[Renal replacement therapy in the elderly]. / Nierenersatztherapie bei Älteren.

Renal function in humans declines with old age. Currently, the normal range for renal function is not differentiated by age but uniformly given, which has evoked criticism. The symptoms of high-grade impairment of renal function are nonspecific. The current data situation does not support early initiation of dialysis: on the contrary, initiation of dialysis should be decided from clinical aspects and not according to the values for the glomerular filtration rate (GFR). Elderly patients should be offered peritoneal dialysis (PD) as well as hemodialysis (HD), which can be performed either at home (PD) or at a dialysis center (HD or PD). Patients and their relatives should be presented with all available information on both therapy options but there are also conservative, palliative therapy options for the very old or for those with a high number of comorbidities. The decision for therapy should be individualized and tailored for each patient. The planning of kidney replacement therapy should be carried out well in advance. A multidisciplinary team should discuss possible barriers to one or the other treatment option and provide assistance for implementation of the individual optimal therapy. In some cases a home-based assisted PD can be a sensible option.

Clindamycin-primaquine for pneumocystis jiroveci pneumonia in renal transplant patients.

BACKGROUND: Trimethoprim/sulfamethoxazole (TMP/SMX) is considered first-line therapy for pneumocystis jiroveci pneumonia (PCP) in renal transplant patients. Alternatives have not been formally studied. Clindamycin-primaquine (C-P) is effective in HIV-associated PCP, but data in renal transplant patients are lacking. PATIENTS AND METHODS: Retrospective cohort study of 57 consecutive renal transplant patients who developed PCP and were treated with C-P (n = 23) or TMP/SMX (n = 34). RESULTS: A non-significantly higher failure rate was observed in patients on C-P due to lack of efficacy (30.4 versus 20.6%, p = 0.545). The difference was more pronounced in severe PCP (60 versus 37.5%, p = 0.611) and a significantly lower efficacy of C-P was seen when used as salvage therapy. The two patients who had received C-P after not responding to TMP/SMX failed this regimen, but all seven patients who had failed initial treatment with C-P and had been switched to TMP/SMX were cured (p = 0.028). No treatment-limiting adverse reactions were reported for patients on C-P while six patients (17.6%) on TMP/SMX developed possibly related treatment-limiting toxicity (p = 0.071). However, in only two patients adverse events were definitely related to TMP/SMX (5.9%). CONCLUSIONS: Clindamycin-primaquine appears to be safe and well tolerated for treating PCP in renal transplant patients but is probably less effective than TMP/SMX, the standard regimen. However, our data indicates that C-P represents an acceptable alternative for patients with contraindications or treatment emergent toxicities during TMP/SMX use. Notably, TMP/SMX was also acceptably tolerated in most patients. TMP/SMX remains an effective salvage regimen in case of C-P failure.

Elimination of middle-sized uremic solutes with high-flux and high-cut-off membranes: a randomized in vivo study.

The elimination of substances between 10 and 50 kDa by conventional high-flux membranes is not satisfactory. We investigated in vivo the elimination of middle-sized uremic solutes by conventional polyflux (PF) and modified high-cut-off (HCO) membranes. All 12 patients underwent four treatments, two with the HCO dialyzer and two with the PF dialyzer, each in either a haemodialysis (HD) or haemodiafiltration (HDF) mode. The reduction ratio of urea, creatinine, ß2-microglobulin (ß2M), leptin, soluble TNF-RI, complement factor D, IL-6, sIL-6 receptor, advanced glycation end-products (AGEs) and albumin was determined. In addition, the amount removed was determined in the dialysate for ß2M, complement factor D, AGEs and albumin. Treatment with HCO removed ß2M, sTNF-RI, factor D, and high molecular AGE significantly better than conventional high-flux membranes. The albumin loss was higher when using HCO membranes. HCO membranes are a promising approach to improve removal of uremic toxins not affected by conventional high-flux membranes.

5-(4-Hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-2,3,6,7-tetrahydro-1,4-thiazepines as compounds with high affinity at the benzodiazepine binding site on GABA(A) receptors.

A series of thiazepines has been studied as new ligands for the benzodiazepine binding site of the GABAA receptor. Compounds with high affinity and weak selectivity regarding alpha beta3gamma2, alpha2beta3gamma2, alpha3beta3gamma2, and alpha5beta3gamma2 subtypes were found. The pharmacophore is discussed based on experimental and theoretical results. The thiazepine sulfur atom was found to be able to act as hydrogen bond acceptor.

Metal pollutant pathways in cohesive coastal catchments: Influence of flocculation and biopolymers on partitioning and flux.

The impacts of the partitioning of potentially toxic metals (PTM) within the estuarine environment is highly complex, but is of key significance owing to increases in populations living within such sensitive environments. Although empirical data exist for the partitioning of metals between the dissolved and particulate phases, little is known regarding the impacts of extracellular polymeric substances (EPS) upon the flocculation of particles within such a dynamic system nor the resultant influence on the distribution of metals between the particulate and dissolved phases. This prevents regulators from fully understanding the fate and risks associated with metals in estuaries. This study provides data associated with the simulation of 3 settlings typical of the turbulent mixing found in estuaries and partitioning of copper, cadmium, nickel, arsenic, lead and zinc for 3 salinities (0, 15, 30 PSU) reflecting the full salinity range from freshwater to seawater. Experiments were completed with and without the presence of EPS, using kaolin as the mineral particulate. The results showed significant differences between salinity, PTMs and turbulence for the experiments with and without EPS present. Overall, salinity was the main factor controlling the PTM partitioning to sediment, however the flocculation process did impact on the PTM distribution and with the addition of EPS the impact was more pronounced. The data highlighted the importance of taking account of EPS within any estuarine sediment process modelling, for relying on simple partitioning with corrections for salinity would likely lead to significant bias.

Impaired insulin sensitivity as an underlying mechanism linking hepatitis C and posttransplant diabetes mellitus in kidney recipients.

Aim of this study was to investigate the mechanism/s associating hepatitis C virus (HCV) infection and posttransplant diabetes mellitus in kidney recipients. Twenty HCV-positive and 22 HCV-negative kidney recipients, 14 HCV-positive nontransplant patients and 24 HCV-negative nontransplant (healthy) subjects were analyzed. A 3-h intravenous glucose tolerance test was performed; peripheral insulin sensitivity was assessed by minimal modeling. Pancreatic insulin secretion, hepatic insulin uptake, pancreatic antibodies and proinflammatory cytokines in serum (tumor necrosis factor-alpha, intereukin-6, high-sensitive C-reactive protein) were also assessed. HCV-positive recipients showed a significantly lower insulin sensitivity as compared to HCV-negative recipients (3.0 +/- 2.1 vs. 4.9 +/- 3.0 min(-1).microU.mL(- 1).10(4), p = 0.02), however, insulin secretion and hepatic insulin uptake were not significantly different. Pancreatic antibodies were negative in all. HCV status was an independent predictor of impaired insulin sensitivity (multivariate analysis, p = 0.008). The decrease of insulin sensitivity due to HCV was comparable for transplant and non-transplant subjects. No significant correlation was found between any of the cytokines and insulin sensitivity. Our results suggest that impaired peripheral insulin sensitivity is associated with HCV infection irrespective of the transplant status, and is the most likely pathogenic mechanism involved in the development of type 2 diabetes mellitus associated with HCV infection.

Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway.

ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. To identify the targeting signals that mediate this recycling, N-glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER-ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC-cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC-53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER-retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant.

Studies on the division cycle of mammalian cells. V. Modifications of time parameters by different steady-state culture conditions.

In cultures of murine neoplastic mast cells, the duration of different phases of the division cycle (G(1), S, G(2), and mitosis [M]) was determined under optimal and several well-defined suboptimal growth conditions. Two methods of evaluation were applied to the same culture system: first, the relative number of G(1), S, G(2), and M cells was determined by pulse labeling of samples with thymidine-(3)H and subsequent radioautography in conjunction with a microfluorometric technique permitting rapid measurements of cellular DNA content; second, after pulse labeling with thymidine-(3)H, the variations with time of the mitotic labeling index were analyzed. Suboptimal culture conditions were obtained by reducing the concentration of single essential medium components (leucine, glucose, or serum) or by the addition of specific metabolic inhibitors (actinomycin D, amethopterin). Growth-limiting culture conditions resulted in increased generation times. Even under control conditions, the cell number doubling time exceeded the generation time, and this difference was more pronounced in suboptimal media. Under most of the suboptimal conditions tested, the increase in generation time was attributable primarily to an extended duration of the G(1) phase. Under certain growth-limiting conditions, however, other phases were also prolonged. In addition, the variabilities of the generation time and of certain cell cycle phases were increased under suboptimal culture conditions. Results obtained by the two methods of evaluation were, in general, in good agreement with each other. Some differences were, however, observed and interpreted in terms of cell death and/or asymmetric frequency distributions of cell cycle parameters.

Determination of rates of DNA synthesis in cultured mammalian cell populations.

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [(3)H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 microM) in combination with hypoxanthine and glycine. If [(3)H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [(3)H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10(6) cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.

Formation of mast cell granules in cell cycle mutants of an undifferentiated mastocytoma line: evidence for two different states of reversible proliferative quiescence.

A heat-sensitive (hs, arrested at 39.5 degrees C, multiplying at 33 degrees C) and a cold-sensitive (cs, arrested at 33 degrees C, multiplying at 39.5 degrees C) cell cycle variant were isolated from an undifferentiated P-815 murine mastocytoma line. At the respective nonpermissive temperature, both the hs and the cs variant cells were reversibly arrested with a DNA content, typical of G1 phase. The cells of two cs variant subclones, when exposed to the nonpermissive temperature of 33 degrees C, formed metachromatically staining granules with an ultrastructure resembling that of mature mast cells. In addition, the cellular 5-hydroxytryptamine content underwent a marked increase, and the cells responded to compound 48/80 by degranulation as described for normal mast cells. On the other hand, in cells of two hs variant subclones, essentially no mast cell granules were detectable at either 33 or 39.5 degrees C. As previously reported, the cs cell cycle variant phenotype is expressed dominantly in heterokaryons obtained by fusing cs with wild-type cells, whereas hs cell cycle variant cells, similar to other hs mutants, were found to behave recessively under these conditions. Thus the state of proliferative quiescence induced in the cs cells at 33 degrees C is qualitatively different from the state of cell cycle arrest observed in hs cells at 39.5 degrees C and may represent a model for proliferative quiescence of differentiated cells in the intact organism.

Plasma Biomarkers of AD Emerging as Essential Tools for Drug Development: An EU/US CTAD Task Force Report.

There is an urgent need to develop reliable and sensitive blood-based biomarkers of Alzheimer's disease (AD) that can be used for screening and to increase the efficiency of clinical trials. The European Union-North American Clinical Trials in Alzheimer's Disease Task Force (EU/US CTAD Task Force) discussed the current status of blood-based AD biomarker development at its 2018 annual meeting in Barcelona, Spain. Recent improvements in technologies to assess plasma levels of amyloid beta indicate that a single sample of blood could provide an accurate estimate of brain amyloid positivity. Plasma neurofilament light protein appears to provide a good marker of neurodegeneration, although not specific for AD. Plasma tau shows some promising results but weak or no correlation with CSF tau levels, which may reflect rapid clearance of tau in the bloodstream. Blood samples analyzed using -omics and other approaches are also in development and may provide important insight into disease mechanisms as well as biomarker profiles for disease prediction. To advance these technologies, international multidisciplinary, multi-stakeholder collaboration is essential.

Impact of acute kidney injury on neurological outcome and long-term survival after cardiac arrest - A 10 year observational follow up.

BACKGROUND: Acute kidney injury (AKI) may be associated with short- and long-term patient morbidity and mortality. Therefore, the impact of AKI after cardiac arrest on survival and neurological outcome was evaluated. METHODS: An observational single center study was conducted and consecutively included all out and in hospital cardiac arrest (OHCA/IHCA) patients treated with therapeutic temperature management between 2006 and 2013. Patient morbidity, mortality and neurological outcome according to the widely used Pittsburgh Cerebral Performance Category (CPC) were assessed. A good neurological outcome was defined as a CPC of 1-2 versus a poor neurological outcome with a CPC of 3-5. AKI was defined by using the KDIGO Guidelines 2012. RESULTS: 503 patients were observed in total. 29.4% (n = 148) developed AKI during their intensive care unit (ICU) stay. 70.6% (n = 355) did not experience AKI. The mean age at admission was 62 years, of those 72.8% were male and 77% experienced an out-of-hospital cardiac arrest (OHCA). AKI occurred with 41.2% more often in the group with poor neurological outcome compared to 17.1% in the group with good neurological outcome. The median survival for patients after cardiac arrest with AKI was 0.07 years compared to 6.5 years for patients without AKI. CONCLUSION: Our data suggest that AKI is a major risk factor for a poor neurological outcome and a higher mortality after cardiac arrest. Further important risk factors were age, time to ROSC and high NSE.

Interleukin 1 (IL-1) gene expression, synthesis, and effect of specific IL-1 receptor blockade in rabbit immune complex colitis.

Interleukin 1 (IL-1) may be a key mediator of inflammation and tissue damage in inflammatory bowel disease (IBD). In rabbits with immune complex-induced colitis, IL-1 alpha and beta mRNA levels were detectable at 4 h, peaked at 12 but were absent at 96 h after the induction of colitis. Colonic IL-1 tissue levels were measured by specific radioimmunoassays. IL-1 alpha was significantly elevated at 4 h (9.4 +/- 1.5 ng/g colon), progressively increased at 48 h (31 +/- 5.8 ng/g) and then decreased by 96 h (11.5 +/- 3.4 ng/g). IL-1 beta levels were 2.0 +/- 0.5 ng/g colon at 4 h, 5.0 +/- 1.6 ng/g at 48 h and undetectable by 96 h. By comparison, colonic levels of PGE2 and LTB4 were unchanged during the first 12 h and did not become elevated until 24 h. IL-1 alpha levels were highly correlated with inflammation (r = 0.885, P less than 0.0001), edema (r = 0.789, P less than 0.0001) and necrosis (r = 0.752, P less than 0.0005). Treatment with a specific IL-1 receptor antagonist (IL-1 ra) before and during the first 33 h after the administration of immune complexes markedly reduced inflammatory cell infiltration index (from 3.2 +/- 0.4 to 1.4 +/- 0.3, P less than 0.02), edema (from 2.2 +/- 0.4 to 0.6 +/- 0.3, P less than 0.01) and necrosis (from 43 +/- 10% to 6.6 +/- 3.2%, P less than 0.03) compared to vehicle-matched colitis animals. These studies demonstrate that (a) IL-1 gene expression and synthesis occur early in the course of immune complex-induced colitis; (b) are significantly elevated for 12 h before the appearance of PGE2 and LTB4; (c) tissue levels of IL-1 correlate with the degree of tissue inflammation and; (d) specific blockade of IL-1 receptors reduces the inflammatory responses associated with experimental colitis.

EU/US/CTAD Task Force: Lessons Learned from Recent and Current Alzheimer's Prevention Trials.

At a meeting of the EU/US/Clinical Trials in Alzheimer's Disease (CTAD) Task Force in December 2016, an international group of investigators from industry, academia, and regulatory agencies reviewed lessons learned from ongoing and planned prevention trials, which will help guide future clinical trials of AD treatments, particularly in the pre-clinical space. The Task Force discussed challenges that need to be addressed across all aspects of clinical trials, calling for innovation in recruitment and retention, infrastructure development, and the selection of outcome measures. While cognitive change provides a marker of disease progression across the disease continuum, there remains a need to identify the optimal assessment tools that provide clinically meaningful endpoints. Patient- and informant-reported assessments of cognition and function may be useful but present additional challenges. Imaging and other biomarkers are also essential to maximize the efficiency of and the information learned from clinical trials.

Amelioration of anemia after kidney transplantation in severe secondary oxalosis.

INTRODUCTION: In small bowel disease such as M. Crohn, the intestinal absorption of oxalate is increased. Severe calcium oxalate deposition in multiple organs as consequence of enteric hyperoxaluria may lead to severe organ dysfunction and chronic renal failure. The management of hemodialyzed patients with short bowel syndrome may be associated with vascular access problems and oxalate infiltration of the bone marrow leading to pancytopenia. Although the risk of recurrence of the disease is very high after renal transplantation, it may be the ultimate therapeutic alternative in secondary hyperoxaluria. CASE: Here, we report a patient with enteric oxalosis due to Crohn's disease. He developed end-stage renal disease, erythropoietin-resistant anemia, oxalate infiltration of the bone marrow and severe vascular access problems. Following high-urgency kidney transplantation, daily hemodiafiltration of 3 hours was performed for 2 weeks to increase oxalate clearance. Despite tubular and interstitial deposition of oxalate in the renal transplant, the patient did not require further hemodialysis and the hematocrit levels normalized. DISCUSSION: Early treatment of hyperoxaluria due to short bowel syndrome is essential to prevent renal impairment. Declining renal function leads to a further increase in oxalate accumulation and consecutive oxalate deposition in the bone marrow or in the vascular wall. If alternative treatments such as special diet or daily hemodialysis are insufficient, kidney transplantation may be a therapeutic alternative in severe cases of enteric oxalosis despite a possible recurrence of the disease.

Living kidney donation: anemia and inflammation in the early postoperative period.

Studies on living donor kidney transplantation primarily address the recipients; few publications focus on kidney donors. The aim of the present study was to detect changes in and compensations of defined parameters of anemia and inflammation in the immediate postnephrectomy period. We included six living kidney donors who underwent an open anterior-extraperitoneal nephrectomy. We excluded donors with complications, such as significant blood loss or infection. Blood samples were taken before nephrectomy as well as on postoperative days 1, 3, 5, and 7, and at discharge for measurements of hemoglobin (Hb), serum erythropoietin (Epo), reticulocytes (Reti), pentraxin 3 (PTX3), and C-reactive protein (CRP). There was a significant decrease in Hb (>3 g/dL), reaching a maximum on day 3 followed by a significant threefold increase in Epo levels on day 5 and a nonsignificant elevation of Reti count. CRP increased approximately 80-fold on day 3. PTX3 showed a similar course, peaking on day 3 with an approximate 70-fold increase. After living donor nephrectomy, there was an unexpectedly pronounced inflammatory reaction in the absence of any signs of bacterial infection simultaneous with a significant decrease in Hb. These parameters improved during the hospital stay, in some cases they achieved the prenephrectomy level at discharge. These data may assist in interpreting laboratory results after nephrectomy among living kidney donors.

Review article: long-term safety of nucleoside and nucleotide analogues in HBV-monoinfected patients.

BACKGROUND: Nucleos(t)ide analogues (NUCs) for chronic hepatitis B treatment achieve high rates of viral suppression and are generally well tolerated. Entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are the currently preferred first-line agents. The safety of these agents in clinical practice is particularly relevant since long-term treatment is usually required. AIM: To summarise and critically discuss recent real-world evidence on the safety of treatment with ETV or TDF in hepatitis B virus (HBV)-monoinfected patients. METHODS: PubMed and conference proceedings up to 15th June 2015 were searched using the terms ((((Hepatitis_B) OR HBV) AND ((tenofovir) OR entecavir)) AND (((lactic_acidosis) OR bone) OR renal)). RESULTS: In selected populations included in registration studies, both ETV and TDF were well tolerated with no clinically significant renal toxicity or lactic acidosis. Growing 'real-world' clinical experience with these agents includes some reports of ETV-associated lactic acidosis and TDF-associated renal impairment; however, evidence from cohort studies appears to be conflicting. In the case of ETV-related lactic acidosis, a small number of cases have been reported, all in patients with decompensated cirrhosis. The degree of association between TDF treatment and changes in markers of renal function varies between studies: discrepancies may result from the use of different definitions and cut-offs for reporting renal toxicities, and differences in patient populations. CONCLUSIONS: Pre-treatment and on-treatment monitoring of eGFR and phosphorus, with prompt appropriate dose adjustment or treatment switch can minimise the impact of NUC renal toxicity. Standardisation of measures of renal impairment and identification of early molecular markers remain an unmet need.

Effects of temperature changes on thymidine kinase in heat- and cold-sensitive cell-cycle mutants and 'wild-type' murine P-815 cells.

Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for 'wild-type' K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3-4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of 'wild-type' cells. In 'wild-type' K 21 cells incubated at 39.5 degrees C, thymidine kinase activity was approx. 30% of that at 33 degrees C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5 degrees C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5 degrees C to 33 degrees C was inhibited by actinomycin D and cycloheximide.

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells.

Heat-sensitive (arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in 'gently lysed cells' (obtained by treatment with 0.01% Brij-58) or 'highly lysed cells' (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [3H]dTTP efficiently, while highly lysed cells exhibited a low level of [3H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extract reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.

Control of DNA polymerase alpha, beta and gamma activities in heat- and cold-sensitive mammalian cell-cycle mutants.

Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase alpha, beta and gamma activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase alpha activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase alpha activities in arrested cells were as high as 15-40% of control values. After return of arrested cells to the permissive temperature, polymerase alpha activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), the decrease of polymerase alpha during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase alpha is tightly coupled with reentry of cells into S phase. For DNA polymerase beta and gamma activities, no obvious correlation with changes in the proliferative state of cells was detected.