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.     

Focus: Preventive Medicine: Pre-Participation Screening of Athletes: Primary Health Care Physicians’ Knowledge,Experience, and Approach in Turkey

Pre-participation screening (PPS) is crucial for assessing the competitive athletes since their risk of sudden death is higher than non-athletes. In Turkey, PPS is performed at the primary health care setting by primary care physicians (PCPs) who are family medicine specialists (FMSs) or general practitioners (GPs). Although there are national guidelines, there is no legal regulation for this process. This study aims to evaluate PCPs’ knowledge, experience, and approach about PPS. We prepared an online survey for PCPs and used non-probabilistic sampling. PPS attitudes and practices were analyzed and compared according to factors such as experience, education, and being GP or FMS. Of the 214 PCPs included in the study, 39.3% were female. The mean age was 44.9 years (SD:8.88). The average work experience was 7.9 years. Most participants were aware of their authorization to perform PPS (89.7%) and had previously prepared it (90.2%). However, 6.5% of them felt confident in performing PPS. Only 13.1% were aware of the guidelines. Almost 25% of the participants stated being informed about the subject at some part of their career, but this did not affect the confidence or referral decisions. In addition to medical history and physical examination, further testing was considered necessary by 96.3% of the participants. Significantly more tests were ordered by GPs than FMSs (p=0.026 and p=0.011, respectively). The accurate referral decision ratio was 59.3%, without difference between FMSs and GPs (p=0.216). We found that awareness of the guidelines was low among PCPs who lack confidence in PPS. These factors collectively increased the tendency for unnecessary further testing and referral. Therefore, the PPS implementation into medical school and residency curriculums and national legal regulation for the process is a necessity in Turkey.     

Genetic Analysis of Turkey Common Bean (Phaseolus vulgaris L.) Genotypes by Simple Sequence Repeats Markers

Russian Journal of Genetics - Common bean (Phaseolus vulgaris L.), besides being an agricultural product that can be consumed as fresh vegetable, is a significant legume widely being planted in...     

Mechanisms of abemaciclib,a CDK4/6 inhibitor,induced apoptotic cell death in prostate cancer cells in vitro

The therapeutic effects of abemaciclib (ABE), an inhibitor of cyclin- dependent kinases (CDK) 4/6, on the proliferation of two types of prostate cancer (PC) cells were revealed. In this study, in vitro cytotoxic and apoptotic effects of ABE on metastatic castration-resistant prostate cancer (mCRPC) androgen receptor (AR) negative PC-3 and AR mutant LNCaP PC cells were analyzed with WST-1, Annexin V, cell cycle, reactive oxygen species (ROS), mitochondrial membrane potential, RT-PCR, western blot, and apoptosis protein array. ABE considerably inhibited the growth of PC cells in a dose-dependent manner (p<0.01) and caused significant apoptotic cell death through the suppression of CDK4/6-Cyclin D complex, ROS generation and depolarization of mitochondria membrane potential. However, PC-3 cells were more sensitive to ABE than LNCaP cells. Furthermore, the expression levels of several pro-apoptotic and cell cycle regulatory proteins were upregulated by ABE in especially PC-3 cells with the downregulation of apoptotic inhibitor proteins. Our results suggest that ABE inhibits PC cell growth and promotes apoptosis and thus ABE treatment may be a promising treatment strategy in especially mCRPC. Further preclinical and clinical studies should be performed to clarify the clinical use of ABE for the treatment of PC.     

Medroxyprogesterone and tamoxifen augment anti-proliferative efficacy and reduce mitochondria-toxicity of epirubicin in FM3A tumor cells in vitro

We have shown that low doses of medroxyprogesterone acetate (MPA- 2.6 microM) and tamoxifen (TAM- 270 nM) could augment the effectiveness of epirubicin in breast tumor cells. In this study, we monitored early cell kinetics (24-96 h plating and S-phase) and mitochondrial morphology during chemo-endocrine treatments to delineate the epirubicin sensitizing mechanism. S-phase fractions with radioactive thymidine uptake, plating efficacy, and transmission electron microscopic analysis were taken for 24-h periods until the 7th day after drug treatments. Despite strongly enhancing the clonogenic killing, both MPA and TAM did not affect epirubicin induced early cytotoxicity. Instead, they augmented the S-phase inhibition, which was even more pronounced for TAM. Epirubicin induced prominent swelling and crista damage of mitochondria and fragmentation of nuclei. Mitochondria were a normal size during a combination of epirubicin with either MPA- or tamoxifen treatment, despite the persistence of chromatin fragmentation and strong synergism on the clonogenic killing of breast tumor cells. Low dosage endocrine agent-induced anthracycline sensitization may be independent of mitochondrial toxicity. Further studies would be worthwhile, since the uncoupling of mitochondrial toxicity from the anti-neoplastic effect may also mean obviated cardiac toxicity in clinic.     

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.     

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.