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Plasma nitriding is an effective surface hardening treatment for austenitic stainless steels. During plasma nitriding, s‐phase formation takes place which is not only responsible for high hardness and wear resistance but also for good corrosion resistance. In order to estimate the thickness of the s‐phase for austenitic stainless steel in a plasma nitriding process, an empirical model is devised. A number of plasma nitriding processes of austenitic stainless steel (304 L) were carried out with varying treatment temperature from 360 °C to 450 °C and process duration ranging from 10 hours to 24 hours with constant pressure, voltage, pulse‐to‐pause‐ratio and gas mixture. A time‐temperature dependent s‐phase formation coefficient is determined by measuring the thickness of the s‐phase using a scanning electron microscope (SEM) and glow discharge optical emission spectroscopy (GDOES). The developed model is verified by three controlled experiments. This model fits the thickness of the s‐phase with an error of less than 6 %. [ABSTRACT FROM AUTHOR]

Plasma nitriding offers great potenzial for improving the wear properties of austenitic steels. Here, the austenitic standard grades 1.4307 and 1.4404, as well as the titanium‐stabilized grades 1.4541 and 1.4571 were investigated regarding the influence of the material condition on the nitriding result and corrosion behavior. Special focus was put on the influence of the Ti‐stabilisation. In addition, it was investigated to what extent corrosion properties are influenced by cold‐forming induced defect structures. In comparison to 1.4307 and 1.4404, less nitrogen is incorporated in areas with forming martensite in titanium‐stabilized austenitic steels and lower nitriding temperatures, while an increased diffusion of nitrogen is observed, when only slip bands are present. The corrosion resistance is generally improved by the plasma nitriding parameters used for this study. In general, a higher thickness of the S‐phase, which forms during the plasma nitriding, results in better corrosion resistance and higher surface hardness. The titanium stabilization inhibits nitrogen diffusion in the presence of deformation induced martensite at lower nitriding temperatures and promotes diffusion in the presence of deformation induced slip bands. [ABSTRACT FROM AUTHOR]

Electron backscatter diffraction (EBSD) and scanning electron microscopy were combined to study the effect of residual defect density on corrosion initiation in aluminium alloy AA2024-T3. EBSD was used to determine the level of misorientation (MO), from pixel to pixel, within individual grains. TheMO can be determined with respect to either the average orientation angle of the grain orwith respect to the average orientation angle of the surrounding pixels (in this instance, amatrix of 7 ×7 surrounding pixels has been applied). Herein, the MO, determined using the surrounding pixels, was used as the means for the assessing the level of defect density within a grain. Itwas found that there was a noteworthy, but not definitive, correlation ofMOwith corrosion initiation after 1 min exposure to 0.1MNaCl solution. Additionally, the S and θ-phase particles were also identified using EBSD, displaying a range of MO and therefore defect density. [ABSTRACT FROM AUTHOR]

The current study focuses on the characterization of the nitrided layer, formed in American Iron and Steel Institute (AISI) 304L and AISI 904L austenitic stainless steels by industrial low-temperature plasma nitriding, using combined analysis techniques. The study highlighted that the evolution of the microstructure of the nitrided layers is influenced by surface finishing prior to nitriding, alloying elements and nitriding conditions, all factors affecting S-phase formation and nitrogen (N) diffusion mechanisms. The chemical bonding characteristics of Cr 2p3/2 and N 1s as revealed by XPS show a shift in binding energy between expanded austenite (S-phase) and CrN-like compounds. S-phase has proven to be more stable in 904L, whereas residual and/or induced ferrite/martensite in 304L acts as a barrier for its development. [ABSTRACT FROM AUTHOR]

Sequential sectioning and sequential imaging by scanning electron microscopy have been employed to obtain three-dimensional images of an AA2024-T3 aluminium alloy after exposure to sodium chloride solution. The three-dimensional arrangement of intermetallic particles within the alloy was disclosed, indicating that groupings of intermetallics are preferential sites for corrosion initiation due to the combined presence of defects such as cracks or voids and local differences in electrochemical potential. Three-dimensional imaging enabled the study of the dealloying process occurring on the S-phase particles (Al-Cu-Mg) during corrosion, revealing the details of a copper-rich sponge-like structure. Initially, dealloying proceeds by initial formation of a copper-rich nanoporous layer containing significant amounts of residual aluminium and magnesium. Subsequently, the layer dealloys further, increasing the free volume of the porous structure. At later stages of dealloying, some copper metal clusters detach from the substrate and oxidize. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Platyhelminthes are highly attractive models for addressing fundamental aspects of stem cell biology in vivo. These organisms possess a unique stem cell system comprised of neoblasts that are the only proliferating cells during adulthood. We have investigated Ts (S-phase duration) of neoblasts during homoeostasis and regeneration in the flatworm, Macrostomum lignano. A double immunohistochemical technique was used, performing sequential pulses with the thymidine analogues CldU (chlorodeoxyuridine) and IdU (iododeoxyuridine), separated by variable chase times in the presence of colchicine. Owing to the localized nature of the fluorescent signals (cell nuclei) and variable levels of autofluorescence, standard intensity-based colocalization analyses could not be applied to accurately determine the colocalization. Therefore, an object-based colocalization approach was devised to score the relative number of double-positive cells. Using this approach, Ts (S-phase duration) in the main population of neoblasts was ∼13 h. During early regeneration, no significant change in Ts was observed. [ABSTRACT FROM AUTHOR]

Cross-sectional transmission electron microscopy and X-ray phase analysis were used to study the structure of a layer formed during nitriding the AISI 316L stainless steel at temperature 440°C. It was found that the applied treatment led to the formation of 6-μm-thick layer of the S-phase. There is no evidence of CrN precipitation. The X-ray diffraction experiments proved that the occurred austenite lattice expansion – due to nitrogen atoms – depended on the crystallographic direction. The cross-sectional transmission electron microscopy studies showed that the layer consisted of a single cubic phase that contained a lot of defects such as dislocations, stacking faults, slip bands and twins. The high-resolution electron microscopy observations were applied to study the defect formation due to the nitriding process. It was shown that the presence of great number of stacking faults leads to formation of nanotwins. Weak, forbidden {100} reflections were still another characteristic feature of the S-phase. These were not detected in the X-ray spectra of the phase. Basing on the high-resolution electron microscopy studies it can be suggested that the short-range ordering of the nitrogen atoms in the octahedral sites inside the f.c.c. matrix lattice takes place and gives rise to appearance of these spots. It is suggested that the cubic lattice undergoes not only expansion but also slight rombohedral distortion that explains differences in the lattice expansion for different crystallographic directions. [ABSTRACT FROM AUTHOR]

Current understanding of the mechanisms involved in osseointegration following implantation of a biomaterial has led to adhesion quantification being implemented as an assay of cytocompatibility. Such measurement can be hindered by intra-sample variation owing to morphological changes associated with the cell cycle. Here we report on a new scanning electron microscopical method for the simultaneous immunogold labelling of cellular focal adhesions and S-phase nuclei identified by BrdU incorporation. Prior to labelling, cellular membranes are removed by tritonization and antigens of non-interest blocked by serum incubation. Adhesion plaque–associated vinculin and S-phase nuclei were both separately labelled with a 1.4 nm gold colloid and visualized by subsequent colloid enhancement via silver deposition. This study is specifically concerned with the effects microgroove topographies have on adhesion formation in S-phase osteoblasts. By combining backscattered electron (BSE) imaging with secondary electron (SE) imaging it was possible to visualize S-phase nuclei and the immunogold-labelled adhesion sites in one energy ‘plane’ and the underlying nanotopography in another. Osteoblast adhesion to these nanotopographies was ascertained by quantification of adhesion complex formation. [ABSTRACT FROM AUTHOR]

Abstract: Prostaglandin F2α (PGF2α) induces cyclin D1 expression and DNA synthesis in Swiss 3T3 cells. In order to assess which signaling mechanisms are implicated in these processes, we have used both a pharmacological approach and interfering mutants. We demonstrate that PGF2α induces extracellular-signal-regulated kinase (ERK1-2) and p38MAPK activation, and inhibition of any of these signaling pathways completely blocks PGF2α-stimulated DNA synthesis. We also show that ERK1-2, but not p38MAPK activation is required to induce cyclin D1 expression, strongly suggesting that the concerted action of cyclin D1 gene expression and other events are required to induce complete phosphorylation of retinoblastoma protein and S-phase entry in response to PGF2α. [Copyright &y& Elsevier]

Abstract. Purpose: The transforming growth factor-β (TGF-β) family includes three multifunctional proteins, TGF-β1, TGF-β2 and TGF-β3, expressed in ocular tissue, which are involved in regulating cell differentiation, cell proliferation and other cell functions. TGF-β is present in aqueous humour and regulates corneal endothelial cells. This study explores the mechanism by which TGF-β regulates the cell cycle in cultured corneal endothelial cells. Methods: The expression of specific receptors for the TGF-β family was investigated at the protein level by affinity cross-linking with radio-iodinated TGF-β1 and immunoprecipitation with specific antibodies to TGF-β receptors. Regulation of entry into the S-phase of the cell cycle was determined by 5-bromo-2′ deoxyuridine (BrdU) incorporation into the cells. The signal transduction pathways were investigated using various blocking agents for protein kinase transducers involved in intracytoplasmic signal transduction. Results: Cultured bovine corneal endothelial cells were confirmed to express TGF-β type 1 and type 2 receptors and endoglin. In the confluent state, TGF-β1 and TGF-β2 stimulated the cells to progress to the S-phase of the cell cycle through platelet-derived growth factor-B (PDGF-B) chain production and protein kinase C. Conclusions: TGF-β accelerated cell cycle progression from the G0/G1 phase to the S-phase in cultured corneal endothelial cells, under our experimental conditions, through pathways involving protein kinase C. These pathways are related to the cross-talk between TGF-β and other cytokines. The conditions employed in the present experiments may be useful for investigating the complex cross-talk between various cytokines and growth factors. [ABSTRACT FROM AUTHOR]

The features of proliferation in brain tumours are related with clinical prognosis for several types of brain tumours, especially gliomas. For childhood central primitive neuro-ectodermal tumours (cPNET), including medulloblastoma, this relation has previously been unclear. The aim of this study is to investigate the relationship between proliferative features of cPNET and in vitro resistance for cytostatic drugs measured with the 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium-bromide (MTT) assay. Tumour material was obtained from 23 surgical specimens of cPNET. The expression of the proliferation markers Ki-67, proliferating cell nuclear antigen (PCNA) and cyclin D1 was determined with immunohistochemistry, while S-phase and DNA ploidy were analysed by flowcytometric analysis cell scan (FACS). The in vitro resistance for 10 cytostatic drugs was determined with the MTT assay. Drug resistance levels were available in 19 (83%) of the 23 samples with a complete profile of 10 cytostatic drugs tested in 14 samples. An excellent correlation in drug resistance scores was found between pharmacologically related drugs. The Ki-67 staining in 20 samples varied from 10 to 60% and from 30 to 100% for PCNA. Cyclin D1 staining was negative in 11 out of 18 samples. The S-phase in 16 samples ranged from 2 to 16%. Increased staining of Ki-67 was related with actinomycin D sensitivity (r -.603; P =0.022), while cells with a higher S-phase percentage were more resistant to ifosfamide (r .952; P <0.0001). In vitro drug resistance testing of central primitive neuro-ectodermal tumours (PNET) is feasible with the MTT assay. Ifosfamide resistance was related with increased Ki-67 and S-phase percentage of the tumour cells, while increased Ki-67 was also related with actinomycin D sensitivity. These findings suggest a cell cycle dependent activity of cytostatic drugs in vitro . [ABSTRACT FROM AUTHOR]

The naturally synchronous plasmodia of myxomycetes synthesize poly(β-l-malic acid), which carries out cell-specific functions. In Physarum polycephalum , poly(β-l-malate) [the salt form of poly(β-l-malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory activity is cell-cycle-dependent, purified DNA polymerase α of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [3 H]thymidine 5′-monophosphate into acid precipitable nick-activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S-phase, equivalent to a minimum of inhibitory activity. To test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed in S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly(β-l-malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(β-l-malate) in plasmodia of Physarum polycephalum . Eur. J. Cell Biol. 70 , 373–380]. To explain the variation in the cell cycle, a periodic competition for poly(β-l-malate) between DNA polymerases and most likely certain histones was assumed. These␣effectors are synthesized in S-phase. By competition␣they displace DNA polymerase from the complex of poly(β-l-malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(β-l-malate) is active in maintaining mitotic synchrony of plasmodia by playing the mediator between the periodic synthesis of... [ABSTRACT FROM AUTHOR]

Cdc45p assembles at replication origins before initiation and is required for origin firing in Saccharomyces cerevisiae. A heat-inducible cdc45 degron mutant was constructed that promotes rapid degradation of Cdc45p at the restrictive temperature. Consistent with a role in initiation, loss of Cdc45p in G1 prevents all detectable DNA replication without preventing subsequent entry into mitosis. Loss of Cdc45p activity during S-phase blocks S-phase completion but not activation of replication checkpoints. Using density substitution, we show that after allowing replication fork establishment, Cdc45p inactivation prevents the subsequent progression of individual replication forks. This provides the first direct functional evidence that Cdc45p plays an essential role during elongation. Thus, like the large T antigen in SV40 replication, Cdc45p plays a central role in both initiation and elongation phases of chromosomal DNA replication. [ABSTRACT FROM AUTHOR]

Assessment of cell proliferation by Ki-67 staining and flow cytometry in fine needle aspirates (FNAs) of reactive lymphadenitis and non-Hodgkin's lymphomasThis study was undertaken to assess cell proliferation in FNAs from a series of 57 non-Hodgkin's lymphomas (NHL) and 11 cases of reactive lymphadenitis using Ki-67 staining and flow cytometry (FCM). The results were compared and correlated to the cytomorphological subgrouping according to Kiel classification. The mean percentages of Ki-67 positivity were 16.6% and 61.1% for low and high grade lymphomas, respectively (P < 0.001). The mean S-phase fraction (SPF) determined by FCM was 4.6% for low grade and 12.9% for high grade lymphomas (P < 0.001). The figures for Ki-67 positivity and S-phase fraction in reactive lymphadenitis were 16.8% and 4%, respectively. We observed a strong correlation in low grade lymphomas between Ki-67 and SPF. A good correlation was also found in reactive lymphadenitis. In high grade lymphomas, however, with highly scattered Ki-67 and S-phase values, this correlation was lost. In some cases this discrepancy can be explained by a rich admixture of non-neoplastic, non-proliferating cells in aspirates from diploid tumours. In addition, the existence of a minor aneuploid tumour cell population of high proliferation such as that in Ki-1 lymphomas will not be accurately analysed by FCM but is easily assessed by Ki-67 staining. However, the main reason seems to be a high variability between the fraction of cells in S-phase and the total number of cells in G1, S and G2 in individual tumours. [ABSTRACT FROM AUTHOR]

Aims:Non-isotopic histone mRNA in-situ hybridization (HmRNA NISH) allows detection of cells in the S phase of the cell cycle in paraffin-embedded tissue. The aim of this study was to evaluate the technique in measuring the proliferative activity of astrocytic neoplasms, and to compare the results with other proliferation estimates and patient survival. Methods and results:The proliferative activity of 71 routinely fixed and paraffin-embedded astrocytomas was studied by light microscopic HmRNA NISH, proliferating cell nuclear antigen (PCNA), Ki67MIB-1 and mitoses. A significant correlation was found between the labelling indices of histone mRNA (the percentage of histone-positive tumour cells; HmRNA-LI), immunohistochemical proliferation marker labelling indices (PCNA-LI: r = 0.64 and Ki67MIB-1-LI: r = 0.44) and mitotic indices (r = 0.45). The results were reproducible as judged by intra- and interobserver agreement (HmRNA/10 HPF (high power fields): r = 0.91 and r = 0.75, respectively, and HmRNA-LI: r = 0.61 and r = 0.62, respectively). The fraction of the cells in the most active cell cycle phases, as suggested by the HmRNA/Ki67 and mitotic index/Ki67 ratios, increased significantly with malignancy grade. In the univariate analysis the association of HmRNA-LI with survival was highly significant (P < 0.0001). Multivariate survival analysis showed that HmRNA-LI was an independent prognostic marker. Conclusions:Non-isotopic histone mRNA in-situ hybridization assay offers an alternative and reproducible method for measuring proliferative activity (S phase) in tumours under morphological control. [ABSTRACT FROM AUTHOR]

Yeast pheromones block cell cycle progression in G1 in order to prepare mating partners for conjugation. We have investigated the mechanism underlying pheromone-induced G1 arrest in the fission yeast Schizosaccharomyces pombe. We find that the G1-specific transcription factor p65cdc10­p72res1/sct1 which controls the expression of S-phase genes is fully activated in pheromone, unlike the analogous control in budding yeast. In contrast, the G1 function of p34cdc2 acting after activation of the G1-specific transcription is blocked. Pheromone inhibits the p34cdc2 kinase associated with both the G1-specific B-type cyclin p45cig2 and the B-type cyclin p56cdc13 and overexpression of p45cig2 or p47cdc13Δ90 overcomes the pheromone-induced G1 arrest. G1 arrest is compromised in enlarged cells. We suggest that onset of S-phase is controlled by pheromone inhibiting the B-cyclin-associated kinase in G1, and that increasing cell size contributes to the mechanism for pheromone adaptation. Thus, pheromone in fission and budding yeast acts similarly in inhibiting the G1 cyclin-dependent kinase (CDK), but differs in its effects on the G1/S transcriptional control, suggesting that inhibition of CDKs may be a more general mechanism for the control of G1 progression compared with G1/S transcriptional control. [ABSTRACT FROM AUTHOR]

Bromodeoxyuridine (BrdUrd), an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase. In this paper a determination of the S-phase in BLV+ cattle with lymphocytosis has been performed by incorporating bromodeoxyuridine in the DNA. This evaluation was compared to the DNA content, demonstrating that i) bromodeoxyuridine incorporation is a reliable marker of S-phase in BLV+ cattle with lymphocytosis and ii) cytofluorimetry is the method of choice, together with immunocytochemistry, to demonstrate bromodeoxyuridine incorporatioin. Zusammenfassung S-Phasen-Auswertung von Lymphozyten aus mit Bovinem Leukämie-Virus (BLV) infizierten Rindern mittels Brom-Deoxyuridin Das Thymidinanalog Brom-Deoxyuridin wird in die DNS eingebaut und kann daher mit monoklonalen Antikörpe***rn nachgewiesen und als Marker für die S-Phase der Zellteilung eingesetzt werden. In der vorliegenden Arbeit wurde dieser Nachweis an Lymphozyten aus Rindern mit durch BLV hervorgerufener Lymphozytose durchgeführt. Der Einbau von Brom-Deoxyuridin wurde mit dem DNS-Gehalt der Zellen verglichen. Dabei konnte festgestellt werden, daß a) der Einbau von Brom-Deoxyuridin ein zuverlässiger Marker für die S-Phase von Lymphozyten aus BLV-positiven Rindern und b) die Zytofluorimetrie zusammen mit Immunozytochemie für den Nachweis des Brom-Deoxyuridineinbaus die beste Auswertungsmethode ist. [ABSTRACT FROM AUTHOR]

Objective To evaluate the prognostic impact of cytogenetic findings in renal cell carcinoma (RCC). Patients and methods Tumour cytogenetics, histomorphology, DNA ploidy and S-phase fraction, stage, size, and grade were related to survival in 50 consecutive patients with RCC. The mean follow-up was 3.9 years (median 4.2, range 0-8.8). Results The predictive probability for recurrence-free survival at 5 years (5-year RFS) for all 50 patients was 0.54. There was a significant association between the degree of cytogenetic complexity and survival, in that patients with five or less aberrations had a better prognosis than those with more than five changes (5-year RFS 0.71 and 0.43, respectively; P<0.05). Patients with del(8p)/-8, +12, and +20 had a significantly worse prognosis compared with those without these aberrations. Of the well-known prognostic variables grade and stage, the former was far better for predicting prognosis. A Spearman correlation test showed a significant covariation of grade with the S-phase fraction, T-stage, and cytogenetic complexity. Conclusion The degree of cytogenetic complexity and recurrent cytogenetic abnormalities affect the prognosis in RCC, although grade was the most reliable independent prognostic factor predicting disease recurrence. [ABSTRACT FROM AUTHOR]