TdR Incorporation Research Articles

Albumin promotes the progression of fibroblasts through late G1 into S-phase in the absence of growth factors

ABSTRACT G1 cell cycle progression is controlled largely by growth factors in early G1 indicating that it is appropriate to divide and by nutrients in late G1 indicating sufficient raw material for cell division. We previously mapped a late G1 cell cycle checkpoint for lipids upstream from a mammalian target of rapamycin complex 1 (mTORC1)-mediated checkpoint and downstream from a mid-G1 checkpoint known as the Restriction point. We therefore investigated a role for lipids in progression through late G1 into S-phase. Quiescent BJ-hTERT human fibroblasts were primed with 10% fetal bovine serum (FBS) for 3.5 h at which time, cells were treated with a mixture of lipids and carrier bovine serum albumin (BSA) along with [3 H]-thymidine deoxyribose ([3 H]-TdR) to monitor progression into S-phase. Surprisingly, BSA by itself was more effective than FBS in promoting progression to S-phase – the lipids had no impact on progression. While insulin strongly stimulated mTORC1 activity, it did not impact on [3 H]-TdR incorporation. Although BSA modestly elevated mTORC1 activity, rapamycin strongly inhibited BSA-induced progression to S-phase. BSA treatment promoted mitosis, but not progression through a second G1. Thus, after priming quiescent cells with FBS, albumin was sufficient to promote progression into S-phase. The BSA was not simply a source of amino acids in that amino acids were present in the culture media. We propose that the presence of albumin – the most abundant protein in serum – reflects a broader availability of essential amino acids needed for cell growth.

A Novel Long Non-Coding RNA KMU15 Promotes Growth and Chemoresistance of Bladder Cancer.

Chemotherapy constitutes one of the most important adjuvant treatments for bladder cancer. However, many patients usually develop chemoresistance during chemotherapy. At present, lncRNA has been confirmed not only to be involved in tumorigenesis and progression, but also in tumor chemoresistance. However, the relationship between lncRNAs and chemoresistance of bladder cancer have been rarely reported. The novel lncRNA-KMU15 was screened by lncRNAs microarray and determination of IC50 in bladder cancer. The expression of KMU15 was evaluated by qRT-PCR. The correlation between KMU15 and clinicopathological parameters was analyzed from clinical cases. The effects of KMU15 on the biological behavior and chemoresistance were investigated by [3H]-TdR incorporation assay and other experiments. The effects of KMU15 on the growth of xenograft tumors and the survival of nude mice under cisplatin were examined in a xenograft mouse model. We confirmed that KMU15 was expressed higher in bladder cancer tissues than paired control tissues. Moreover, the expression of KMU15 was significantly positively correlated with the grade, stage, metastasis, and recurrence of bladder cancer and was significantly negatively correlated with the prognosis. In addition, KMU15 knockdown could significantly inhibit bladder cell proliferation, adhesion, migration, and chemoresistance and promoted apoptosis. Knockdown of KMU15 inhibited the growth of xenografts in nude mice and significantly prolonged the survival of tumor-bearing mice under cisplatin. The novel lncRNA KMU15, which is highly expressed in bladder cancer tissues, could promote the proliferation and progression and was closely related to the malignant degree of bladder cancer. It could also significantly enhance the chemoresistance of bladder cancer cells. Therefore, it was expected to be a new therapy target for bladder cancer and a potential prognosis biomarker for chemotherapy.

Reinvestigation of the T-Cell Inactivation Achieved for the Prevention of TA GV HD By 2500 Gy of Gamma Irradiation, a Comparison with Pathogen Reduction

▪Background: Transfusion Associated Graft vs. Host Disease (TA GVHD) results in high morbidity and mortality, caused by contaminating T-cells present in transfused blood products. Gamma irradiation (GI) of platelet components (PC) with a dose of 2500 cGy is the approved methodology to prevent TA GVHD in high-risk patients. That methodology has been established based on in vitro Limiting Dilution Assays (LDA) that quantify the inactivation of T-cells. The limited natural abundance of T-cells in blood products and the number of T-cells that can be reproducibly cultured in single wells have defined LDA assay sensitivity. LDA assays usually have a dynamic range of 4-5 log10. Even though that methodology has been used for decades with presumable success, cases of GVHD recognized as transfusion associated, have to be connected temporally and phenotypically to the blood product in use, in order to be characterized as such. That leaves open the possibility that some atypical cases of GVHD in blood product recipients may be miss-assigned and go unrecognized.An alternative methodology to prevent TA GVHD is the photochemical treatment of PC with amotosalen/UVA (PCT; INTERCEPTTM Blood System). PCT has replaced GI in Europe for pathoen inactivated PC for more than 10 years. In this study, we evaluated both GI and PCT on inactivation of T-cells using a highly sensitive approach that includes the use of higher numbers of PBMCs isolated by leukapheresis, and an increased number of cells cultured in a single well (107/well) by using larger wells and optimizing culture conditions.Methods: PBMCs harvested by leukapheresis from individual donors (AllCells, Alameda, CA) were spiked (106/mL) into identical units of human plasma and inactivated using either GI with 2500 cGy, PCT, or were retained as untreated control. For the LDA assay, PCT- or GI-treated cells isolated post treatment, were incubated in individual wells for 14 days in the presence of pooled allostimulator cells from 10 unrelated donors (5X106 treated with 7500 cGy) and growth stimulating factors (PHA and IL-2) under standard culture conditions. Proliferation was assessed by tritiated thymidine ([3H]TdR6.7 Ci/mmol) incorporation into PBMC, as well as by the observation of bright large clusters that were clearly observed at 40x magnification.Results: Initial experiments showed that 107PBMCs/well or 106PBMCs/well resulted in proliferation after GI at 2500 cGy, while 105 PBMCs/well did not. On the other hand, 107 PCT treated-PBMCs/well were found neither to proliferate, nor to incorporate [3H]TdRabove background. T-cell precursor frequency was measured for each donor by incubation of serial dilutions of viable PBMCs (50 - 1 /well; 12 wells per dilution) in the presence of 107 inactivated PBMCs. The experiment was repeated 10 times with PBMCs from different donors.For GI, [3H]TdR incorporation above background indicative of T-cell growth, as well as T-cell proliferating colonies were observed in 4 of 10 experiments when 106PBMCs/ well were cultured. No T-cell growth was detected by [3H]TdR incorporation when 105 GI PBMCs/well were cultured, while proliferating colonies were observed in 1 of 10 experiments. No T-cell proliferation was detected by either criterion, when 107 PCT-treated PBMCs/well were cultured.Conclusions: The accepted dose of 2500 cGy gamma irradiation for prevention of TA GVHD results in more than 4.2log10 but less than 6.2 log10 T-cell inactivation. However, it still allows the proliferation of T-cell clones from some donors. Treatment of T-cells with amotosalen/UVA results in complete inactivation of T-cells (>6.2 log10) with no break though proliferation detected.The data in this study have not been reviewed by the FDA. DisclosuresMerkel:Cerus Corporation: Other: fee for service with Cerus Corporation. Giclas:Cerus Corporation: Other: fee for service with Cerus Corporation. Gibula:Cerus Corporation: Other: fee for service with Cerus Corporation. Andersen:Cerus Corporation: Other: fee for service with Cerus Corporation. Knight:Cerus Corporation: Other: fee for service with Cerus Corporation. Lin:Cerus corp: Employment. Castro:Cerus Corporation: Employment. Green:Cerus Corporation: Employment. Stassinopoulos:Cerus Corporation: Employment.

Inhibition of Survival Pathways MAPK and NF-kB Triggers Apoptosis in Pancreatic Ductal Adenocarcinoma Cells via Suppression of Autophagy.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a survival rate of 4-6 months from diagnosis. PDAC is the fourth leading cause of cancer-related death in the Western world, with a mortality rate of 10 cases per 100,000 population. Chemotherapy constitutes only a palliative strategy, with limited effects on life expectancy. To investigate the biological response of PDAC to mitogen-activated protein kinase (MAPK) and NF-kappaB (NF-kB) inhibitors and the role of autophagy in the modulation of these signaling pathways in order to address the challenge of developing improved medical protocols for patients with PDAC. Two ATCC cell lines, MIAPaCa-2 and PANC-1, were used as PDAC models. Cells were exposed to inhibitors of MAPK or NF-kB survival pathways alone or after autophagy inhibition. Several aspects were analyzed, as follows: cell proliferation, by [(3)H]TdR incorporation; cell death, by TUNEL assay, regulation of autophagy by LC3-II expression level and modulation of pro-and anti-apoptotic proteins by Western blot. We demonstrated that the inhibition of the MAPK and NF-kB survival pathways with U0126 and caffeic acid phenethyl ester (CAPE), respectively, produced strong inhibition of pancreatic tumor cell growth without inducing apoptotic death. Interestingly, U0126 and CAPE induced apoptosis after autophagy inhibition in a caspase-dependent manner in MIA PaCa-2 cells and in a caspase-independent manner in PANC-1 cells. Here we present evidence that allows us to consider a combined therapy regimen comprising an autophagy inhibitor and a MAPK or NF-kB pathway inhibitor as a possible treatment strategy for pancreatic cancer.

Lipoxin A4 Inhibits NF-κB Activation and Cell Cycle Progression in RAW264.7 Cells

Lipoxins (LXs), including lipoxin A4 (LXA4), etc., have been approved for potent anti-inflammatory and immunomodulatory properties. Based on the important roles of macrophages in inflammation and immunomodulation, we investigate the effects of LXA4 on lipopolysaccharide (LPS)-induced proliferation and the possible signal transduction pathways in RAW264.7 macrophages. RAW264.7 cells were treated in vitro with or without LPS in the absence or presence of LXA4. [(3)H]-TdR incorporation assay and flow cytometry were used for detecting cell proliferation and cycle, respectively. Moreover, Western blot was applied to evaluate the protein expression levels of Cyclin E, IκBα, nuclear factor-κB (NF-κB), and IκB kinase (IKK). Our research showed that LXA4 suppressed LPS-induced proliferation, increased the proportion of the G0/G1 phase, decreased the proportion of the S phase, and downregulated the expression of Cyclin E. Besides these, LXA4 suppressed LPS-induced IκBα degradation, NF-κB translocation, and the expression of IKK. The data suggested that LXA4 inhibited LPS-induced proliferation through the G0/G1 phase arrest in RAW264.7 macrophages, and the inhibitory effect might depend on NF-κB signaling transduction pathway.

Puerarin Attenuates Cerebral Damage by Improving Cerebral Microcirculation in Spontaneously Hypertensive Rats

Puerariae Lobatae Radix (Gegen in Chinese) is the dried root of Pueraria lobata, a semiwoody, perennial, and leguminous vine native to China. Puerarin is one of the effective components of isoflavones isolated from the root of Pueraria lobata. Previous studies showed that extracts derived from the root of Pueraria lobata possessed antihypertensive effect. Our study is to investigate whether puerarin contributes to prevention of stroke by improving cerebral microcirculation in rats. Materials and Methods. Video microscopy and laser Doppler perfusion imaging on the pia mater were used to measure the diameter of microvessel and blood perfusion in 12-week old spontaneously hypertensive rats (SHRs) and age-matched normotensive WKY rats. Histological alterations were observed by hematoxylin and eosin staining, and microvessel density in cerebral tissue was measured by immunohistochemical analysis with anti-Factor VIII antibody. Cell proliferation was detected by [3H]-TdR incorporation, and activities of p42/44 mitogen activated protein kinases (p42/44 MAPKs) were detected by western blot analysis in cultured cerebral microvascular endothelial cells (MECs). Results. Intravenous injection of puerarin relaxed arterioles and increased the blood flow perfusion in the pia mater in SHRs. Puerarin treatment for 14 days reduced the blood pressure to a normal level in SHRs (P < 0.05) and increased the arteriole diameter in the pia mater significantly as compared with vehicle treatment. Arteriole remodeling, edema, and ischemia in cerebral tissue were attenuated in puerarin-treated SHRs. Microvessel density in cerebral tissue was greater with puerarin than with vehicle treatment. Puerarin-treated MECs showed greater proliferation and p42/44 MAPKs activities than vehicle treatment. Conclusions. Puerarin possesses effects of antihypertension and stroke prevention by improved microcirculation in SHRs, which results from the increase in cerebral blood perfusion both by arteriole relaxation and p42/44 MAPKs-mediated angiogenesis.

Requirement of the expression of 3-phosphoglycerate dehydrogenase for traversing S phase in murine T lymphocytes following immobilized anti-CD3 activation

Murine resting (G0) T lymphocytes contained no detectable mRNA of 3-phosphoglycerate dehydrogenase (PHGDH) catalyzing the first step in the phosphorylated pathway of l-serine biosynthesis. Immobilized anti-CD3 activation of G0 T cells expressed the PHGDH mRNA in G1 with a maximum level in S phase. G0 T cells activated with either immobilized anti-CD3 plus CsA or PBu2, which failed to drive the activated T cells to enter S phase, did not express the PHGDH mRNA unless exogenous rIL-2 was added. Blocking of IL-2R signaling by adding anti-IL-2 and anti-IL-2Rα resulted in no expression of the PHGDH mRNA during immobilized anti-CD3 activation of G0 T cells. Deprivation of l-serine from culture medium or addition of antisense PHGDH oligonucleotide significantly reduced [3H]TdR incorporation of activated T cells. These results indicate that the PHGDH gene expression, dictated by IL-2R signaling, is a crucial event for DNA synthesis during S phase of activated T cells.

Virus dynamics in a large epishelf lake (Beaver Lake, Antarctica)

Summary Virus concentrations, virus‐to‐bacterium ratios (VBRs) and levels of lysogenic phage were measured throughout the 110‐m water column of epishelf Beaver Lake during the austral summer of 2003/2004. The aim was to determine which factor(s) controlled viral dynamics using detailed concomitant published data on inorganic nutrients, chlorophyll a, dissolved organic carbon (DOC) and bacterial production for Beaver Lake (Freshwater Biology, 51, 2006, 1119). Denaturing Gradient Gel Electrophoresis (DGGE) analysis of the bacterial community was also undertaken to investigate the potential relationship between bacterial community composition and viral dynamics. Virus concentration ranged between 1.43 × 104 and 302.4 × 104 viruses mL−1 and showed a clear increase over the summer, while bacterial concentrations exhibited no seasonal pattern (range: 9.6 × 104 to 44.6 × 104 cells mL−1). Consequently, VBR varied with highest values in January (3.32–7.33). The percentage of lysogenic phage was low, ranging from 0 to 11.69%, suggesting that other life cycles (i.e. lytic or possibly pseudolysogeny) were taking place. Attempts to measure viral production using both the TdR incorporation technique and the dilution technique failed to produce consistent results, in common with other ultra‐oligotrophic Antarctic lakes. There were significant correlations between viral concentration and each of chlorophyll a, DOC, ammonium and nitrate concentrations and bacterial production, but not between bacterial abundance and either soluble reactive phosphorus or temperature. Lack of change in the dominant bacterial community composition over summer suggests that changes in viral concentrations were a function of changes in other biological and physicochemical factors, rather than changes in host/phage infection. The results suggest a significant connection between viruses and bacteria, with the DOC pool acting as a conduit for the movement of carbon between the two components.

Combined use of etanercept and MTX restores CD4+/CD8+ ratio and Tregs in spleen and thymus in collagen-induced arthritis

To further explore the mechanism of etanercept (ENT, rhTNFR:Fc) and methotrexate (MTX) in the combined treatment of rheumatoid arthritis (RA), we investigated whether thymic and splenic T-cell subsets and their related cytokines imbalance could be restored by ETN/MTX treatment. The effect of ETN/MTX on collagen-induced arthritis (CIA) was evaluated by arthritis scores, joint and spleen histopathology, as well as indices of thymus and spleen. T lymphocytes proliferation was determined by [(3)H]-TdR incorporation. Levels of TNF-α, LT-α, IL-1β, RANKL, IL-10, IL-17, IFN-γ and IL-6 were detected by enzyme linked immunosorbent assay. The subsets of T lymphocytes including CD4(+), CD8(+), CD3(+)CD4(+), CD4(+)CD25(+), CD4(+)CD62L(+) and CD4(+)CD25(+)Foxp3(+) cells were quantified using flow cytometry. Combined administration of ETN/MTX significantly inhibited the proliferation of T lymphocytes, decreased serum IL-6, TNF-α, IL-1β, RANKL and macrophage supernatant IL-17, LT-α, increased serum IFN-γ and macrophage supernatant IL-10. Moreover, the combined administration could restore CD4(+)/CD8(+) ratio and Treg cells of CIA thymus and spleen. Taken together, our findings suggest that ENT/MTX may modify the abnormal T lymphocytes balance from central to peripheral lymphoid organs, which may partially, explained the mechanism of the combined administration.

Effective Prevention of Acute Graft-Verses-Host Disease by Targeting PKC Alpha and PKC Theta in Mice

Abstract 1898Protein kinase C theta (PKCθ), a T cell signaling molecule, has been implicated as a therapeutic target for several autoimmune diseases as well as graft-versus-host disease (GVHD). PKCθ plays a vital role in stabilization of the immunologic synapse between T effector cells and antigen presenting cells (APC), but has been shown to be excluded from the immunologic synapse in T regulatory cells (T reg). PKCθ inhibition reduces the alloreactivity of donor T cells responsible for induction of GVHD while preserving graft-versus-leukemia (GVL) responses. The roles of PKCθ and the potential compensatory alpha isoform (PKCα) are not clearly defined with regard to alloresponses or T cell mediated responses in GVHD.In this context, we measured PKCθ and PKCα/θ gene deficient T cell activation upon TCR-ligation in vitro using [3H]-TdR incorporation and CSFE labeling assays. T cells from PKCθ and PKCα/θ gene deficient donor mice were utilized in vivo in a pre-clinical allogenic murine model of myeloablative bone marrow transplantation (BMT). The development of GVHD was monitored in recipient mice with or without injection of A20-luciferase cells to observe the progression of GVL in vivo.Combined blockade of PKCα and PKCθ causes a significant decrease in T cell proliferation compared to blocking PKCθ alone in vitro. Deficiency in PKCα and PKCθ had no effect on immune reconstitution following irradiation and BMT in vivo. Even with a high transplant load of 5×106 CD4+ and CD8+ T cells, PKCα/θ deficient (PKCα/θ−/−) T cells failed to induce acute GVHD. Our data suggest that the ability of double deficient T cells to induce GVHD was further reduced than PKCθ-deficient T cells. Additionally, a greater number and percentage of B220+ B cells and FoxP3+ T regs were isolated from the spleens of PKCα/θ−/− T cell recipient mice 120 after BMT than were isolated from wild type (WT) or PKCθ−/− T cell recipients. Fewer CD4+ or CD8+ T effector cells were isolated from the spleens of PKCα/θ−/− T cell recipient mice 120 after BMT than were isolated from wild type or PKCθ−/− T cell recipients. Importantly, the activity of B cells isolated from PKCα/θ−/− T cell recipient mice 120 after BMT was greater on a per cell basis, while the activity of T effector cells isolated from these mice was greatly reduced compared to WT or PKCθ−/− T cell recipients. While not absent, GVL was reduced in PKCα/θ−/− T cell recipient mice when compared to WT or PKCθ−/− T cell recipients.This work demonstrates the requirement of PKCα and θ for optimal activation and function of T cells in vitro. These experiments highlight a potential compensatory role for PKCα in the absence of PKCθ in T cell signaling and activation. Combined deficiency of PKCα and θ prevents induction of acute GVHD while improving the maintenance of splenic cellularity in PKCα/θ T cell recipient mice. Additionally, PKCα/θ dual deficient T cell transplant shifts the splenic balance toward a greater number and percentage of T reg and B cells and away from T effector cells following BMT. The reduced and sub-optimally active T effector cells isolated from PKCα/θ−/− T cell recipient mice in combination with reduced GVL stresses the importance of PKCα and θ molecules and their roles in T cell activity in the context of both GVHD and GVL. Dual deficiency of PKCα/θ is associated with a decline of T effector function that is optimal for the amelioration of GVHD, but is perhaps too reduced to substantially maintain effective GVL. Modulation of PKCα and θ signaling presents a valid avenue of investigation as a therapeutic option for GVHD. Disclosures:No relevant conflicts of interest to declare.

Identification and Characterization of Human Aspergillus Fumigatus-Specific Tr1-(Like) Cells

Abstract 181 Introduction:The ubiquitous mold Aspergillus fumigatus (A. fumigatus) induces two forms of pathogenesis: invasive aspergillosis in neutropenic patients and allergic aspergillosis in patients with chronic obstructive lung disease as well as in immunosuppressed patients. Mouse models of aspergillosis suggest that not only effector T cells (Teff) but also regulatory T cells (Treg) play a crucial role for the regulation of a protective T cell-mediated immunity to A. fumigatus. However, it is little-known about the involvement of Treg during A. fumigatus infection in humans. In order to develop new therapeutical strategies for the treatment of aspergillosis this project aims to understand the influence of regulatory T cells on A. fumigatus infection in humans.Material/Methods: A. fumigatus-specific CD4+ T cell clones were established from PBMC of healthy donors. Based on this clone pool Treg clones were identified due to their inability to proliferate in the absence of costimulation assessed by 3[H]-TdR incorporation as well as their Ag-specific cytokine production and phenotype determined by flow cytometry. Treg function was analyzed by their ability to suppress proliferation of autologous CD4+ T cells using CFSE dilution. Results:We identified A. fumigatus-specific T cell clones that exhibited marginal detectable proliferation after restimulation with immobilized anti-CD3 mAb in the absence of costimulation. However, these T cell clones vigorously proliferated in response to restimulation with their cognate antigen. A more detailed characterization showed that these suppressor T cell clones produced high amounts of IL-10 and moderate levels of IFN-gamma upon Ag-specific restimulation and expressed low amounts of Foxp3 but not Helios, a transcription factor that had recently been linked to natural occurring Treg. Most importantly, these T cell clones suppressed Ag-specific expansion of CD4+ Teff. This effect was contact-independent since suppression of Ag-specific CD4+ T cell expansion detected in transwell experiments was comparable to cocultures that enabled cellular-contact. Furthermore, anti-CD3/CD28-induced proliferation of naïve CD4+ T cells was not reduced in the presence of culture supernatants obtained from suppressor T cell clones after their antigen-specific restimulation in the absence of DCs. Conclusions:We identified for the first time A. fumigatus-specific CD4+ T cell clones with a Tr1(-like) IL-10+IFN-gamma+Foxp3lowHelios− phenotype. These cells suppressed expansion of A. fumigatus-specific Teff in an Ag-specific manner mediated by soluble factors released from Tr1(-like) cell clones. Since these factors did not affect CD4+ T cell proliferation in the absence of DCs our data suggest, that Tr1(-like) cell clones rather negatively regulate the stimulatory capacity of DCs leading to a reduced expansion of Ag-specific CD4+ T cells. Therefore these Tr1(-like) cells might play a protective role during A. fumigatus infection in humans. Thus, adoptive transfer of A. fumigatus-specific Treg could be useful to enhance protective immunity in patients with chronic A. fumigatus infection. Disclosures:Topp:Micromet: Consultancy, Honoraria.

Effect of 8-methoxypsoralen in the dark on proliferation of stimulated lymphocytes

The effect of 8-methoxypsoralen (8-MOP) in the dark on proliferation of phytohaemagglutinin-stimulated human lymphocytes has been examined, using total blood leucocytes, the lymphocyte fraction and the T-lymphocyte subpopulation. 8-MOP (1 microgram mL-1) increased [3H]TdR incorporation and caused a rise in the blastic and mitotic index in all experimental cultures. The lymphocyte proliferation in these experiments was effected by a comitogenic effect of 8-MOP on T-lymphocytes.

Relationships Between Coastal Bacterioplankton Growth Rates and Biomass Production: Comparison of Leucine and Thymidine Uptake with Single-Cell Physiological Characteristics

Specific growth rates of heterotrophic bacterioplankton have been frequently estimated from in situ bacterial production (BP) to biomass (BB) ratios, using a series of assumptions that may result in serious discrepancies with values obtained from predator-free cultures. Here, we used both types of approaches together with a comprehensive assessment of single-cell physiological characteristics (membrane integrity, nucleic acid content, and active respiration) of coastal bacterioplankton during a complete annual cycle (February 2007-January 2008) in the southern Bay of Biscay off Xixón, Spain. Both leucine and thymidine incorporation rates were used in conjunction with empirical tracer to carbon or cells conversion factors (eCFs) to accurately derive BP. Leu and TdR incorporation rates covaried year-round, as did the corresponding eCFs at 0 and 50 m depth. eCFs peaked in autumn, with mean annual values close to the theoretical ones (3.4 kg C mol Leu(-1) and 2.0 × 10(18) cells mol TdR(-1)). Bacterial abundance (0.2-1.5 × 10(6) cells L(-1)) showed a bimodal distribution with maxima in May and October and minima in March. Live (membrane-intact) cells dominated year-round (79-97%), with high nucleic acid cells (42-88%) and actively respiring bacteria (CTC+, 1-16%) showing distinct surface maxima in April and July, respectively. BB (557-1,558 mg C m(-2)) and BP (7-139 mg C m(-2) day(-1)) presented two distinct peaks in spring and autumn, both of similar size due to a strong upwelling event observed in September. Specific growth rates (0.35-3.8 day(-1)) were one order of magnitude higher in predator-free incubations than bacterial turnover rates derived from integrated BP:BB ratios (0.01-0.16 and 0.01-0.09 day(-1), for Leu and TdR, respectively) and were not correlated, probably due to a significant contribution of low activity cells to total standing stocks. The Leu:TdR molar ratio averaged for the water column (6.6-25.5) decreased significantly with higher integrated BB, indicating that low standing stocks tend to present unbalanced growth. Discrepancies about the true magnitude of specific growth rates must be solved before fully appreciating the role of bacteria in the ocean carbon cycle.

The effects of solar radiation on the stability of 3H‐thymidine and 3H‐leucine during bacterioplankton production measurements

Whereas [3H]thymidine ([3H]TdR) and [3H]leucine ([3H]leu) are commonly used to measure bacterial production, their stability during ultraviolet radiation (UVR) exposure has been questioned. Because these molecules prove invaluable in studies examining UVR effects on bacterial production, molecule integrity during and after exposure to UVR is critical. The stability of both radiolabeled and non‐radiolabeled leucine and thymidine stability was examined after UVR exposure. Solutions of [3H]TdR and [3H]leu were prepared in deionized water, 0.2 µm‐filtered estuarine and marine waters followed by sunlight exposure for 4 h centered around solar noon. Incorporation of each irradiated substrate by bacterioplankton was then determined in Pensacola Bay and Gulf of Mexico waters and compared with incorporation using nonirradiated replicate [3H]TdR and [3H]leu solutions. Differences in [3H]TdR incorporation rates for irradiated versus nonirradiated compounds were of marginal statistical difference when irradiated in seawater but not in distilled water whereas no statistical differences were observed for [3H]leu. However, nonradiolabeled samples analyzed after solar exposure using high performance liquid chromatography (HPLC) showed no significant difference for irradiated samples versus dark controls. Therefore, UVR exposure has no effect on compound integrity. Furthermore, there is no reason to discontinue use or question results obtained by employing [3H]TdR or [3H]leu during simultaneous exposure and incubation experiments measuring bacterial production. This type of incubation can lead to a more realistic view of the cumulative effect of UVR on bacterial production over the day rather than short incubations performed in the dark following exposure to sunlight.

Microcalorimetric study of the opposing effects of ginsenosides Rg1 and Rb1 on the growth of mice splenic lymphocytes

Splenic lymphocytes play an important role in host acute or chronic diseases. The abnormality of these cells in the spleens of humans might lead to some riskful diseases for human. Hence, in this study, the effects of two ginsenosides Rg1 and Rb1 on splenic lymphocytes growth were studied by microcalorimetry. Some qualitative and quantitative information, such as the metabolic power-time curves, growth rate constant k, maximum heat-output power of the exponential phase P max, total heat output Q t of splenic lymphocytes were obtained to present the effects of Rg1 and Rb1 on these cells. The values of k, P max, and Q t from the thermogenic growth curves of splenic lymphocytes were found to increase in the presence of Rg1, while the change was adverse for Rb1, illustrating that Rg1 had promotion effect and Rb1 had inhibitory effect on splenic lymphocytes growth and these promotion or inhibitory effects were enhanced with increasing the concentration of the two compounds, respectively. The microcalorimetric results were confirmed by MTT assay for determining the MTT optical density (OD) value and [3H] Thymidine incorporation assay ([3H]-TdR) for determining the count per minute (cpm) value: Rg1 could increase the MTT OD value and the cpm value of [3H]-TdR incorporation into splenic lymphocytes, and these values were increased with increasing the concentration of this compound, while Rb1 had the adverse results. The structure–activity relationships showed that the glucopyranoside and hydroxyl groups at the dammarane-type mother nucleus skeleton might play a crucial role for the opposing effects of the two ginsenosides on splenic lymphocytes. Compared with the other two assay methods, the microcalorimetric method provided more useful and reliable information for quickly and objectively evaluating the effects of drugs or compounds on the living cells, which would be a highly promising analytical tool for the characterization of the biological process and the estimation of the drugs’ efficiency.

Growth response of the bacterial community to pH in soils differing in pH

The effect of pH on the instantaneous growth of soil bacterial communities was studied in five soils with different pH (4.5-7.8) using leucine (Leu) and thymidine (TdR) incorporation. The pH dependency of bacterial growth was modelled using three different unimodal functions, and the pH(opt) for growth and the pH range in which growth was >50% of the optimal growth were compared. Leu and TdR incorporation yielded very similar results. The best fits were obtained using a third-degree polynomial function and the cardinal pH model. However, a simple second-degree function was adequate in most cases, yielding very similar pH(opt) values to the other two models. Bacterial growth was highly influenced by pH, showing optimum growth at a pH related to the soil pH. The lowest pH(opt) was found in the most acidic soil and the highest pH(opt) in the soil with the highest pH. The pH(opt) for bacterial growth was close to the soil pH measured in water, but higher (0.7-2.1 units) than the pH measured with 0.1 M KCl. The pH range in which bacterial growth was >50% of that at optimum was, on average, 1.7 units below and above the optimum pH.

Hexarelin suppresses high lipid diet and vitamin D 3-induced atherosclerosis in the rat

Growth hormone-releasing peptides (GHRP) and ghrelin are synthetic and natural ligands of growth hormone secretagogue receptor (GHSR) respectively and are shown to exert protective actions on cardiac dysfunction. Because ghrelin has been reported to inhibit proinflammatory responses in human endothelium and GHSR has been identified in blood vessels, we hypothesized that GHRP could alleviate the development of atherosclerosis (As). Atherosclearosis was induced by a short period (4 days) of vitamin D 3 and chronic (three months) intragastric feeding of high fat emulsion (containing 0.5% propylthiouracil) in adult SD rats. Some As rats received chronic hexarelin (a variant of GHRP) injection (SC BID, 30 days) and normal rats received placebo as control. Significant atherosclerosis developed in animals fed with the emulsion. Serum total cholesterol and LDL-c increased, and HDL-c and aortic nitric oxide (NO) decreased significantly in As group. Hexarelin suppressed the formation of atherosclerotic plaques and neointima, partially reversed serum HDL-c/LDL-c ratio and increased the levels of serum NO and aortic mRNAs of eNOS, GHSR and CD36 in As rats. Hexarelin also decreased [ 3H]-TdR incorporation in cultured vascular smooth muscle cell (VSMC) and calcium sedimentation in aortic wall. Furthermore, foam cell formation induced by ox-LDL was decreased by hexarelin. In conclusion, hexarelin suppresses high lipid diet and vitamin D3-induced atherosclerosis in rats, possibly through upregulating HDL-c/LDL-c ratio, vascular NO production and downregulating the VSMC proliferation, aortic calcium sedimentation and foam cell formation. These novel anti-atherosclerotic actions of hexarelin suggest that the peptide might have a clinical potential in treating atherosclerosis.

Effects of D1-like dopamine receptor on insulin receptor-mediated proliferation of vascular smooth muscle cells

Objective To investigate the role of D1-like dopamine receptor on insulin receptor-mediated proliferation of vascular smooth muscle cells. Methods A10 cells were used in this study and the mechanisms of D1-like receptor on insulin receptor were determined by immunoblotting. The proliferation of VSMC cells was determined by [3H]-TdR incorporation. Results Insulin increased proliferation of A10 cells in a concentration-dependent manner. D1-like receptor, by itself, had no effect on cell proliferation, but blocked the insulin-mediated proliferation of A10 cells.

Lipoxin A4 inhibited hepatocyte growth factor‐induced invasion of human hepatoma cells

Inflammation is a critical component of tumor progression. Lipoxin A(4) (LXA(4)) has been approved for potent anti-inflammatory properties. Recently, it was reported that LXA(4) repressed the expression and activity of cyclooxygenase-2 (COX-2), which is essential for invasion. However, there are few reports dealing with its effects on cancer. To explore whether LXA(4) regulate invasion, the effects of LXA(4) and its receptor agonist BML-111 on hepatocyte growth factor (HGF)-induced invasion of hepatoma cells and the possible mechanisms were researched. Lipoxin A(4) receptor (ALX) expression in HepG2 cells were measured through reverse transcription polymerase chain reaction and western blot. Cytotoxicity of LXA(4) and BML-111 to HepG2 cells was detected by MTT and ((3)H)-TdR incorporation assay. Cell migration and invasion assays were performed using a Boyden chemotaxis chamber. COX-2 expression was detected by real-time polymerase chain reaction and western blot, respectively. Moreover, the expressions of matrix metalloproteinases (MMP)-2, MMP-9, IkappaBalpha and nuclear factor-kappaB (NF-kappaB) p65 were observed via western blot, and NF-kappaB transcriptional activity was tested by transfections and luciferase activities assay. ALX expression was detected in HepG2 cells, and suitable concentrations of LXA(4) and BML-111 had no cytotoxicity to cells. LXA(4) and BML-111 inhibited HGF-induced migration and invasion; downregulated COX-2, MMP-2 and -9; restrained HGF-induced IkappaBalpha degradation, NF-kappaB translocation and the transcriptional activity of NF-kappaB in HepG2 cells. Furthermore, exogenous PGE2 could reverse the inhibitory effects of LXA(4) also BML-111 on HGF-induced invasion and migration partially. LXA(4) inhibited HGF-induced invasion of HepG2 cells through NF-kappaB/COX-2 signaling pathway partially.

Loss of Protein Inhibitors of Activated STAT-3 Expression in Glioblastoma Multiforme Tumors: Implications for STAT-3 Activation and Gene Expression

STATs activate transcription in response to numerous cytokines, controlling proliferation, gene expression, and apoptosis. Aberrant activation of STAT proteins, particularly STAT-3, is implicated in the pathogenesis of many cancers, including GBM, by promoting cell cycle progression, stimulating angiogenesis, and impairing tumor immune surveillance. Little is known about the endogenous STAT inhibitors, the PIAS proteins, in human malignancies. The objective of this study was to examine the expression of STAT-3 and its negative regulator, PIAS3, in human tissue samples from control and GBM brains. Control and GBM human tissues were analyzed by immunoblotting and immunohistochemistry to determine the activation status of STAT-3 and expression of the PIAS3 protein. The functional consequence of PIAS3 inhibition by small interfering RNA or PIAS3 overexpression in GBM cells was determined by examining cell proliferation, STAT-3 transcriptional activity, and STAT-3 target gene expression. This was accomplished using [(3)H]TdR incorporation, STAT-3 dominant-negative constructs, reverse transcription-PCR, and immunoblotting. STAT-3 activation, as assessed by tyrosine and serine phosphorylation, was elevated in GBM tissue compared with control tissue. Interestingly, we observed expression of PIAS3 in control tissue, whereas PIAS3 protein expression in GBM tissue was greatly reduced. Inhibition of PIAS3 resulted in enhanced glioblastoma cellular proliferation. Conversely, PIAS3 overexpression inhibited STAT-3 transcriptional activity, expression of STAT-3-regulated genes, and cell proliferation. We propose that the loss of PIAS3 in GBM contributes to enhanced STAT-3 transcriptional activity and subsequent cell proliferation.