproduction of NPY (AAV-NPY) to produce weight gain and thus leptin excess in adult Y2(-/-) mice. Cancellous bone volume and bone cell function were assessed. Results: Osteoblast activity was comparably elevated in oblob, Y2(-/-), and Y2(-/-) oblob mice. However, greater bone resorption in oblob and Y2(-/-) oblob mice reduced cancellous bone volume compared with Y2(-/-). Both wildtype and Y2(-/-) AAV-NPY mice exhibited marked elevation of white adipose tissue accumulation and hence leptin expression, thereby reducing osteoblast activity. Despite this anti-osteogenic leptin effect in the obese AAV-NPY model, osteoblast activity in Y2(-/-) AAV-NPY mice remained significantly greater than in wildtype AAV-NPY mice. Conclusions: This study suggests that NPY is not a key regulator of the leptin-dependent osteoblast activity, because both the leptin-deficient stimulation of bone formation and the excess leptin inhibition of bone formation can occur in the presence of high hypothalamic NPY. The Y2(-/-) pathway acts consistently to stimulate bone formation; in contrast, leptin continues to suppress bone formation as circulating levels increase. As a result, they act increasingly in opposition as obesity becomes more marked. Thus, in the absence of leptin, the cancellous bone response to loss of Y2 receptor and leptin activity can not be distinguished. However, as leptin levels increase to physiological levels, distinct signaling pathways are revealed.

Inhibitors of cyclo-oxygenase-2 and secretory phospholipase A2 preserve bone architecture following ovariectomy in adult rats

Epidemiological evidence and in vitro data suggest that COX-2 is a key regulator of accelerated remodeling. Accelerated states of osteoblast and osteoclast activity are regulated by prostaglandins in vitro, but experimental evidence for specific roles of cyclooxygenase-2 (COX-2) and secretory phospholipase A(2) (sPLA(2)) in activated states of remodeling in vivo is lacking. The aim of this study was to determine the effect of specific inhibitors of sPLA(2)-IIa and COX-2 on bone remodeling activated by estrogen deficiency in adult female rats. One hundred and twenty-four adult female Wistar rats were ovariectomized (OVX) or sham-operated. Rats commenced treatment 14 days after surgery with either vehicle, a COX-2 inhibitor (DFU at 0.02 mg/kg/day and 2.0 mg/kg/day) or a sPLA(2)-group-IIa inhibitor (KH064 at 0.4 mg/kg/day and 4.0 mg/kg/day). Treatment continued daily until rats were sacrificed at 70 days or 98 days post-OVX. The right tibiae were harvested, fixed and embedded in methylmethacrylate for structural histomorphometric bone analysis at the proximal tibial metaphysis. The specific COX-2 or sPLA(2) inhibitors prevented ovariectomy-induced (OVX-induced) decreases in trabecular connectivity (P < 0.05); suppressed the acceleration of bone resorption; and maintained bone turnover at SHAM levels following OVX in the rat. The sPLA2 inhibitor significantly suppressed increases in osteoclast surface induced by OVX (P < 0.05), while the effect of COX-2 inhibition was less marked. These findings demonstrate that inhibitors of COX-2 and sPLA(2)-IIa can effectively suppress OVX-induced bone loss in the adult rat by conserving trabecular bone mass and architecture through reduced bone remodeling and decreased resorptive activity. Moreover, we report an important role of sPLA(2)-IIa in osteoclastogenesis that may be independent of the COX-2 metabolic pathway in the OVX rat in vivo. (c) 2006 Elsevier Inc. All rights reserved.

Mechanisms of hematopoietic stem cell mobilization: When innate immunity assails the cells that make blood and bone

Mobilization is now used worldwide to collect large numbers of hematopoietic stem and progenitor cells (HSPCs) for transplantation. Although the first mobilizing agents were discovered largely by accident, discovery of more efficient mobilizing agents will require a better understanding of the molecular mechanisms responsible. During the past 5 years, a number of mechanisms have been identified, shedding new light on the dynamics of the hematopoietic system in vivo and on the intricate relationship between hematopoiesis, innate immunity, and bone. After briefly reviewing the mechanisms by which circulating HSPCs home into the bone marrow and what keeps them there, the current knowledge of mechanisms responsible for HSPC mobilization in response to hematopoietic growth factors such as granulocyte colony-stimulating factor, chemotherapy, chemokines, and polyanions will be discussed together with current strategies developed to further increase HSPC mobilization. (c) 2006 International Society for Experimental Hematology.

Overexpression of transforming growth factor-beta is associated with increased hyaluronan content and impairment of repair in Marfan syndrome aortic aneurysm

Background-Marfan syndrome (MFS), a condition caused by fibrillin-1 gene mutation is associated with aortic aneurysm that shows elastic lamellae disruption, accumulation of glycosaminoglycans, and vascular smooth muscle cell (VSMC) apoptosis with minimal inflammatory response. We examined aneurysm tissue and cultured cells for expression of transforming growth factor-beta1 to -beta3 (TGF beta 1 to 3), hyaluronan content, apoptosis, markers of cell migration, and infiltration of vascular progenitor cells (CD34). Methods and Results-MFS aortic aneurysm (6 males, 5 females; age 8 to 78 years) and normal aorta (5 males, 3 females; age 22 to 56 years) were used. Immunohistochemistry showed increased expression of TGF beta 1 to 3, hyaluronan, and CD34-positive microcapillaries in MFS aneurysm compared with control. There was increased expression of TGF beta 1 to 3 and hyaluronan in MFS cultured VSMCs, adventitial fibroblasts (AF), and skin fibroblasts (SF). Apoptosis was increased in MFS (VSMC: mean cell loss in MFS 29%, n of subjects = 5, versus control 8%, n = 3, P < 0.05; AF: 28%, n = 5 versus 7%, n = 5, P < 0.05; SF: 29%, n = 3 versus 4%, n = 3, not significant). In MFS, there was a 2-fold increase in adventitial microcapillaries containing CD34-positive cells compared with control tissue. Scratch wound assay showed absence of CD44, MT1-MMP, and beta-3 integrin at the leading edge of migration in MFS indicating altered directional migration. Western blot showed increased expression of TGF beta 1 to 3 in MFS but no change in expression of CD44, MT1-MMP, or beta-3 integrin compared with controls. Conclusions-There was overexpression of TGF-beta in MFS associated with altered hyaluronan synthesis, increased apoptosis, impaired progenitor cell recruitment, and abnormal directional migration. These factors limit tissue repair and are likely to contribute to aneurysm development.

Regulation of bone biology by prostaglandin endoperoxide H synthases (PGHS): A rose by any other name...

It is well established that prostaglandins are essential mediators of bone resorption and formation. In the early 1990s, it was discovered that enzymatic reactions producing prostaglandins were regulated by two cyclooxygenase enzymes, one producing prostaglandins constitutively in tissues like the stomach, prostaglandin endoperoxide H synthase-1 (PGHS-1 or COX-1), and another induced by mitogens or inflammatory mediators (PGHS-2 or COX-2). This neat distinction has not been maintained because both enzymes act in different cell systems to provide physiological signaling, constitutively or by induction under certain conditions. For example, the regulation patterns of PGHS-1 and PGHS-2 are distinct, but the evidence shows that PGHS-2 functions constitutively in the skeleton. PGHS-2 hits quickly been established, therefore, as a key regulator of bone biology, capable of rapid and transient expression in bone cells, and mediating osteoclastogenesis, mechanotransduction, bone formation and fracture repair. The goal of this review is to Summarize the current state of our knowledge of PGHS regulation of bone metabolism and to identify some of the key unresolved challenges and questions that require further study. (c) 2006 Elsevier Ltd. All rights reserved.

Relationship between bicarbonate retention and bone characteristics in broiler chickens

Determination of the bicarbonate retention factor (BRF) is an important step during development of the indicator amino acid oxidation technique for use in a new model. A series of 4-h oxidation experiments were performed to determine the BRF of broilers aged 7, 14, 21, 28, 35, and 42 d using 4 birds per age group. A priming dose of 1.2 mu Ci of (NaHCO3)-C-14, followed by eight half-hourly doses of 1 mu Ci of (NaHCO3)-C-14 were given orally to each of 4 birds per age. The percentage of 14 C dose expired by the bird at a steady state was measured. These birds, as well as 12 additional birds matched for age and BW, were killed, and femur bone mineral density was measured by quantitative computed tomography to determine the relationship between bone development and bicarbonate retention at each age. There was a correlation (r = 0.50; P < 0.05) between total cross-sectional femur bone mineral density and bicarbonate retention at each age. A prediction equation (Y = 6.95 x 10(-2) X - 3.51 x 10(5)X(2) + 27.58; P < 0.0001, R-2 = 0.79) where Y = bicarbonate retention and X = BW was generated to predict Y as a function of X. Bicarbonate retention values peaked at 28 d, during the stage of the most rapid bone deposition and the highest growth rate. A constant BRF was found from 1,900 to 2,700 g of BW of 35.15 +/- 1.095% (mean SEM). This retention factor will allow the accurate correction of oxidation of C-14-labeled substrates in broilers of different ages and BW in future indicator amino acid oxidation studies.