A structure–function analysis in patients with prekallikrein deficiency

Objective: To investigate the structure–function relation in prekallikrein (PK) deficiency. PK is one of the proteins of the contact phase of blood coagulation which at the present time is the object of a revival of interest.

Methods: All patients with PK deficiency who had been investigated by molecular biology techniques are the object of the present investigation. Details of patients were obtained from personal files and a time-unlimited PubMed search. Only cases with a molecular-biology-based diagnosis were included.

Results: Twelve families were included. The total number of missense mutation was 10, together with 3 stop codons and 2 insertions. These mutations involved mainly exons 11 and 14. There were eight proved homozygotes and three compound heterozygotes. In one instance, homozygosity was probable but not proved. In nine cases, the defect was Type I, whereas it was Type II in the remaining three. No bleeding manifestations were present in 11 of the 12 probands. One proband had epistaxis, but she had hypertension. Altogether, four patients had hypertension and one of them had also two myocardial infarctions.

Conclusions: Despite the paucity of cases, it was established that the majority of mutations involved the catalytic domain. It is auspicable that future reports of patients with this disorder should include molecular studies. This would certainly contribute to the understanding of the contact phase of blood coagulation.     

Estrogen receptor-α in osteocytes is important for trabecular bone formation in male mice

The bone-sparing effect of estrogen in both males and females is primarily mediated via estrogen receptor-α (ERα), encoded by the Esr1 gene. ERα in osteoclasts is crucial for the trabecular bone-sparing effect of estrogen in females, but it is dispensable for trabecular bone in male mice and for cortical bone in both genders. We hypothesized that ERα in osteocytes is important for trabecular bone in male mice and for cortical bone in both males and females. Dmp1-Cre mice were crossed with ERα^flox/flox mice to generate mice lacking ERα protein expression specifically in osteocytes (Dmp1-ERα^−/−). Male Dmp1-ERα^−/− mice displayed a substantial reduction in trabecular bone volume (−20%, P < 0.01) compared with controls. Dynamic histomorphometry revealed reduced bone formation rate (−45%, P < 0.01) but the number of osteoclasts per bone surface was unaffected in the male Dmp1-ERα^−/− mice. The male Dmp1-ERα^−/− mice had reduced expression of several osteoblast/osteocyte markers in bone, including Runx2, Sp7, and Dmp1 (P < 0.05). Gonadal intact Dmp1-ERα^−/− female mice had no significant reduction in trabecular bone volume but ovariectomized Dmp1-ERα^−/− female mice displayed an attenuated trabecular bone response to supraphysiological E2 treatment. Dmp1-ERα^−/− mice of both genders had unaffected cortical bone. In conclusion, ERα in osteocytes regulates trabecular bone formation and thereby trabecular bone volume in male mice but it is dispensable for the trabecular bone in female mice and the cortical bone in both genders. We propose that the physiological trabecular bone-sparing effect of estrogen is mediated via ERα in osteocytes in males, but via ERα in osteoclasts in females.Bone mass is maintained by highly regulated processes involving osteoblastic bone formation and osteoclastic bone resorption. Estrogen is the major sex hormone involved in the regulation of bone mass in females and several studies demonstrate that estrogen is also of importance for the male skeleton (1–6). The biological effects of estradiol (E2) are mainly mediated by the nuclear estrogen receptors (ERs), ERα encoded by the Esr1 gene, and ERβ encoded by the Esr2 gene. The bone-sparing effect of estrogen in both males and females is primarily mediated via ERα (6–8), although the effect of ERα activation in bone might be slightly modulated by ERβ in female mice (9–12).Two studies using different strategies for the inactivation of ERα in osteoclasts have determined the role of ERα in osteoclasts for the bone-sparing effect of estrogen (13, 14). Nakamura et al. used Ctsk (Cathepsin K)-Cre mice to inactivate ERα in mature osteoclasts, resulting in trabecular bone loss caused by increased bone resorption in female but not male mice (14). In a separate study Martin-Millan et al. inactivated ERα in monocytes/osteoclast precursors using LysM-Cre mice and found that estrogen attenuates osteoclast generation and life span via cell-autonomous effects and that ERα in osteoclasts mediates the protective effect of estrogens on trabecular but not cortical bone in female mice (13). Collectively, these two studies clearly demonstrated that ERα in osteoclasts is crucial for trabecular bone in females, but it is dispensable for trabecular bone in male mice and for cortical bone in both males and females. However, not only osteoclasts but also osteoblasts/osteocytes express ERs (15–17). Several in vitro studies have suggested that ERα in osteoblasts/osteocytes is of importance for the regulation of bone metabolism, but this has not yet been demonstrated in vivo (2, 16, 18).It is well established that mechanical loading is a major regulator of cortical bone dimensions (19, 20). Previous studies have demonstrated that female but not male mice with ERα inactivation display reduced cortical osteogenic bone response to mechanical loading (19, 21–24). In addition, we recently showed that ERα is required for the cortical osteogenic response to mechanical loading in a ligand-independent manner, involving activation function-1 but not activation function-2 in ERα in female mice (20). The primary ERα target cell for this role of ERα in the osteogenic bone response to mechanical loading is not yet characterized, but a plausible candidate is the osteocytes, which are the mechanosensors in bone.Because ERα in osteoclasts is required for the bone-sparing effect of estrogen specifically in trabecular bone in female mice, we hypothesized that ERα in osteocytes might be crucial for trabecular bone in male mice, for cortical bone in both genders, and for the cortical osteogenic response to mechanical loading in female mice (13, 14). Therefore, dentin matrix protein (Dmp)1-Cre [Tg(Dmp1-cre)1Jqfe] mice were crossed with ERα^flox/flox (Esr1^tm1.1Gust) mice to generate mice lacking ERα protein expression specifically in osteocytes (Dmp1-ERα^−/−). The main finding from this in vivo study shows that ERα in osteocytes is important for trabecular bone formation in male mice.     

Thyroid structure and function in bovine β-mannosidosis

Summary In bovine -mannosidosis, the thyroid in the affected newborn shows marked cytoplasmic vacuolation. There is an associated reduction in the serum concentrations of thyroxine and tri-iodothyronine.     

Age-related changes in liver structure and function: Implications for disease ?

The geriatric populations of many countries are growing rapidly and they present major problems to healthcare infrastructures from both medical and economic perspectives. The elderly are predisposed to a variety of diseases, which contribute to a marked increase in morbidity in this subpopulation. The incidence of liver disease increases in the elderly, but the cellular and subcellular perturbations that underlie this suspected predisposition to pathology remain unresolved. Several age-related changes have been documented, including (a) a decline in liver volume, (b) an increase in the hepatic dense body compartment (lipofuscin), (c) moderate declines in the Phase I metabolism of certain drugs, (d) shifts in the expression of a variety of proteins and (e) diminished hepatobiliary functions. Other more subtle changes (e.g., muted responses to oxidative stress, reduced expression of growth regulatory genes, diminished rates of DNA repair, telomere shortening) may contribute to reduced hepatic regenerative capacity, shorter post-liver transplant survival and increased susceptibility to certain liver diseases in the elderly.     

Lung function abnormalities after bone marrow transplantation in children: has the trend recently changed?

STUDY OBJECTIVES: To evaluate early and late lung function abnormalities and their predictors in a large sample of children who underwent bone marrow transplantation (BMT) for leukemias in the 1990s, highlighting changes with respect to the 1980s. DESIGNS: Prospective cohort. SETTING: A university department of pediatrics. PARTICIPANTS: Seventy-five consecutive children who underwent BMT were enrolled in the study (median age, 11 years; range, 6 to 19 years; 45 male and 30 female children). Twenty-three children received autologous BMT, and 52 children received allogeneic BMT; 50 children completed the study. MEASUREMENTS: Clinical examinations and lung function tests were performed before BMT, and 3 to 6 months, 12 months, and 24 months after BMT. RESULTS: Before BMT, at 3 to 6 months after BMT, and at 24 months after BMT, 44%, 85%, and 62% of children, respectively, had altered lung function in the absence of persistent respiratory symptoms. Between 3 months and 6 months after BMT, a restrictive pattern was the most frequent abnormality. The only predictive factors for late abnormalities were transplantation performed in the advanced disease phase (odds ratio [OR], 6.75; p = 0.005) and bronchopulmonary infections (OR, 3.9; p < 0.05). CONCLUSIONS: These data suggest that a significant proportion of children who undergo BMT, especially if for leukemia in advanced phase, have early and late pulmonary abnormalities. These abnormalities, especially the late ones, seem to be more severe than patients reported in studies analyzing children undergoing BMT in the 1980s. This could be due to the more intensive front-line treatment protocols employed for treatment of children with acute leukemia in the 1990s.     

Importance of lipid–pore loop interface for potassium channel structure and function

Potassium (i.e., K⁺) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K⁺ channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K⁺ channel inactivation and exhibits a remarkable structural plasticity that correlates to K⁺ channel inactivation. The transmembrane helix 1 unwinds when the K⁺ channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K⁺ ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K⁺ channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K⁺ channel pore.     

Effects of experimentally induced hyperthyroidism on central hypothalamic–pituitary–adrenal axis function in rats: in vitro and in situ studies

Hyperthyroidism is associated with hypercorticosteronemia, although the locus that is principally responsible for the hypercorticosteronism remains unclear. The purpose of this study was to assess the effects of hyperthyroidism on the functional integrity of the hypothalamic–pituitary–adrenal (HPA) axis, to identify the locus in the HPA axis that is principally affected, and address the time-dependent effects of alterations in thyroid status. The functional integrity of each component of the HPA axis was examined in vitro and in situ in sham-thyroidectomized male Sprague–Dawley rats given placebo or in thyroidectomized rats given pharmacological dose (50 μg) of thyroxin for 7 or 60 days. Basal plasma corticosterone and corticosterone binding globulin (CBG) concentrations were significantly increased in short- and long-term hyperthyroid rats, and by 60 days. Basal plasma ACTH levels were similar to controls. Both hypothalamic CRH content and the magnitude of KCL- and arginine vasopressin (AVP)-induced CRH release from hypothalamic culture were increased in long-term hyperthyroid rats. There was a significant increase in the content of both ACTH and β-endorphin in the anterior pituitaries of both short- and long-term hyperthyroid animals. Short-term hyperthyroid rats showed a significant increase in basal POMC mRNA expression in the anterior pituitary, and chronically hyperthyroid animals showed increased stress-induced POMC mRNA expression. Adrenal cultures taken from short-term hyperthyroid rats responded to exogenous ACTH with an exaggerated corticosterone response, while those taken from 60-day hyperthyroid animals showed responses similar to controls. The findings show that hyperthyroidism is associated with hypercorticosteronemia and HPA axis dysfunction that becomes more pronounced as the duration of hyperthyroidism increases. The evidence suggests that experimentally induced hyperthyroidism is associated with central hyperactivity of the HPA axis.     

Blood–brain barrier structure and function and the challenges for CNS drug delivery

The neurons of the central nervous system (CNS) require precise control of their bathing microenvironment for optimal function, and an important element in this control is the blood–brain barrier (BBB). The BBB is formed by the endothelial cells lining the brain microvessels, under the inductive influence of neighbouring cell types within the ‘neurovascular unit’ (NVU) including astrocytes and pericytes. The endothelium forms the major interface between the blood and the CNS, and by a combination of low passive permeability and presence of specific transport systems, enzymes and receptors regulates molecular and cellular traffic across the barrier layer. A number of methods and models are available for examining BBB permeation in vivo and in vitro, and can give valuable information on the mechanisms by which therapeutic agents and constructs permeate, ways to optimize permeation, and implications for drug discovery, delivery and toxicity. For treating lysosomal storage diseases (LSDs), models can be included that mimic aspects of the disease, including genetically-modified animals, and in vitro models can be used to examine the effects of cells of the NVU on the BBB under pathological conditions. For testing CNS drug delivery, several in vitro models now provide reliable prediction of penetration of drugs including large molecules and artificial constructs with promising potential in treating LSDs. For many of these diseases it is still not clear how best to deliver appropriate drugs to the CNS, and a concerted approach using a variety of models and methods can give critical insights and indicate practical solutions.     

Administration of insulin–like growth factor–1 (IGF–1) improves both structure and function of delta–sarcoglycan deficient cardiac muscle in the hamster

Dilated cardiomyopathies (DCM) are due to progressive dilatation of the cardiac cavities and thinning of the ventricular walls and lead unavoidably to heart failure. They represent a major cause for heart transplantation and, therefore, defining an efficient symptomatic treatment for DCM remains a challenge. We have taken advantage of the hamster strain CHF147 that displays progressive cardiomyopathy leading to heart failure to test whether stimulation of a hypertrophic pathway could delay the process of dilatation.Six month old CHF147 hamsters were treated with IGF–1 so that we could compare the efficacy of systemic administration of human recombinant IGF–1 protein (rh IGF–1) at low dose to that of direct myocardial injections of a plasmid DNA containing IGF–1 cDNA (pCMV–IGF1).IGF-1 treatment did not induce a significant variation of ventricle mass, but preserved left ventricular (LV) wall thickness and delayed dilatation of cardiac cavities when compared to non–treated hamsters. Together with this reduction of dilatation, we also noted a reduction in the amount of interstitial collagen. Furthermore, IGF–1 treatment induced beneficial effects on cardiac function since treated hamsters presented improved cardiac output and stroke volume, decreased end diastolic pressure when compared to nontreated hamsters and also showed a trend towards increased contractility (dP/dt_max).This study provides evidence that IGF–1 treatment induces beneficial structural and functional effects on DCM of CHF147 hamsters, hence making this molecule a promising candidate for future gene therapy of heart failure due to DCM.     

Role of helix 8 in G protein-coupled receptors based on structure–function studies on the type 1 angiotensin receptor

G protein-coupled receptors (GPCRs) are transmembrane receptors that convert extracellular stimuli to intracellular signals. The type 1 angiotensin II receptor is a widely studied GPCR with roles in blood pressure regulation, water and salt balance and cell growth. The complex molecular and structural changes that underpin receptor activation and signaling are the focus of intense research. Increasingly, there is an appreciation that the plasma membrane participates in receptor function via direct, physical interactions that reciprocally modulate both lipid and receptor and provide microdomains for specialized activities. Reversible protein:lipid interactions are commonly mediated by amphipathic α-helices in proteins and one such motif – a short helix, referred to as helix VIII/8 (H8), located at the start of the carboxyl (C)-terminus of GPCRs – is gaining recognition for its importance to GPCR function. Here, we review the identification of H8 in GPCRs and examine its capacity to sense and interact with diverse proteins and lipid environment, most notably with acidic lipids that include phosphatidylinositol phosphates.     

Sexual function and patients' perceptions in inflammatory bowel disease: a case–control survey

Background

Sexuality is important when assessing quality of life (QoL), which is often disturbed in inflammatory bowel disease (IBD). However, sexuality is not addressed in most QoL questionnaires.

Aims

To evaluate the prevalence and predisposing factors of sexual dysfunction among IBD patients, and their own perception.

Methods

A postal survey was conducted in IBD patients 25–65 years of age from two tertiary centres. Patients were asked to provide a control of the same gender and age without IBD. The questionnaire assessed patient perception of the impact of IBD on their sexuality, and also allowed calculation of the Erectile Function International Index or the Female Sexual Function Index.

Results

A total of 355 patients and 200 controls were available for the final analysis. Both groups were comparable except for a higher proportion of individuals who had been treated for depression among patients. Half of the female and one-third of the male patients considered that both sexual desire and satisfaction worsened after IBD diagnosis. As compared to controls, both men and women with IBD showed significantly lower scores in sexual function indexes, but a higher prevalence of sexual dysfunction was only noticed among women. Independent predictors of sexual dysfunction among IBD patients were the use of corticosteroids in women, and the use of biological agents, depression and diabetes in men.

Conclusions

Sexuality is often disturbed in IBD patients, particularly among women. Many factors seem to contribute to worsened intimacy. Sexuality should be considered when QoL is assessed in these patients.     

Trends in atrial fibrillation ablation: have we maximized the current paradigms?

Purpose

The purpose of this study was to evaluate how atrial fibrillation (AF) ablation has evolved over time with regards to patient characteristics, procedural variables, complications, and outcomes.

Methods

We evaluated trends over time from 2003 to 2010 in clinical characteristics, procedural variables, complications, and Kaplan?CMeier AF-free rates after the initial and final AF ablation in 1,125 patients undergoing 1,504 ablations.

Results

Evaluating trends from 2003 to 2010, we found that patients undergoing AF ablation became older (P?2 scores (P?P?=?0.021), persistent AF (P?P?P?=?0.005). Procedure times decreased from 256?±?49 to 122?±?28?min (P?P?P?=?0.023), minor (P?=?0.023), and total complications (P?=?0.001) decreased over time. The learning curve to minimize complications was 6?years. For paroxysmal AF, initial ablation AF-free rates improved over time (P?=?0.015) but improvement plateaued in recent years. For persistent AF, initial ablation AF-free rates trended toward improvement over time (P?=?0.062) but also plateaued in recent years. For long-standing persistent AF (P?=?0.995), there was no outcome improvement after initial ablation over time. There was no trend for improved final outcomes (including repeat ablations) over time for paroxysmal, persistent, or long-standing AF (P?=?0.150 to P?=?0.978).

Conclusions

Despite decreased procedural and fluoroscopy times and reduced complication rates, post-ablation freedom from AF has not improved commensurately in recent years. A better understanding of AF initiation and maintenance may be required to devise personalized approaches to AF ablation and further improve outcomes.     

Thioredoxin-interacting protein and myocardial mitochondrial function in ischemia–reperfusion injury

Cellular metabolism and reactive oxygen species (ROS) formation are interrelated processes in mitochondria and are implicated in a variety of human diseases including ischemic heart disease. During ischemia, mitochondrial respiration rates fall. Though seemingly paradoxical, reduced respiration has been observed to be cardioprotective due in part to reduced generation of ROS. Enhanced myocardial glucose uptake is considered beneficial for the myocardium under stress, as glucose is the primary substrate to support anaerobic metabolism. Thus, inhibition of mitochondrial respiration and uncoupling oxidative phosphorylation can protect the myocardium from irreversible ischemic damage. Growing evidence now positions the TXNIP/thioredoxin system at a nodal point linking pathways of antioxidant defense, cell survival, and energy metabolism. This emerging picture reveals TXNIP’s function as a regulator of glucose homeostasis and may prove central to regulation of mitochondrial function during ischemia. In this review, we summarize how TXNIP and its binding partner thioredoxin act as regulators of mitochondrial metabolism. While the precise mechanism remains incompletely defined, the TXNIP–thioredoxin interaction has the potential to affect signaling that regulates mitochondrial bioenergetics and respiratory function with potential cardioprotection against ischemic injury.     

Churg–Strauss syndrome presenting with eosinophilic myocarditis: A diagnostic challenge

Churg–Strauss syndrome (CSS) is an unusual disease that presents as systemic vasculitis and peripheral eosinophilia in patients with an atopic constitution. Cardiac involvement is unusual and often not prominent on initial presentation, but is an important cause of morbidity and mortality in patients with CSS. We report the case of a young woman with severe acute myocarditis. Coronary arteriography demonstrated extensive focal vasculopathy, consistent with coronary vasculitis, and myocardial biopsy showed eosinophilic myocarditis. This presentation led to an initial diagnosis of CSS in this patient and appropriate therapy resulted in a spectacular remission of disease activity.