RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay.

Early and high-throughput individual dose estimates are essential following large-scale radiation exposure events. In the context of the Running the European Network for Biodosimetry and Physical Dosimetry (RENEB) 2021 exercise, gene expression assays were conducted and their corresponding performance for dose-assessment is presented in this publication. Three blinded, coded whole blood samples from healthy donors were exposed to 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 1 Gy/min) using the X-ray source Yxlon. These exposures correspond to clinically relevant groups of unexposed, low dose (no severe acute health effects expected) and high dose exposed individuals (requiring early intensive medical health care). Samples were sent to eight teams for dose estimation and identification of clinically relevant groups. For quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray analyses, samples were lysed, stored at 20°C and shipped on wet ice. RNA isolations and assays were run in each laboratory according to locally established protocols. The time-to-result for both rough early and more precise later reports has been documented where possible. Accuracy of dose estimates was calculated as the difference between estimated and reference doses for all doses (summed absolute difference, SAD) and by determining the number of correctly reported dose estimates that were defined as ±0.5 Gy for reference doses <2.5 Gy and ±1.0 Gy for reference doses >3 Gy, as recommended for triage dosimetry. We also examined the allocation of dose estimates to clinically/diagnostically relevant exposure groups. Altogether, 105 dose estimates were reported by the eight teams, and the earliest report times on dose categories and estimates were 5 h and 9 h, respectively. The coefficient of variation for 85% of all 436 qRT-PCR measurements did not exceed 10%. One team reported dose estimates that systematically deviated several-fold from reported dose estimates, and these outliers were excluded from further analysis. Teams employing a combination of several genes generated about two-times lower median SADs (0.8 Gy) compared to dose estimates based on single genes only (1.7 Gy). When considering the uncertainty intervals for triage dosimetry, dose estimates of all teams together were correctly reported in 100% of the 0 Gy, 50% of the 1.2 Gy and 50% of the 3.5 Gy exposed samples. The order of dose estimates (from lowest to highest) corresponding to three dose categories (unexposed, low dose and highest exposure) were correctly reported by all teams and all chosen genes or gene combinations. Furthermore, if teams reported no exposure or an exposure >3.5 Gy, it was always correctly allocated to the unexposed and the highly exposed group, while low exposed (1.2 Gy) samples sometimes could not be discriminated from highly (3.5 Gy) exposed samples. All teams used FDXR and 78.1% of correct dose estimates used FDXR as one of the predictors. Still, the accuracy of reported dose estimates based on FDXR differed considerably among teams with one team's SAD (0.5 Gy) being comparable to the dose accuracy employing a combination of genes. Using the workflow of this reference team, we performed additional experiments after the exercise on residual RNA and cDNA sent by six teams to the reference team. All samples were processed similarly with the intention to improve the accuracy of dose estimates when employing the same workflow. Re-evaluated dose estimates improved for half of the samples and worsened for the others. In conclusion, this inter-laboratory comparison exercise enabled (1) identification of technical problems and corrections in preparations for future events, (2) confirmed the early and high-throughput capabilities of gene expression, (3) emphasized different biodosimetry approaches using either only FDXR or a gene combination, (4) indicated some improvements in dose estimation with FDXR when employing a similar methodology, which requires further research for the final conclusion and (5) underlined the applicability of gene expression for identification of unexposed and highly exposed samples, supporting medical management in radiological or nuclear scenarios.

The estrogen receptor α-selective agonist propyl pyrazole triol improves glucose tolerance in ob/ob mice: potential molecular mechanisms

The authors and journal apologise for an error in the above paper, which appeared in volume 199 part 2, pages 275­286. The error relates to Fig. 10, given on page 283.

Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest.

Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal γH2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Krüppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC TICs are possible factors contributing to their therapy resistance, which may be exploited for DNA damage-sensitizing purposes.

Activation of estrogen response elements is mediated both via estrogen and muscle contractions in rat skeletal muscle myotubes.

The aim of the present study was to investigate the activation of estrogen response elements (EREs) by estrogen and muscle contractions in rat myotubes in culture and to assess whether the activation is dependent on the estrogen receptors (ERs). In addition, the effect of estrogen and contraction on the mRNA levels of ERalpha and ERbeta was studied to determine the functional consequence of the transactivation. Myoblasts were isolated from rat skeletal muscle and transfected with a vector consisting of sequences of EREs coupled to the gene for luciferase. The transfected myoblasts were then differentiated into myotubes and subjected to either estrogen or electrical stimulation. Activation of the ERE sequence was determined by measurement of luciferase activity. The results show that both ERalpha and ERbeta are expressed in myotubes from rats. Both estrogen stimulation and muscle contraction increased (P < 0.05) transactivation of the ERE sequence and enhanced ERbeta mRNA, whereas ERalpha was unaffected by estrogen and attenuated (P < 0.05) by muscle contraction. Use of ER antagonists showed that, whereas the estrogen-induced transactivation is mediated via ERs, the effect of muscle contraction is ER independent. The muscle contraction-induced transactivation of ERE and increase in ERbeta mRNA were instead found to be MAP kinase (MAPK) dependent. This study demonstrates for the first time that muscle contractions have a similar functional effect as estrogen in skeletal muscle myotubes, causing ERE activation and an enhancement in ERbeta mRNA. However, in contrast to estrogen, the effect is independent of ERs and dependent on MAPK, suggesting activation via the estrogen related receptor (ERR).

The estrogen receptor {alpha}-selective agonist propyl pyrazole triol improves glucose tolerance in ob/ob mice; potential molecular mechanisms.

The aim of this study was to validate the role of estrogen receptor alpha (ERalpha) signaling in the regulation of glucose metabolism, and to compare the molecular events upon treatment with the ERalpha-selective agonist propyl pyrazole triol (PPT) or 17beta-estradiol (E(2)) in ob/ob mice. Female ob/ob mice were treated with PPT, E(2) or vehicle for 7 or 30 days. Intraperitoneal glucose and insulin tolerance tests were performed, and insulin secretion was determined from isolated islets. Glucose uptake was assayed in isolated skeletal muscle and adipocytes. Gene expression profiling in the liver was performed using Affymetrix microarrays, and the expression of selected genes was studied by real-time PCR analysis. PPT and E(2) treatment improved glucose tolerance and insulin sensitivity. Fasting blood glucose levels decreased after 30 days of PPT and E(2) treatment. However, PPT and E(2) had no effect on insulin secretion from isolated islets. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissue were similar in PPT and vehicle-treated ob/ob mice. Hepatic lipid content was decreased after E(2) treatment. In the liver, treatment with E(2) and PPT increased and decreased the respective expression levels of the transcription factor signal transducer and activator of transcription 3, and of glucose-6-phosphatase. In summary, our data demonstrate that PPT exerts anti-diabetic effects, and these effects are mediated via ERalpha.

Gene expression profiling identifies liver X receptor alpha as an estrogen-regulated gene in mouse adipose tissue.

Estrogens reduce adipose tissue mass in both humans and animals. The molecular mechanisms for this effect are, however, not well characterized. We took a gene expression profiling approach to study the direct effects of estrogen on mouse white adipose tissue (WAT). Female ovariectomized mice were treated for 10, 24 and 48 h with 17beta-estradiol or vehicle. RNA was extracted from gonadal fat and hybridized to Affymetrix MG-U74Av2 arrays. 17beta-Estradiol was shown to decrease mRNA expression of liver X receptor (LXR) alpha after 10 h of treatment compared with the vehicle control. The expression of several LXRalpha target genes, such as sterol regulatory element-binding protein 1c, apolipoprotein E, phospholipid transfer protein, ATP-binding cassette A1 and ATP-binding cassette G1, was similarly decreased. We furthermore identified a 1.5 kb LXRalpha promoter fragment that is negatively regulated by estrogen. Several genes involved in lipogenesis and lipolysis were identified as novel targets that could mediate estrogenic effects on adipose tissue. Finally, we show that ERalpha is the main estrogen receptor expressed in mouse white adipose tissue (WAT) with mRNA levels several hundred times higher than those of ERbeta mRNA.

Rituximab, a chimaeric anti-CD20 monoclonal antibody, in the treatment of hairy cell leukaemia.

The introduction of first alpha-interferon and later the purine analogues has revolutionized the treatment of hairy cell leukaemia (HCL). However, there are still some patients that initially or eventually fail to respond and, thus, there is a need for alternative treatment modalities. We have treated 11 HCL patients (eight relapsing and three newly diagnosed) with a chimaeric monoclonal antibody, rituximab, in a dose of 375 mg/m2 once a week for 4 weeks. The response rate was seven out of eleven (64%) with six complete remissions and one partial remission, all which have lasted between 0 and 34 months (median 14 months). Rituximab appears promising in the treatment of HCL and warrants further studies.

Slips, trips and falls in different work groups--with reference to age and from a preventive perspective.

Inter-record linkage between two Swedish databases on population and injury was effected to provide information on occupational slip, trip and fall (STF) accidents. The text descriptions in more than 1600 accident reports from occupational groups with high incidence rates of STF accidents were categorised by gender and age and the factors contributing to the accidents studied. Both older male and female workers had higher rates of reported STF accidents than younger workers, but it was established that within any one occupation the workplace hazards were common to all. Both for men and for women, the initial approach to the prevention of STF accidents should be to improve orderliness in the workplace.

Injuries after falls at work in the United Kingdom and Sweden with special reference to fractures in women over 45.

OBJECTIVES: To describe the relation with age of risk of reported injury after a fall among women at work in two countries, the United Kingdom and Sweden, with particular emphasis on fractures, and to interpret these data. METHODS: Rates of accidents compiled under the national reporting regulations of each country during a two year period were described by age, sex, cause (fall on the level, fall from a height, other), and occurrence of fracture, with emphasis on the relative risk (RR) in workers aged 45 years and over compared with those aged under 45. For fractures (major fractures only in the United Kingdom) among women, RRs were calculated for all occupations, with the three digit occupational classification schemes of each country. Summary RRs for older versus younger women, directly standardised for occupation, were derived. RESULTS: Among women, RRs for injury after a fall on the level and fall from a height were 2.77 and 1.77 respectively in Sweden and 2.28 and 1.54 in the United Kingdom. When restricted to fractures, the RRs became 4.75 and 3.66 respectively in Sweden and 3.35 and 1.97 in the United Kingdom. Standardisation for occupation gave RRs for fractures of 4.87 and 3.75 in Sweden and 3.43 and 2.16 in the United Kingdom. Almost all occupational groups with enough fractures for analysis showed an excess of fractures related to falls among older women. A different age pattern was seen for all injuries or fracture after other types of accidents and for all types of accident in men. CONCLUSION: It is argued that, for fractures at least, the results for women are unlikely to be due to reporting bias and unlikely to be explained by a greater exposure to workplace hazards among older women. Whether there is an increased risk of falling, as distinct from sustaining a fracture, is not clear. The generality of the increased risk suggest that efforts should be made to minimise hazards in all occupational sectors, particularly those using many women.

The age-related risk of occupational accidents: the case of Swedish iron-ore miners.

The paper examines age-related accident risks faced by Swedish male iron-ore miners. A retrospective longitudinal analysis of national registers was conducted over a ten-year period using three times periods of five years and five age categories. Age-related accident frequency, characteristics and severity were examined. High accident ratios were rare among older miners whatever the time period, but some accident patterns became substantially more frequent in some older age cohorts over the years. Injuries tended to be more severe in older age groups, all accidents aggregated as well as by accident pattern. It is concluded that inequality in risk exposure between age groups may explain the lower accident ratios found among older workers, but also that the aging of a working population may lead to the application of task-assignment principles that penalize older workers, at least with regard to certain specific accident risks.

Effect of wall tension on DNA and protein synthesis in bovine mesenteric arteries in vitro.

The aim of this investigation was to study the effect of wall tension and calcium antagonists on DNA and protein synthesis in bovine mesenteric arteries in vitro. The wall tension of the bovine mesenteric arteries was raised by stretching the vessel wall perpendicular to the length axis of the vessel. DNA and protein synthesis were determined by measuring incorporation of 3H-thymidine into DNA and incorporation of 14C-leucine into protein respectively. Elevating the wall tension from 0.05 N to 0.5 N significantly increased 3H-thymidine incorporation and 14C-leucine incorporation after an incubation period of 3 hr. Stretch had no effect on the distribution of 3H-thymidine. The distribution of 14C-leucine was increased by stretch in regular medium and to a less extent in calcium-free medium, which suggest that stretch stimulates the membrane transport of 14C-leucine. When the tension was increased from 0.05 N to 0.5 N for 10 min. before the incubation with 3H-thymidine, no effect was found. One microM nifedipine or felodipine inhibited the increase in 3H-thymidine incorporation caused by stretching, while no effect was found on 14C-leucine incorporation. In calcium-free medium, stretch-induced DNA synthesis was completely abolished. 14C-Leucine incorporation was impaired in calcium-free medium but the stretch-induced increase still remained. The results suggest that mechanical force may play an important role in DNA synthesis and protein metabolism of vascular smooth muscle.

Factors influencing ergonomic conditions and employment rate after an occupational musculoskeletal injury.

In 195 cases of musculoskeletal occupational injury individual and work related factors and their relationship with reduction of physical work load and active employment was studied. Data concerning the injuries were obtained and after 18 months the work places were assessed. Information on employment status was obtained by a postal questionnaire after 3 years. Multiple logistic regression was used to explain the two outcome measures. Injuries classified as diseases and informative injury reports were factors positively associated with reduction of work load. Male gender, higher education, and a sick-leave shorter than 6 months were factors positively associated with employment.

Relationship between coronary atherosclerosis and "sudden cardiac death". Effects of age and calcium blockers in hypercholesterolemic mini-pigs.

Marked atherosclerosis was produced in the coronary arteries (c.a.) of full-grown ("old") mini-pigs by combination of protracted hypercholesterolemia with two repetitions of irradiation of the heart region. "Sudden cardiac death" with occlusion of some peripheral c.a. occurred to 40% of the pigs within 15 to 21 weeks from the last irradiation. Growing ("young") pigs, after the same treatment, developed more marked atherosclerotic lesions in the c.a. than "old" pigs. There was a trend to a situation in which mortality (58%; p = 0.2) in "young" pigs was higher than in "old". When "old" pigs were treated with the calcium-channel blocker nifedipine (2 x 20 mg/day; mean body weight 66 kg), there was some trend to reduced mortality from 40% to 25% (p = 0.25). If the effects of age and nifedipine were combined, the difference in mortality (58% or 25%) was significant (p less than 0.05). In pigs that had died a "sudden cardiac death", the content of cholesteryl esters in the c.a. rose to values of greater than 60 x 10(-6) mol/g prot. In age-matched control pigs, the mean ester content was 2.8 or 1.2 x 10(-6) mol/g prot. in "young" and "old" pigs. In nonirradiated hypercholesterolemic pigs, the mean ester content in "young" animals was 39 x 10(-6) mol/g prot. but in "old" pigs it came to 4.8 x 10(-6) mol/g prot. 12 months after the "sudden cardiac death" period had ended, the ester content in the surviving pigs was 25 x 10(-6) mol/g prot. in "young" and 3.5 x 10(-6) mol/g prot. in "old" animals. Irradiation had produced some kind of healing effect. This regress in cholesteryl ester content was significantly and moderately delayed by nifedipine treatment. It did not otherwise change the cholesterol metabolism of the arteries. A probable explanation for the partly marked and rapid changes in cholesterol metabolism of c.a. was that there had been a change in phenotype of vascular smooth muscle cells, mainly localised to the intima of the c.a. The vascular endothelial cells influence the phenotype of vascular smooth muscle cells by stimulating proliferation and accumulation of cholesterol by growth factors (PDGF) which act via thrombospondin. By releasing heparin and/or heparin-like glycosaminoglycans, endothelium may inhibit the effect of thrombospondin on vascular smooth muscle cells. An additional contribution is possibly made by cholesterol from high-lipid macrophages.

Contraction and cyclic AMP-related relaxation of the intimal and medial smooth muscle layers of pig thoracic aorta.

The contractile responses of an alpha-adrenoceptor agonist, phenylephrine, and of histamine were compared in the intimal and medial smooth muscle layers of the pig aortic arch. Further, the relaxant effects evoked by some compounds influencing the cyclic AMP system were compared in the two muscle layers, as well as their effects on the cyclic AMP content and phosphodiesterase activity. Phenylephrine and histamine induced contraction of the smooth muscle layers. The increase in tension was faster in the intimal than in the medial layer. The alpha-adrenoceptor agonist phenylephrine was a more potent contractile agent in the intimal than in the medial smooth muscle. With histamine, no significant difference in the dose-response curves between the two muscle layers was found. Histamine-contracted muscle preparations were relaxed in a dose-dependent manner by the phosphodiesterase-inhibiting compound 3-isobutyl-1-methylaxanthine (MIX) and by 8-bromo-cyclic AMP. The two substances were more potent relaxants in the medial than in the intimal smooth muscle layer. The content of cyclic AMP in the intimal and the medial smooth muscle was increased by MIX. Isoprenaline had no relaxing effect on the muscle preparations and did not change the content of cyclic AMP. There were no differences in the basal levels of cyclic AMP in the intima and media. Vmax of phosphodiesterase activities differed, however, between the two preparations. This study demonstrates that the intimal layer is characterized by a larger contractile responsiveness to phenylephrine and a lower relaxant response to compounds influencing the cyclic AMP-system than those of the medial layer.

Pasteur effect in vascular and intestinal smooth muscle.

The increase in lactate production on changing from aerobic to anaerobic conditions, i.e. the Pasteur effect, has been reported to be small in vascular muscle and especially in aorta. It has been suggested that this may be an artefact caused by damage to the intimal endothelium. We have compared the Pasteur effect in different kinds of pig arteries, but also in rabbit colon. The aerobic lactate production in 60 min was 11-15 mumol/g in the aorta and the carotid artery, but 3 mumol/g in the mesenteric and renal arteries and 4 mumol/g in the rabbit colon. The increase in lactate production under anaerobic conditions was 12-20 mumol/g/60 min in the carotid artery, aorta and rabbit colon and 10 mumol/g/60 min in the mesenteric and renal arteries. When calculated in per cent, the Pasteur effect was greater in the mesenteric artery than in the aorta, but the actual rise in lactate production in mumol/g was higher in the aorta and carotid artery. The high aerobic lactate production of smooth muscle in vitro may be related to its low ability to oxidize glucose; some other substrates may be preferentially oxidized when present in vitro or in vivo.

Sudden death related to advanced coronary atherosclerosis in mini-pigs: influence of some drugs.

Advanced coronary atherosclerosis was produced in 30 mini-pigs by a combination of a hypercholesterolaemic diet and X-irradiation to the precordial region. Within 11-25 weeks after the irradiation, 13 of the 30 animals died a sudden death probably caused by coronary atherosclerosis. The contents of free and ester-bound cholesterol in the right coronary artery were significantly higher in the animals which died spontaneously than in surviving animals. In an untreated group of 12 animals 7 died whereas in a group treated with beta-pyridylcarbinol only 1 out of 5 died. In the coronary arteries, the contents of both free and ester-bound cholesterol were significantly lower in the beta-pyridylcarbinol-treated animals. In a sulfinpyrazone-treated group 3 out of 8, and in a metoprolol-treated group 2 out of 5 animals died. None of these drugs reduced the accumulation of cholesterol in the coronary arteries. The rate of sudden death was 26 +/- 6% (P less than 0.05) lower in the combined group of treated animals than in the untreated ones. By regular ECG recordings, signs which could predict the fatal outcome of the experiment were looked for. Although depressed ST segments were present before death in a few animals, this was not a regular phenomenon. It is concluded that advanced coronary atherosclerosis in mini-pigs often leads to sudden death and that this animal model seems suitable for testing the potential therapeutic effects of drugs.

Influence of anoxia and dinitrophenol on the phosphorylase a activity and the cyclic nucleotide content of smooth muscle.

The effect of anoxia or 2,4-dinitrophenol (DNP) on the phosphorylase activity and the cyclic AMP and the cyclic GMP content was studied in smooth muscle preparations. When the aerobic conditions were changed to anaerobic in experiments on bovine mesenteric artery, there was a significant increase in the activity of phosphorylase a during the first 60 min. We had observed a reduction of the glycogen content of the artery during this time period, which accounted for about 2/3 of the increase in lactate production (Pasteur effect). Under anaerobic conditions the content of cyclic AMP in the vessel was not changed, and the increase in phosphorylase a activity was not inhibited by a blockade of adrenergic beta-receptors. DNP, which like anoxia inhibits the mitochondrial production of ATP, increased the phosphorylase a activity to the same extent as anoxia. Anoxia and DNP also enhanced the activity of phosphorylase a in pig thoracic aorta and rabbit colon smooth muscle. In thoracic aorta both anoxia and DNP produced a more transient and smaller increase in the phosphorylase a activity than in the mesenteric artery. The Pasteur effect was also relatively smaller (100%) in thoracic aorta than in mesenteric artery (400%). It is suggested that an anoxic increase in the phosphorylase a activity participates in the Pasteur effect in smooth muscle.

Experimental atherosclerosis in hypercholesterolemic mini-pigs. Regression of cholesterol ester accumulation in aorta and coronary arteries after treatment with clofibrate, beta-pyridylcarbinol or a normo-lipidemic diet.

Studies have been performed on groups of mini-pigs 21-23 months of age, which after 18 months of hypercholesterolemia (approximately 10 mmol) had developed raised atherosclerotic lesions with high levels of cholesterol esters, especially in the abdominal aorta and the coronary arteries. If the hypercholesterolemia was continued for 18 months, no significant change in the cholesterol ester content in the aorta occurred; in the coronary arteries there was a significant decrease in these older pigs. If the hypercholesterolemic pigs also were treated with beta-pyridylcarbinol the findings were very similar to the first. When hypercholesterolemic pigs were treated with clofibrate, or when the hypercholesterolemic diet was replaced with the basal food for 18 months, the plasma cholesterol level was normalized (approximately 2 mmol) within 1-2 months. The cholesterol ester content in the thoracic aorta was reduced in both groups but not that in the abdominal aorta. Clofibrate decreased the cholesterol ester level in the coronary arteries when compared to the hypercholesterolemic group; the drug also reduced the free cholesterol level when compared to the basal group. We suggest that an increased plasma cholesterol level initiated the development of the atherosclerotic lesions; their later development was only partly dependent on the plasma cholesterol level.

Some properties of Ca-binding microsomal subfractions isolated from rabbit colon muscle.

From a homogenate of rabbit colon smooth muscle a microsomal fraction was isolated, which was divided into subfractions by centrifugation on a discontinuous sucrose gradient. The Ca-binding properties of the subfractions were investigated under different conditions. In the presence of 0.35 mM ATP the Ca binding of the fractions amounted to 4--8 nmol/mg protein. The 35% fraction bound more Ca per mg protein than the 35--45% fraction. The Ca accumulation was comparatively higher both in the presence of 5 mM ATP and in the presence of 5 mM oxalate. The two fractions showed about the same sensitivity for oxalate. This substance stimulated the Ca uptake at 5 mM but not at lower concentrations. The amount and the rate of Ca binding were more dependent on variations in the exogenous ATP concentration in the 35% fraction than was the case for the 35--45% fraction. The Ca binding was completely inhibited by salyrgan when the microsomal fractions were pretreated with this agent. Sodium azide did not influence the Ca-binding capacity of the fractions. It is suggested that the microsomal subfractions of the rabbit colon muscle represent physiologically important parts of the Ca sequestering system of the muscle, since Ca binding takes place at Ca- and ATP-concentrations which are believed to be present in the myoplasm.