The Effect of Cell Cycle and Expression of Cyclin B1 and Cyclin C Protein in Hepatocellular Carcinoma Cell Line HepG2 and SMMC-7721 after of Silencing β-Catenin Gene

Background/Aims: Abnormalities in cell cycle regulation are reported to be strongly associated with tumorigenesis and progression of tumors. Wnt/β-catenin signaling pathway and cell cycle play key roles during the genesis and development of hepatocellular carcinoma (HCC). Current studies indicated that expressions of cyclin A, E and D1 were affected after silencing of β-catenin gene in HCC, but it is unclear if other cyclins are affected. Methodology: To determine the relation, small interference RNA (siRNA) against β-catenin was transfected into HCC cell lines HepG2 and SMMC-7721, and cell cycle and cyclin B1 and cyclin C protein expression were detected. Results: Cell cycle was arrested in G0/G1 at 72h after transfection and the cell cycle began to transfer from G0/G1 to G2/M through S and had a trend to revert at 96h. In addition, β-catenin protein expression was decreased at both 72 and 96h, although the level was slightly higher at 96h than that at 72h. However, cyclin B1 expression decreased at 72h and increased at 96h, cyclin C expression increased at 72h and decreased at 96h. Conclusions: These findings suggest that silencing β-catenin gene may induce the changes of cell cycle and cyclin B1 and cyclin C protein expression. Wnt/β-catenin signaling pathway probably takes part in the genesis and development of HCC through regulating cell cycle and the expression of cyclin B1 and cyclin C.     

Peripheral Blood Lymphocyte β2 Integrin and ICAM Expression in Inflammatory Bowel Disease

The 2 integrin intercellular adhesionmolecule (ICAM) adhesion pathway is likely pivotal inthe immunopathogenesis of inflammatory bowel disease(IBD). We have undertaken a comprehensive study ofperipheral blood lymphocyte (PBL) expression of all2 integrins and ICAMs in patients with IBD usingflow cytometry and assessed our data on the basis of IBDdiagnosis, disease state of activity, and use ofcorticosteroids. Blood was collected from patients with Crohn'sdisease (N = 49), ulcerative colitis (N = 43), andnormal control volunteers (N = 15). Mononuclear cellswere separated using a Ficoll-Hypaque gradient and prepared for flow cytometry. The data wereanalyzed for percentage expression, mean fluorescentintensity (MFI) as well as for histogram patterns. Theanalysis was stratified for disease diagnosis, disease activity level, and for use of prednisone amongpatients with active disease. There was decreasedpercentage expression of CD11a, CD18, and ICAM-3 inCrohn's disease and ulcerative colitis compared with normal, but an increased MFI for thesemolecules among patients with Crohn's disease. ActiveCrohn's disease showed a greater change in this patterncompared with both inactive disease and activeulcerative colitis. CD11a and CD18 histograms typicallyhad two peaks of expression. The predominance of onepeak over the other varied with disease diagnosis andactivity. CD11b and alphad expression patterns were not different in IBD compared with normal.CD11c was not expressed by PBLs and, ICAM-2, typicallyan endothelial ligand, was expressed on PBLs. There werechanges in the expression of 2 integrins in IBD, which were more evident in Crohn's diseasethan ulcerative colitis. We hypothesize that thedecreased percentage expression and increased MFI ofCD11a, CD18, and ICAM-3 may suggest that cellsup-regulate these ligands following activation and areegressing into tissue.     

Growth Arrest and Decrease of α-SMA and Type I Collagen Expression by Palmitic Acid in the Rat Hepatic Stellate Cell Line PAV-1

Liver fibrosis is characterized by an activation of hepatic stellate cells (HSC). During primary culture HSC evolve from a quiescent into an activated phenotype which is characterized by α-smooth muscle actin (α-SMA) up-regulation, increase in cell growth, and extracellular matrix secretion. HSC culture with trans-resveratrol can lead to deactivation of myofibroblast-like HSC. We used an HSC line, PAV-1, to check the role of retinol and palmitic acid in the deactivation process of HSC. Using mass and metabolic-based methods, Western blot and immunocytochemistry assays, we demonstrated that treatment with palmitic acid (75 μM) alone or in combination with retinol (2 μM) significantly decreased cell proliferation and α-SMA expression. We also established that the association of both compounds strongly decreased collagen type I expression. Our results suggest the potential use of palmitic acid alone or in combination with retinol to induce HSC deactivation.     

Biomarkers of β-Cell Stress and Death in Type 1 Diabetes

The hallmark of type 1 diabetes (T1D) is a decline in functional β-cell mass arising as a result of autoimmunity. Immunomodulatory interventions at disease onset have resulted in partial stabilization of β-cell function, but full recovery of insulin secretion has remained elusive. Revised efforts have focused on disease prevention through interventions administered at earlier disease stages. To support this paradigm, there is a parallel effort ongoing to identify circulating biomarkers that have the potential to identify stress and death of the islet β-cells. Whereas no definitive biomarker(s) have been fully validated, several approaches hold promise that T1D can be reliably identified in the pre-symptomatic phase, such that either β-cell preservation or immunomodulatory agents might be employed in at-risk populations. This review summarizes the most promising protein- and nucleic acid-based biomarkers discovered to date and reviews the context in which they have been studied.     

Assessment of Myeloperoxidase and Oxidative α1-Antitrypsin in Patients on Hemodialysis

Background and objectives: The present study assesses the effects of the oxidative stress marker, myeloperoxidase (MPO), and the possible MPO-related oxidative stress marker, oxidative α₁-antitrypsin (oxAT), on carotid intima-media thickness (CIMT) and protein-energy wasting (PEW) in patients on hemodialysis (HD).Design, setting, participants, & measurements: Blood samples were obtained from 383 patients before HD to measure WBC count, serum albumin, lipids, high-sensitivity C-reactive protein (CRP), α₁-antitrypsin (AT), interleukin-6, oxidative LDL-C, MPO, and oxAT. We assessed both CIMT and the geriatric nutritional risk index (GNRI) in this cross-sectional competitive study.Results: Levels of MPO and oxAT correlated. Myeloperoxidase was associated with max-CIMT, and oxAT correlated with max-CIMT and GNRI. Multivariate linear regression models showed that MPO and oxAT were independent predictors of increasing max-CIMT, whereas oxAT, but not MPO, independently correlated with GNRI. In four combined MPO and oxAT groups classified according to median values, a multinomial logistic regression model showed that high MPO together with high oxAT was independently associated with increased max-CIMT. Moreover, the OR for max-CIMT with positive PEW and high MPO was significantly increased in the four groups with combined MPO and PEW.Conclusions: High MPO with high oxAT and high MPO with PEW seem to contribute to plaque formation in patients on HD, whereas elevated MPO or oxAT alone might not predict increasing CIMT. In contrast, a high oxAT value seems to be an independent predictor of PEW in patients on HD.Ahigh prevalence of cardiovascular disease (CVD) events and of protein-energy wasting (PEW) is significantly associated with increased mortality and morbidity among patients undergoing hemodialysis (HD) (1). The progression of atherosclerosis and PEW in such patients is accelerated by various factors, in particular oxidative stress and inflammation. Oxidative stress is closely associated with advancing atherosclerosis (2) and is a principal risk factor for cardiovascular mortality in patients on HD (3). Such patients develop uremic syndrome per se or factors associated with uremia and an excessively inflamed state with marked hypoalbuminemia (4). Increased oxidative stress and higher levels of C-reactive protein (CRP) and pro-inflammatory cytokines contribute to increased protein catabolism in these states.Myeloperoxidase (MPO) is a hemoprotein that is secreted during the activation of neutrophils and of reactive species-generating enzyme, which acts in host defense by catalyzing the production of hypochloric acid (HOCl) (5). Neutrophil-derived MPO induces vascular injury responses such as endothelial dysfunction and thus MPO might play an important role in vascular injury and atherogenesis (6). Elevated levels of MPO are associated with coronary heart disease and predict risk in patients with acute coronary syndromes (7,8). Myeloperoxidase influences atherosclerosis not only in nonuremic patients, but also in patients with end-stage renal disease (ESRD), especially when under HD therapy. During HD sessions, MPO might be released in association with biocompatibility factors such as deleterious effects induced in the blood during dialysis (9). A high MPO value, as well as hepatic acute phase proteins and increased expression of pro-inflammatory interleukins, is associated with CVD among patients on HD (10), and increased levels of MPO are closely linked to mortality in such patients (11). Moreover, MPO influences PEW in patients on HD (12).However, MPO levels are not associated with atherosclerosis progression in nonuremic patients (13), which suggests that serum MPO levels do not always reflect MPO catalysis of HOCl.Free radicals released from activated neutrophils oxidize the 52-kD acute-phase protein α₁-antitrypsin, which is a typical serine proteinase inhibitor (14). Thus, oxidized α₁-antitrypsin (oxAT) is a potential marker of activated neutrophil-associated oxidation including MPO catalyzing HOCl production (15).The present competitive study was designed to estimate MPO and oxAT values to predict carotid intima-media thickness (CIMT) and PEW in patients on HD.     

The Impact of Transfusion and Chelation on Oxidative Stress in Immigrant Syrian Children with β-Thalassemia

Iron overload in β-thalassemia major and intermedia patients leads to oxidative stress and causes to formation of lipid hydroperoxides. Thiobarbituric acid reactive substances (TBARS) are a well established method for screening and monitoring of lipid peroxidation. We aimed to investigate serum TBARS and its relationship with biochemical and hematologic parameters of Turkish and immigrant Syrian β-thalassemia children reflecting the effects of this socioeconomic condition on follow up of these patients. Lipid peroxidation products (TBARS) of Turkish (TR) (n = 62, from the cities of Gaziantep and Sivas, Turkey) and Syrian (SYR) (n = 34, from Gaziantep, Turkey) β-thalassemia patients aged 2–17 years and 58 healthy subjects aged 2–16 years were studied. Liver and renal function tests, serum ferritin levels, white blood cell, absolute neutrophil and platelet counts, hemoglobin (Hb) levels of the patients were analyzed. Serum TBARS concentrations were found to be elevated in β-thalassemia patients compared to healthy subjects (mean: 12.47 ± 8.53 vs. 9.78 ± 7.09, p = 0.045). In SYR patients mean pretransfusional Hb level (7.26.2.04 vs. 8.49 ± 1.01, p = 0.002) was lower and ferritin levels (5983.56 ± 5065.56 vs. 3234.60 ± 2237.82, p = 0.001), liver enzymes (ALT: 77.82 ± 76.48 vs. 42.13 ± 51.50, p = 0.005) were higher when compared to TR group. Positive correlation between TBARS and ferritin levels (p = 0.029, r = 0.231) and liver enzymes (for ALT p < 0.001, r = 0.373) was observed. β-thalassemia patients are under more oxidative stress than healthy subjects. Liver is one of the major organs which are mainly affected by oxidative stress. War and migration might have caused inappropriate transfusion conditions and insufficient chelation therapy in the SYR group.     

Diverse Regulation of Cardiac Expression of Relaxin Receptor by α1- and β1-Adrenoceptors

Purpose

Relaxin, a new drug for heart failure therapy, exerts its cardiac actions through relaxin family peptide receptor 1 (RXFP1). Factors regulating RXFP1 expression remain unknown. We have investigated effects of activation of adrenoceptors (AR), an important modulator in the development and prognosis of heart failure, on expression of RXFP1 in rat cardiomyocytes and mouse left ventricles (LV).

Methods

Expression of RXFP1 at mRNA (real-time PCR) and protein levels (immunoblotting) was measured in cardiomyocytes treated with α- and β-AR agonists or antagonists. RXFP1 expression was also determined in the LV of transgenic mouse strains with cardiac-restricted overexpression of α_1A-, α_1B- or β₂-AR. Specific inhibitors were used to explore signal pathways involved in α₁-AR mediated regulation of RXFP1 in cardiomyocytes.

Results

In cultured cardiomyocytes, α₁-AR stimulation resulted in 2–3 fold increase in RXFP1 mRNA (P?<?0.001), which was blocked by specific inhibitors for protein kinase C (PKC) or mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK). Activation of β₁-, but not β₂-AR, significantly inhibited RXFP1 expression (P?<?0.001). Relative to respective wild-type controls, RXFP1 mRNA levels in the LV of mice overexpressing α_1A- or α_1B-AR were increased by 3- or 10-fold, respectively, but unchanged in β₂-AR transgenic hearts. Upregulation by α₁-AR stimulation RXFP1 expression was confirmed at protein levels both in vitro and in vivo.

Conclusions

Expression of RXFP1 was up-regulated by α₁-AR but suppressed by β-AR, mainly β₁-AR subtype, in cardiomyocytes. Future studies are warranted to characterize the functional significance of such regulation, especially in the setting of heart failure.     

Egr-1 Promotes Cell Proliferation and Invasion by Increasing β-Catenin Expression in Gastric Cancer

Background

Abnormal expression of early growth response gene 1 (Egr-1) and β-catenin may play a crucial role in the development and progression of human cancer. However, little is known about the expression and underlying molecular mechanisms in which Egr-1 and β-catenin are involved in the development and progression of gastric cancer.

Aims

The purpose of this study was to elucidate the potential relationship between Egr-1 and β-catenin expression in gastric cancer, which contributes to finding new molecular carcinogenesis as a potential therapeutic target for gastric cancer.

Methods

In a sample of 102 cases of human gastric cancer, the expression of Egr-1 and β-catenin was detected using immunohistochemistry. Egr-1 gene was transfected into gastric cancer SGC7901 cells and its role in proliferation and cell invasion was detected by MTT assay, flow cytometry, wound-healing and transwell invasion assay. Western blot analysis was used to study the expression of β-catenin and cyclin D1 proteins.

Results

Upregulated Egr-1 and β-catenin protein expression were strongly correlated with cancer progression and depth of invasion in gastric cancer. β-catenin, present mainly in cytoplasmic and nucleus of gastric cancer cells, was also positively correlated with Egr-1 expression in gastric cancer. Furthermore, the overexpression of Egr-1 upregulated β-catenin expression level, promoted cell proliferation, increased cell population in S-phase and enhanced gastric cancer cell migration and invasion in vitro.

Conclusions

Egr-1 might contribute to gastric cancer proliferation and invasion through activation of the β-catenin signaling pathway.     

Postsynaptic αAl-Adrenoceptors in the Hypertensive Rat: Studies on Vascular Reactivity In Vivo and Receptor Binding In Vitro

An increase in vascular responsiveness (increase in diastolic blood pressure) to phenylephrine (PE), noradrenaline (NA) or angiotensin (AII) was seen in pithed SHR when compared with normotensive controls (WKY). This increase in vasoconstrictor effect was not seen with the postsynaptic α₂-adrenoceptor agonists BHT 933 or TL 99. In SHR and DOCA-salt hypertensive rats prazosin was significantly more potent as an antagonist of α₁-receptor mediated vasoconstriction (PE, or the endogenous release of NA by electrical stimulation of the spinal cord), and was also more potent against NA administered by i.v. injection, compared with normotensive controls. The potency of yohimbine against the postsynaptic α₂-receptor mediated pressor responses to TL 99 was not significantly different in SHR or normotensive rats. Although these results indicate an apparent shift in the ratio of postsynaptic α₁/α₂-receptor mediated effects in hypertension in vivo, these effects were not correlated with any changes in α-receptor density (B max) nor affinity (K_D) of the ligands 3H-prazosin or 3H-yohimbine in membranes prepared from SHR. The     

Prenatal Diagnosis of Hemoglobinopathies: Detection of α-Thalassemia Trait and of Sickle Cell Disease In Utero

We recently examined a 16-year-old high school student who participated in a survey for Hemoglobinopathies in an Anatolian city in Turkey. Analysis if his hemoglobin (Hb) showed the presence of an electrophoretically slow-moving variant (at pH 8.6, about as Hb S) which, upon further examination, was also seen in his father, one brother, and one sister.     

Differential expression of estrogen receptor α, β1, and β2 in lobular and ductal breast cancer

The role of estrogen receptor (ER) α as a target in treatment of breast cancer is clear, but those of ERβ1 and ERβ2 in the breast remain unclear. We have examined expression of all three receptors in surgically excised breast samples from two archives: (i): 187 invasive ductal breast cancer from a Japanese study; and (ii) 20 lobular and 24 ductal cancers from the Imperial College. Samples contained normal areas, areas of hyperplasia, and in situ and invasive cancer. In the normal areas, ERα was expressed in not more than 10% of epithelium, whereas approximately 80% of epithelial cells expressed ERβ. We found that whereas ductal cancer is a highly proliferative, ERα-positive, ERβ-negative disease, lobular cancer expresses both ERα and ERβ but with very few Ki67-positive cells. ERβ2 was expressed in 32% of the ductal cancers, of which 83% were postmenopausal. In all ERβ2-positive cancers the interductal space was filled with dense collagen, and cell nuclei expressed hypoxia-inducible factor 1α. ERβ2 expression was not confined to malignant cells but was strong in stromal, immune, and endothelial cells. In most of the high-grade invasive ductal cancers neither ERα nor ERβ was expressed, but in the high-grade lobular cancer ERβ was lost and ERα and Ki67 expression were abundant. The data show a clear difference in ER expression between lobular and ductal breast cancer and suggest (i) that tamoxifen may be more effective in late than in early lobular cancer and (ii) a potential role for ERβ agonists in preventing in situ ductal cancers from becoming invasive.Despite decades of research, the etiology of breast cancer remains unclear. It is currently thought that most breast cancers occur in the normal terminal duct lobular unit and progress in a stepwise fashion over time (1). Ductal carcinoma in situ (DCIS) means the cancer has not spread beyond the duct into any normal surrounding breast tissue and is thought by some to be the direct precursor of invasive ductal carcinoma (IDC).Estrogens play an important role in normal breast development as well as breast cancer progression (2). Most of the effects of estrogen are mediated through its two receptors: estrogen receptor α (ERα) and β (ERβ) (3). ERα is expressed in 50–80% of breast tumors, and its presence is the main indicator for antihormonal therapy (4). ERβ was first discovered in 1996, and its role in breast cancer is still being explored (5–7).The first step in understanding the role of ERβ in breast cancer was to define the expression pattern of ERβ in the normal human breast and in various stages of cancer. Since its discovery, several laboratories have reported ERβ expression in clinical samples (8–28). Most of these studies investigated the expression of ERβ in invasive breast cancer samples (12–15, 17, 19, 21–23). Some studies have reported ERβ expression in invasive breast cancer and normal breast tissue (11, 18, 26–28), but few have compared the expression of ERβ in the normal tissue, DCIS, and IDC within the same sample. Usually tumor samples are taken from one patient and normal tissue from another patient (8–10). Samples taken from different patients have intrinsic limitation (i.e., they cannot account for variations between different patients). In addition, because tumors are heterogeneous, core biopsies do not fully reflect the histological and biological diversity of breast tumors (29).The roles of ERβ1 and its splice variant ERβ2 in breast cancer are still unclear. As reviewed by Murphy and Leygue (30), some studies show a loss of ERβ1 as ductal cancer progresses, but others do not. Some studies show ERβ2 as a marker of bad prognosis (31), and others not (19). Some of these differences may be due to differences in antibody use and differences in tissue fixation and handling.When ERα and ERβ are coexpressed in breast cancer it is unclear whether tamoxifen treatment will be successful. This is because tamoxifen acts as an agonist of ERβ at activator protein 1 (AP-1) sites (32) and thus should oppose the antiproliferative effects of the tamoxifen–ERα complex. Yan et al. (33) have found that expression of ERβ predicts tamoxifen benefit in patients with ERα-negative early breast cancer, whereas Esslimani-Sahla et al (23) have found that low ERβ level is an independent marker, better than ERα level, to predict tamoxifen resistance. Although apparently saying different things, these two results actually agree with each other: in ERα-negative breast cancer, estrogen is not driving proliferation, so tamoxifen via ERβ may interfere with another growth signaling pathway. In ERα-positive cancers whose proliferation is driven by E2, tamoxifen with ERβ would oppose the antiproliferative effects of the ERα–tamoxifen complex.Investigation of the expression pattern of ERβ in normal tissue, DCIS, and IDC is important to understand the function of this receptor in the progression of breast cancer. We have a set of samples obtained from surgical excision of breast tumors from women before pharmacological intervention. The cohorts include lobular cancer, which has not yet been thoroughly studied for ERβ expression. Lobular cancer is an ERα-positive form of breast cancer characterized by loss of E-cadherin and relatively low proliferation rate. It is accompanied by a resistance to anoikis (34). It accounts for 10–15% of diagnosed breast cancer, and there are still many questions about the optimal therapeutic approach to this cancer. We have explored the changes in expression of the two ERs using identical protocols and reagents in different developmental stages of breast cancer within each patient.     

Expression of Transforming Growth Factor β1 in Bronchial Biopsies in Asthma and COPD

The role of transforming growth factor β₁ (TGF β₁) in airway remodeling in asthma and chronic obstructive pulmonary disease (COPD) has not been fully described. To evaluate the possible pathogenetic role of TGF β₁ in asthma and COPD, immunohistochemical expression of TGF β₁ was described in bronchial biopsies from patients with asthma and COPD compared with healthy individuals. Twelve subjects with asthma, 13 subjects with COPD, and 10 healthy individuals enrolled in the study. Bronchial biopsies were stained with hematoxylin and eosin and anti‐TGF β₁ antibody. As a result, immunoreactive TGF β₁ was mainly localized in association with connective tissue in all groups. The staining intensity was not statistically different among the groups in bronchial epithelium, whereas it was significantly higher in the group of asthma in the submucosa. Because there is evidence showing a significant increase of staining intensity in the submucosa from asthmatics but not from subjects with COPD, we may conclude that TGF β₁ may play a significant role in pathogenesis of asthma but not in COPD.     

Expression and localization of the AT1 and AT2 angiotensin II receptors and α1A and α1D adrenergic receptors in aorta of hypertensive and diabetic rats

Hypertension and diabetes are multifactorial diseases that frequently coexist and exacerbate each another. During the development of diabetes, the impairment of noradrenergic and renin-angiotensin systems has been reported in the response mediated by α₁-AR and AT₁ receptors. Although their participation in the development of cardiovascular complications is still controversial, some studies have found increased or diminished response to the vasoconstrictive effect of noradrenaline or angiotensin II in a time-dependent manner of diabetes. Thus, the aim of this work was to investigate the possible changes in the expression or localization of α₁-AR (α_1A and α_1D) and angiotensin II receptors (AT₁ and AT₂) in aorta of rats after 4 weeks of the onset of diabetes. In order to be able to examine the expression of these receptors, immunofluorescence procedure was performed in tunica intima and tunica media of histological sections of aorta. Fluorescence was detected by a confocal microscopy. Our results showed that the receptors are expressed in both tunics, where adrenergic receptors have a higher density in tunica intima and tunica media of SHR compared with WKY; meanwhile, the expression of angiotensin II receptors is not modified in both groups of rats. On the other hand, the results showed that diabetes produced an increase or a decrease in the expression of receptors that is not associated to a specific type of receptor, vascular region, or strain of rat. In conclusion, diabetes and hypertension modify the expression of the receptors in tunica intima and tunica media of aorta in a different way.     

Analysis of Gene Mutation Types of α- and β-Thalassemia in Fuzhou,Fujian Province in China

Abstract

The aim of this study was to explore the type and distribution of α- and β-thalassemia (α- and β-thal) mutations/deletions in Fuzhou, Fujian Province, People’s Republic of China (PRC). For patients whose mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) conforms to the characteristics of thalassemia, gap-polymerase chain reaction (gap-PCR) and PCR-reverse dot-blot assays were used to detect α- and β-thal mutations/deletions. Four hundred and twenty-eight cases of thalassemia were detected in 752 suspected cases of thalassemia, including 248 cases of α-thal (57.9%), 175 cases of β-thal (40.9%), and five cases of compound α/β-thal (1.2%). The major genetic types of α-thal were – –^SEA (Southeast Asian; HBA1) and –α^3.7 (rightward; HBA2) deletions, and the carrier rate was 24.9 and 3.3%, respectively. The major genetic types of β-thal were IVS-II-654 (C>T) (HBB: c.316-197C>T), codons 41/42 (–TTCT) (HBB: c.126_129delCTTT), codon 17 (A>T) (HBB: c.52A>T), codons 27/28 (+C) (HBB: c.84_85insC) and –28 (A>G) (HBB: c.-78A>G), and the carrier rate was 9.8, 7.3, 3.5, 1.1 and 0.8%, respectively. The genotypes of α- and β-thal in Fuzhou (Fujian Province, PRC) are characterized by a wide range of distribution, high carrier rate, genetic diversity and genetic heterogeneity. Therefore, it is necessary to detect the thalassemia mutations in the suspected population of this area.     

Quadrupole-Time-of-Flight Mass Spectrometric Identification of Hemoglobin Subunits α, β, γ and δ in Unknown Peaks of High Performance Liquid Chromatography of Hemoglobin in β-Thalassemias

Abstract

This is the first report of quadrupole time-of-flight (Q-TOF) mass spectrometric identification of the hemoglobin (Hb) subunits, α, β, δ and γ peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the cation exchange chromatography of Hb. The objectives were to identify the unknown high performance liquid chromatography (HPLC) peaks in healthy subjects and in patients with β-thalassemia (β-thal). The results demonstrate the existence of pools of free globin chains in red blood cells (RBCs). The α-, β-, δ- and γ-globin peptides were identified in the unknown HPLC peaks. The quantification and role of the free globin pool in patients with β-thal requires further investigation. Identification of all types of Hb subunits in the retention time (RT) before 1?min. suggests that altered Hbs is the nature of these fast-eluting peaks. Relevancy of thalassemias to the protein-aggregation disorders will require review of the role of free globin in the pathology of the disease.     

Transcriptional Upregulation of α2δ-1 Elevates Arterial Smooth Muscle Cell Voltage-Dependent Ca2+ Channel Surface Expression and Cerebrovascular Constriction in Genetic Hypertension

A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca(2+) (Ca(V)1.2) currents that induces pathological vasoconstriction. Ca(V)1.2 channels are heteromeric complexes composed of a pore-forming Ca(V)1.2α(1) with auxiliary α(2)δ and β subunits. Molecular mechanisms that elevate Ca(V)1.2 currents during hypertension and the potential contribution of Ca(V)1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α(2)δ subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats was associated with an unequal elevation in α(2)δ-1 and Ca(V)1.2α(1) mRNA and protein in cerebral artery myocytes, with α(2)δ-1 increasing more than Ca(V)1.2α(1). Other α(2)δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive spontaneously hypertensive rats displayed higher surface-localized α(2)δ-1 and Ca(V)1.2α(1) proteins, surface α(2)δ-1:Ca(V)1.2α(1) ratio, Ca(V)1.2 current density and noninactivating current, and pressure- and depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α(2)δ-1 ligand, did not alter α(2)δ-1 or Ca(V)1.2α(1) total protein but normalized α(2)δ-1 and Ca(V)1.2α(1) surface expression, surface α(2)δ-1:Ca(V)1.2α(1), Ca(V)1.2 current density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in α(2)δ-1 expression that promotes surface trafficking of Ca(V)1.2 channels in cerebral artery myocytes. This leads to an increase in Ca(V)1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α(2)δ-1 targeting is a novel strategy that may be used to reverse pathological Ca(V)1.2 channel trafficking to induce cerebrovascular dilation in hypertension.