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.     

A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.     

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.     

l7Rn6 encodes a novel protein required for clara cell function in mouse lung development

The highly secretory Clara cells play a pivotal role in protecting the lung against inflammation and oxidative stress. This study reports the positional cloning of a novel protein required for Clara cell physiology in mouse lung development. The perinatal lethal N-ethyl-N-nitrosourea-induced l7Rn6(4234SB) allele contained a nonsense mutation in the previously hypothetical gene NM_026304 on chromosome 7. Whereas l7Rn6 mRNA levels were indistinguishable from wild type, l7Rn6(4234SB) homozygotes exhibited decreased expression of the truncated protein, suggesting protein instability. During late gestation, l7Rn6 was widely expressed in the cytoplasm of lung epithelial cells, whereas perinatal expression was restricted to the bronchiolar epithelium. Homozygosity for the l7Rn6(4234SB) allele did not affect early steps in lung patterning, growth, or cellular differentiation. Rather, mutant lungs demonstrated severe emphysematous enlargement of the distal respiratory sacs at birth. Clara cell pathophysiology was evident from decreased cytoplasmic CCSP and SP-B protein levels, enlargement and disorganization of the Golgi complex, and formation of aberrant vesicular structures. Additional support for a role in the secretory pathway derived from l7Rn6 localization to the endoplasmic reticulum. Thus, l7Rn6 represents a novel protein required for organization and/or function of the secretory apparatus in Clara cells in mouse lung.     

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.     

Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes

To meet the new challenge of generating the draft sequences of mammalian genomes, we describe the development of a novel high throughput 96-well method for the purification of plasmid DNA template using size-fractionated, acid-washed glass beads. Unlike most previously described approaches, the current method has been designed and optimized to facilitate the direct binding of alcohol-precipitated plasmid DNA to glass beads from alkaline lysed bacterial cells containing the insoluble cellular aggregate material. Eliminating the tedious step of separating the cleared lysate significantly simplifies the method and improves throughput and reliability. During a 4 month period of 96-capillary DNA sequencing of the Rattus norvegicus genome at the Baylor College of Medicine Human Genome Sequencing Center, the average success rate and read length derived from >1 800 000 plasmid DNA templates prepared by the direct lysis/glass bead method were 82.2% and 516 bases, respectively. The cost of this direct lysis/glass bead method in September 2001 was ~10 cents per clone, which is a significant cost saving in high throughput genomic sequencing efforts.     

Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.

Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.