Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Thyroid membrane ADP ribosyltransferase activity. Stimulation by thyrotropin and activity in functioning and nonfunctioning rat thyroid cells in culture

Bovine thyroid membranes possess both ADP ribosyltransferase and NAD glycohydrolase activities with the same Km values for NAD and the same pH optima. In intact membranes, the ADP ribosyltransferase is limited in its extent by the amount of available membrane acceptor which can be ADP-ribosylated; in membranes solubilized with lithium diiodosalicylate, an artificial acceptor, L-arginine methyl ester, can be substituted to eliminate this limitation. The product of the ADP ribosyltransferase is a mono-ADP-ribosylated acceptor whether the intact or solubilized membrane provides the enzyme activity and whether membrane or exogenous acceptor, L-arginine methyl ester, is utilized. The intact membranes and the solubilized preparation also have an enzyme activity which can release AMP from the mono-ADP-ribosylated acceptor whether formed by the action of the membrane ADP ribosyltransferase or the A promoter of cholera toxin. The NAD glycohydrolase activity appears to represent the half-reaction of the ADP ribosyltransferase, i.e. an activity measurable substituting water for a membrane acceptor or L-arginine methyl ester. Membranes from functional rat thyroid cells in culture, i.e. cells chronically stimulated by thyrotropin and unresponsive to further additions of thyrotropin, have low ADP-ribosylation but high NAD glycohydrolase activities. In contrast, membranes from nonfunctional rat thyroid cells, i.e. cells unresponsive to thyrotropin, have high ADP-ribosylation and low NAD glycohydrolase activities. NAD hydrolysis by the NAD glycohydrolase activity cannot account for the low ADP-ribosylation activity in membranes from the functioning cells, and its low level of ADP-ribosylation can be eliminated by solubilizing the membranes and substituting an artificial acceptor, L-arginine methyl ester. The ADP ribosyltransferase activity of rat thyroid cell membrane preparations can be enhanced by thyrotropin in a dose-dependent manner but not by insulin, glucagon, hydrocortisone, adrenocorticotropin, or its glycoprotein hormone analog, human chorionic gonadotropin. It is thus suggested (i) that, in analogy to cholera toxin, thyrotropin-stimulated ADP-ribosylation may be important in the regulation of the adenylate cyclase response and (ii) that the level of membrane acceptor available for ADP-ribosylation may relate both to a stable "'activated" state of the adenylate cyclase system in cells chronically stimulated with thyrotropin and/or to a desensitized state with regard to a failure of more thyrotropin to elicit additional functional responses.     

Thyroglobulin interactions with thyroid membranes. Relationship between receptor recognition of N-acetylglucosamine residues and the iodine content of thyroglobulin preparations

Bovine thyroglobulin has been subjected to sequential glycohydrolase treatment in order to define further the components of the carbohydrate chain which are important in binding of the glycoprotein to bovine thyroid membranes. Preparations of asialoagalactothyroglobulin exhibit the best binding, suggesting that exposed N-acetylglucosamine residues on the B carbohydrate chain of thyroglobulin play an important role in the interaction of thyroglobulin with the thyroid membranes. Enhanced binding of asialoagalactothyroglobulin to microsomal, lysosomal, and Golgi membranes, as well as to thyroid cells in culture, was also observed. Isopycnic rubidium chloride gradient centrifugation, a procedure used in the isolation of thyroglobulin molecules with a low iodine content, also isolates thyroglobulin molecules with a low sialic acid content and with an increased ability to interact with wheat germ agglutinin, a lectin which recognizes exposed N-acetylglucosamine residues. The studies further indicate that there is a correlation between iodine content, exposed N-acetylglucosamine residues, and the binding of thyroglobulin to thyroid membranes.     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

Background

Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.

Results

Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al³⁺ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.

Conclusions

High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-153) contains supplementary material, which is available to authorized users.     

Cell cycle related proteins in hyperplasia of usual type in breast specimens of patients with and without breast cancer

Background  

Hyperplasia of usual type (HUT) is a common proliferative lesion associated with a slight elevated risk for subsequent development of breast cancer. Cell cycle-related proteins would be helpful to determine the putative role of these markers in the process of mammary carcinogenesis. The aim of this study was to analyze the expression of cell cycle related proteins in HUT of breast specimens of patients with and without breast cancer, and compare this expression with areas of invasive carcinomas.     

New in vitro model to study high glucose-dependent endothelial dysfunctions

Several thrombogenic abnormalities are associated with diabetes. Since endothelial dysfunction occurs at early stages of disease, it may reflect pathophysiological changes that are responsible for alterations in vascular structure, growth and modifications of adhesivity to platelets and leukocytes, leading to atherosclerosis and thrombosis. Predisposing factors of vascular diseases, such as diabetes, are also associated with endothelial dysfunction. Restoration or replacement of endothelium-related factors like nitric oxide impede the progression of vascular thrombogenic diseases, and prevent the action of vasoconstrictor factors such as endothelin or other prothrombotic factors such as plasminogen-activator inhibitor-1. Since high glucose concentration in blood is the hallmark of diabetes and because the vascular lesions of atherosclerosis are localized in large artheries, we have cultured endothelial cells from the human aorta. Two endothelial cell strains from the same aortic tract that show different characteristics and behavior in high glucose were isolated. Such findings reflect the importance to have well characterized and standardized cell culture systems to carry out experiments to study the glucose-dependent atherosclerotic process in vitro. Our cell strains may represent a useful in vitro model to study the complex pathophysiology of diabetes-related atherosclerosis.     

Evaluation of APOE polymorphisms and the risk for age-related macular degeneration in a Southeastern Brazilian population

This study aimed to evaluate the role of APOE polymorphisms (rs429358 and rs7412) in the risk of age-related macular degeneration in a sample of the Southeastern Brazilian population. Seven hundred and five unrelated individuals were analyzed, 334 with age-related macular degeneration (case group), and 371 without the disease (control group). In the case group, patients were further stratified according to disease phenotypes, divided into dry and wet age-related macular degeneration, and non-advanced and advanced age-related macular degeneration. APOE polymorphisms (rs429358 and rs7412) were evaluated through polymerase chain reaction and direct sequencing. In the comparison of cases vs. controls, none of the associations reached statistical significance, considering the Bonferroni-adjusted P-value, although there was a suggestive protection for the E3/E4 genotype (OR = 0.626; P-value = 0.037) and E4 carriers (OR = 0.6515; P-value = 0.047). Statistically significant protection for both the E3/E4 genotype and E4 carriers was observed in the comparisons: advanced age-related macular degeneration vs. controls (OR = 0.3665, P-value = 0.491 × 10⁻³ and OR = 0.4031, P-value = 0.814 × 10⁻³, respectively), advanced age-related macular degeneration vs. non-advanced age-related macular degeneration (OR = 0.2529, P-value = 0.659 × 10⁻⁴ and OR = 0.2692, P-value = 0.631 × 10⁻⁴, respectively). In the comparison of wet age-related macular degeneration vs. control, protection was statistically significant only for E3/E4 (OR = 0.4052, P-value = 0.001). None of the comparisons demonstrated any significant association for E2 genotypes or E2 carriers in age-related macular degeneration risk in this study. Findings suggest a protective role of the E4 haplotype in the APOE gene in the risk for advanced and wet forms of age-related macular degeneration, in a sample of the Brazilian population. To our knowledge, this is the first Brazilian study to show the association between APOE polymorphisms and age-related macular degeneration.