A calmodulin dependent protein kinase activity associated with rabbit heart sarcolemma

Summary Sarcolemmal vesicles prepared from rabbit heart muscle by differential and discontinuous sucrose density gradient centrifugation, exhibited high marker enzyme activities: Na⁺ + K⁺ ATPase 22 µMol Pi × mg^–1 × h^–1. Adenylate cyclase 500 pmole CAMP × mg^–1 × min^–1, calcium antagonist receptors 0.7 pmoles × mg^–1. Calmodulin in the presence of calcium and -ATP³² stimulated rapidly and specifically the ³²P incorporation into two membrane proteins of 54 and 44 kDa. Calmodulin stimulated the phosphorylation of the 44 kDa to a greater extent (17.9 pmol ³²P × mg^–1 protein) than the 54 kDa protein (1.3 pmoles ³²P x mg^–1 protein). Removal of endogenous calmodulin from the membrane by EGTA extraction resulted in a 2.5 fold increase in calmodulin dependent ³²P incorporation into the two proteins in the presence of exogenous calmodulin. It is suggested that the calmodulin dependent protein kinase activity in heart sarcolemma may mediate the effects of calmodulin in the regulation of Ca²⁺ transport across the membrane.     

Biosynthesis of tetrahydrobiopterin: a sensitive assay of 6-pyruvoyltetrahydropterin synthase using [2'-3H]dihydroneopterin 3'-triphosphate as substrate

Pyruvoyltetrahydropterin synthase catalyzes the release of tritiated water from [2'-3H]dihydroneopterin 3'-triphosphate. The tritiated water formed during the enzymatic reaction is separated from substrate by adsorption of the latter to activated charcoal. The sensitivity and specificity of the assay allows the determination of the enzyme in crude cell extract.     

Effects of modulated microwave radiation at cellular telephone frequency (1.95 GHz) on X-ray-induced chromosome aberrations in human lymphocytes in vitro

The case for a DNA-damaging action produced by radiofrequency (RF) signals remains controversial despite extensive research. With the advent of the Universal Mobile Telecommunication System (UMTS) the number of RF-radiation-exposed individuals is likely to escalate. Since the epigenetic effects of RF radiation are poorly understood and since the potential modifications of repair efficiency after exposure to known cytotoxic agents such as ionizing radiation have been investigated infrequently thus far, we studied the influence of UMTS exposure on the yield of chromosome aberrations induced by X rays. Human peripheral blood lymphocytes were exposed in vitro to a UMTS signal (frequency carrier of 1.95 GHz) for 24 h at 0.5 and 2.0 W/kg specific absorption rate (SAR) using a previously characterized waveguide system. The frequency of chromosome aberrations was measured on metaphase spreads from cells given 4 Gy of X rays immediately before RF radiation or sham exposures by fluorescence in situ hybridization. Unirradiated controls were RF-radiation- or sham-exposed. No significant variations due to the UMTS exposure were found in the fraction of aberrant cells. However, the frequency of exchanges per cell was affected by the SAR, showing a small but statistically significant increase of 0.11 exchange per cell compared to 0 W/kg SAR. We conclude that, although the 1.95 GHz signal (UMTS modulated) does not exacerbate the yield of aberrant cells caused by ionizing radiation, the overall burden of X-ray-induced chromosomal damage per cell in first-mitosis lymphocytes may be enhanced at 2.0 W/kg SAR. Hence the SAR may either influence the repair of X-ray-induced DNA breaks or alter the cell death pathways of the damage response.     

CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection

Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense Reader. Performance of the ElectraSense platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense platform was also compared to the standard fluorescent detection, and good consistency was observed between these two different detection techniques. A lower detection limit of 0.75 pM was obtained for ElectraSense as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus).     

After the crash: How do predators adjust following the invasion of a novel toxic prey type?

The ability of a native predator to adjust to a dangerously toxic invasive species is key to avoiding an ongoing suppression of the predator's population and the trophic cascade of effects that can result. Many species of anurophagous predators have suffered population declines due to the cane toad's (Rhinella marina: Bufonidae) invasion of Australia; these predators can be fatally poisoned from attempting to consume the toxic toad. We studied one such toad‐vulnerable predator, the yellow‐spotted monitor (Varanus panoptes: Varanidae), testing whether changes to the predator's feeding behaviour could explain how the species persists following toad invasion. Wild, free‐roaming lizards from (1) toad‐naïve and (2) toad‐exposed populations were offered non‐toxic native frogs and slightly toxic cane toads (with parotoid glands removed) in standardized feeding trials. Toad‐naïve lizards readily consumed both frogs and toads, with some lizards displaying overt signs of illness after consuming toads. In contrast, lizards from toad‐exposed populations consumed frogs but avoided toads. Repeated encounters with toads did not modify feeding responses by lizards from the toad‐naïve populations, suggesting that aversion learning is limited (but may nonetheless occur). Our results suggest that this vulnerable predator can adjust to toad invasion by developing an aversion to feeding on the toxic invader, but it remains unclear as to whether the lizard's toad‐aversion arises via adaptation or learning.     

Does KRAS Testing in Metastatic Colorectal Cancer Impact Overall Survival? A Comparative Effectiveness Study in a Population-Based Sample

Purpose

Epidermal growth factor receptor (EGFR) inhibitors are approved for treating metastatic colorectal cancer (CRC); KRAS mutation testing is recommended prior to treatment. We conducted a non-inferiority analysis to examine whether KRAS testing has impacted survival in CRC patients.

Patients and Methods

We included 1186 metastatic CRC cases from seven health plans. A cutpoint of July, 2008, was used to define two KRAS testing time period groups: “pre-testing” (n = 760 cases) and “post-testing” (n = 426 cases). Overall survival (OS) was estimated, and the difference in median OS between the groups was calculated. The lower bound of the one-sided 95% confidence interval (CI) for the difference in survival was used to test the null hypothesis of post-testing inferiority. Multivariable Cox regression models were constructed to adjust for covariates.

Results

The median unadjusted OS was 15.4 months (95% CI: 14.0–17.5) and 12.8 months (95% CI: 10.0–15.2) in the pre- and post-testing groups, respectively. The OS difference was −2.6 months with one-sided 95% lower confidence bound of −5.13 months, which was less than the non-inferiority margin (−5.0 months, unadjusted p = 0.06), leading to a failure to reject inferiority of OS in the post-testing period. In contrast, in the adjusted analysis, OS non-inferiority was identified in the post-testing period (p = 0.001). Sensitivity analyses using cutpoints before and after July, 2008, also met the criteria for non-inferiority.

Conclusion

Implementation of KRAS testing did not influence CRC OS. Our data support the use of KRAS testing to guide administration of EGFR inhibitors for treatment of metastatic CRC without diminished OS.     

Similar Outcomes Achieved Between Anterior and Posterior Approach Total Hip Arthroplasty Using Dual Mobility Implants

BackgroundDual mobility (DM) bearings for total hip arthroplasty (THA) have been proposed to reduce the risk of instability in high-risk patients; however, their utility in primary THA remains relatively unexplored. No previous reports have described whether surgical approach influences outcomes associated with DM implant systems. This study aims to compare patient reported outcomes and post-operative groin pain between patients undergoing anterior approach versus posterior approach following primary THA with DM implants.MethodsWe retrospectively reviewed all patients who underwent primary THA and received a DM implant between 2011-2021. Patients were stratified into two cohorts based on surgical approach (anterior vs. posterior approach). Primary outcomes included the presence of substantial postoperative groin pain as well as readmission and revision rates. Demographic differences were assessed using chi-square and independent sample t-tests. Outcomes were compared using multilinear and logistic regressions.ResultsOf the 495 patients identified, 55 (11%) underwent THA via the anterior approach and 440 (89%) via the posterior approach. Surgical time (100.24 vs. 109.42 minutes, p=0.070), length of stay (2.19vs.2.67 days,p=0.072), discharge disposition (p=0.151), and significant postoperative groin pain (1.8%vs.0.7%,p=0.966) did not statistically differ between the cohorts. 90-day readmission (9.1%vs.7.7%,p=0.823) and revision rate (0.0%vs.3.0%,p=0.993) did not significantly differ as well. Specifically, readmission (p=0.993) and revision (p=0.998) for instability did not significantly differ between the cohorts. We found no statistical difference in HOOS, JR (p=0.425), VR-12 PCS (p=0.718), and VR-12 MCS (p=0.257) delta score improvement from preoperative to 1-year follow-up between the two groups.ConclusionComparable outcomes following implantation of DM constructs may be achievable irrespective of the surgical approach employed. The incidence of iliopsoas injections for groin pain did not significantly differ between anterior and posterior approaches. Future investigation is needed to determine whether surgical approach influences long-term outcomes in patients receiving DM implants. Level of Evidence: III