Rate Of 3H-thymidine Incorporation Research Articles

Kras Is Critical for B Cell Lymphopoiesis

Small GTPases of the Ras subfamily regulate multiple signaling pathways and control numerous biological functions. Although the three major Ras members, Kras, Hras and Nras, are highly homologous, individual Ras gene can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological role of Kras. Here, we generated and examined mice with hematopoietic- and B cell-specific deletion of Kras. In VavCreKrasfl/fl mice with hematopoietic deletion of Kras, the populations of bone marrow (BM) pre-, immature and mature B cells were reduced. The population of peripheral follicular (FO), marginal zone (MZ) and B1 mature B cells were also reduced in VavCreKrasfl/fl relative to control mice. In addition, BM chimeric mice with B cell-specific deficiency of Kras generated by transplantation of T- and B-null Rag1-deficient mice with a mixture of BM cells from VavCreKrasfl/fl mice and B cell-deficient μMT mice displayed a marked reduction of pre-, immature and mature B cells in the BM and mature B cells in the spleen. Thus, Kras deficiency intrinsically impairs early B cell development and late B cell maturation. Further, the effect of Kras deficiency on B-cell proliferation and survival was examined. The 3H-thymidine incorporation rate of Kras-deficient, relative to control, mature B cells in response to anti-IgM or anti-IgM plus IL-4 was markedly reduced. In addition, Kras-deficient B cells displayed a marked decrease of cell cycle entry and increase of cell apoptosis upon anti-IgM or anti-IgM plus IL-4 stimulation. Thus, Kras deficiency impairs BCR-induced B cell proliferation and survival. Lastly, the role of Kras in BCR signaling was studied. The level of total BCR-activated Ras (Ras-GTP) was largely reduced in Kras-deficient B cells. BCR-induced Ca2+ flux was comparable between Kras-deficient and control B cells. BCR-induced phosphorylation of Akt and IkBa was normal in mutant relative to control B cells. However, BCR-induced activation of ERK1/2 but not JNK or p38 was impaired in Kras-deficient relative to control B cells. Consistently, BCR-induced activation of Raf-1 and MEK1/2, the upstream activators of ERK1/2, was markedly reduced in mutant B cells. In addition, pre-BCR-induced ERK1/2 activation was impaired in Kras-deficient pre-B cells. Pre-BCR-induced activation of AP-1, the transcription factors downstream of ERK1/2, was decreased in mutant pre-B cells. Thus, Kras plays an important role in BCR- and pre-BCR-mediated activation of the ERK pathway in B cells. Taken together, these findings demonstrate that Kras is the Ras family member that critically regulates early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway. DisclosuresNo relevant conflicts of interest to declare.

Diel and seasonal variations in bacterioplankton production in a hypertrophic shallow lagoon

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 72:255-267 (2014) - DOI: https://doi.org/10.3354/ame01701 Diel and seasonal variations in bacterioplankton production in a hypertrophic shallow lagoon Gabriela Onandia*, Maria Rosa Miracle, Cristina Blasco, Eduardo Vicente Department of Microbiology and Ecology, ICBiBE, University of Valencia, Calle Dr. Moliner 50, 46100 Burjassot, Valencia, Spain *Corresponding author: gabriela.onandia@uv.es ABSTRACT: Diel and seasonal variations in bacterial secondary production (BP) were investigated in the hypertrophic Albufera de Valencia (Spain) by measuring 3H-leucine and 3H-thymidine incorporation rates (LIR and TIR). LIR and TIR were positively correlated. Seasonal variations in the LIR:TIR ratio seemed to be related to dissolved organic carbon (DOC) availability, with higher concentrations favoring protein synthesis. The light:dark ratio of the incorporation rates of both radiotracers was close to 1. However, this ratio tended to be >1 when DOC was lower, namely in winter. A clear diel pattern in bacterial activity was found, with an increasing trend during the night hours. The opposite diel pattern was observed for DOC. BP remained similar across seasons but showed a decrease in late winter during a period of high water discharge through the lagoon and concurrent minimum chlorophyll a concentrations. A comparison between hypertrophic systems aiming to explore the upper BP limit indicated that our BP estimates were considerably high but did not surpass previous values in hypertrophic lakes. KEY WORDS: Bacterioplankton production · Carbon flux · Shallow hypertrophic lake Full text in pdf format PreviousNextCite this article as: Onandia G, Miracle MR, Blasco C, Vicente E (2014) Diel and seasonal variations in bacterioplankton production in a hypertrophic shallow lagoon. Aquat Microb Ecol 72:255-267. https://doi.org/10.3354/ame01701 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 72, No. 3. Online publication date: July 10, 2014 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2014 Inter-Research.

Potential Healing of Meniscal Injuries in the Avascular Zone after Radiofrequency Stimulation (SS-34)

Meniscal repair in the white-white zone has a high incidence of failure due to poor vascularity. Radiofrequency application has been shown to increase the angiogenic capabilities of fibroblasts in chronic tendinopathies. The purpose of this study was to evaluate the effect of radiofrequency (RF) stimulation, in conjunction with suture repair, on the healing of tears in the white-white zone of the meniscus. 54 New Zealand white rabbits underwent surgically induced meniscal injuries within the avascular (white-white) region. Rabbits were then divided into three treatment groups: no repair (N=6), suture repair only (N=15) and suture repair in conjunction with RF treatment (N=33). Radiofrequency was applied using a 0.8 mm radiofrequency wand at level 4 for 500 milliseconds. Rabbits were then sacrificed at 9, 28 and 84 days for gross, histological and biochemical examination by two blinded observers. Biochemical analyses included evaluation of cell proliferation (3H-thymidine) as well as mitogenic (IGF-1, bFGF) and angiogenic (VEGF, αV) factors. Of specimens repaired with radiofrequency combined with suture, 19 (58%) showed increased gross morphologic and histological healing compared to controls. Two meniscal explants treated with RF had complete histological healing. No significant healing was seen in specimens that had either no repair or repair with suture alone. Biochemically, at nine days, the rate of 3H-thymidine for meniscal tears treated with suture repair + RF was 590±80 cpm/mg dry tissue. This value was approximately 40% greater than the menisci treated with suture repair only, which had a value of 380±30 cpm/mg (p < 0.05). Normal, unrepaired meniscal tissue had a 3H-thymidine incorporation rate of 250±35 cpm/mg. Semiquantitative RT-PCR analysis showed that at nine days post-repair, the RF-treated menisci had significantly increased mR.N.A expression of IGF-1, bFGF, VEGF and αV relative to untreated repairs. By day 28 and 84, the levels appeared equivalent between all treatment groups (p > 0.05). This study demonstrated that in our animal model, radiofrequency application in adjunct to suture repair of white-white meniscal tears led to a significantly increased healing response both in vitro and in vivo. Although the mechanism by which RF stimulation improves the healing response is not completely understood, our biochemical analyses suggest that it may enhance cellular proliferation as well as the mitogenic and angiogenic capability of the meniscal fibroblast.

Uncoupled viral and bacterial distributions in coral reef waters of Tuamotu Archipelago (French Polynesia)

This study examined the distribution of virioplankton and bacterioplankton in two coral reef systems (Ahe and Takaroa atolls) in the Tuamotu Archipelago, in comparison with the surrounding oligotrophic ocean. Mean concentrations of 4.8×105 and 6.2×105cellsml−1 for bacteria and 8.1×106 and 4.3×106VLP(virus-like particle) ml−1 were recorded in Ahe and Takaroa lagoons, respectively. Chlorophyll-a concentrations and dissolved organic matter were higher in Ahe whereas 3H thymidine incorporation rates were higher in Takaroa. First data on lytic and lysogenic strategies of phages in coral reef environments were discussed in this paper. The fraction of visibly infected cells by viruses was negligible regardless of the lagoon station (mean=0.15%). However, the fraction of lysogenic cells ranged between 2.5% and 88.9%. Our results suggest that the distribution patterns of virioplankton are apparently not coupled to the spatial dynamics of the bacterioplankton communities.

Protective effects of metallothionein I and II against metal- and ultraviolet radiation-induced damage in cultured lens epithelial cells

Few quantitative analyses of the induction of metallothioneins (MTs) have been conducted, and there are no reports on the contribution of MTs to the protective mechanism against ultraviolet (UV) radiation in the lens. In this study, we quantitatively analyzed the induction of MTs and analyzed the resulting protective effects against both metal- and UV radiation-induced damage in the cultured lens epithelial cell line, alphaTN4-1. The induction profiles of MTs by ZnCl(2) treatment in alphaTN4-1 cells were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The cells in which MTs were induced were either treated with high concentrations of ZnCl(2) or CdCl(2), or irradiated with UV-C, UV-B, or UV-A radiation, followed by analysis of cell viability. The (3)H-thymidine incorporation rate was used as an indicator of cell viability. mRNA expression of MT-I and MT-II, the main MT isoform classes, was induced by ZnCl(2) treatment in a dose-dependent manner. MT induction increased the protective effects against both metaland UV-A radiation-induced cell damage. Our results suggest that MTs play an important role in the protection against damage induced by both toxic metals and UV-A radiation in lens epithelial cells.

Conversion of vitamin D 3 to hormonally active 1α,25-dihydroxyvitamin D 3 in cultured keratinocytes: Relevance to cell growth and differentiation

The regulatory potential of intracellularly generated calcitriol on growth and differentiation of cultured keratinocytes is determined by the degree of cell confluence and availability of the highly lipophilic substrate vitamin D 3 to these cells. The enzymatic conversion of vitamin D 3 to calcitriol is considerably elevated in the presence of the nontoxic surfactant copolymer pluronic ® F127 (120 μg/ml medium) compared to the control without this agent. We found a positive correlation between the formation rate of calcitriol and inhibition of the 3H-thymidine incorporation rate into the DNA of keratinocytes. Intracellularly generated calcitriol causes a clear increase of the cell diameter, and thus has a prodifferentiating effect on keratinocytes in vitro. These findings corroborate the hypothesis that UVB-induced production of vitamin D 3 in human skin results in formation of substantial amounts of calcitriol in keratinocytes which suppress the growth and initiate differentiation of these cells.

Immunological Actions of Sophy β‐Glucan (β‐1,3‐1,6 Glucan), Currently Available Commercially as a Health Food Supplement

We examined the immunological actions of Sophy beta-glucan(Ikewaki N., et al. United States Patent 6956120 and Japan Patent 2004-329077), a type of beta-1,3-1,6 glucan produced by the black yeast Aureobasidium pullulans (A. pullulans) strain AFO-202, currently available commercially as a health food supplement, using different human in vitro experimental systems. Sophy beta-glucan significantly (P<0.01) stimulated the (3)H-thymidine incorporation rates (marker of DNA synthesis) in human peripheral blood mononuclear cells (PBMCs) obtained from normal adult donors, in vitro. Enzyme-linked immunoassays (EIAs) revealed that Sophy beta-glucan stimulated the production of interleukin-8 (IL-8) or soluble Fas (sFas), but not that of IL-1beta, IL-2, IL-6, IL-12 (p70+40), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) or soluble Fas ligand (sFasL), in either cultured PBMCs or cells of the human monocyte-like cell line, U937. The induction by Sophy beta-glucan of DNA synthesis in PBMCs was completely blocked by the addition of monoclonal antibodies (mAbs) to CD11a, CD54, human leukocyte antigen-class II (HLA-class II), Toll-like receptor-2 (TLR-2), and Toll-like receptor-4 (TLR-4). In these blocking experiments using the mAbs, the main differences in the results between PBMCs and U937 cells were that the mAbs against TLR-2 and TLR-4 did not block the Sophy beta-glucan-induced production of IL-8 in the U937 cells. Furthermore, a mAb to the beta-glucan receptor, Dectin-1, significantly (P<0.05) blocked the Sophy beta-glucan induced DNA synthesis in the PBMCs, and Sophy beta-glucan-induced production of IL-8 in the U937 cells. The Sophy beta-glucan-induced production of IL-8 in the U937 cells was significantly (P<0.01) blocked by the conventional protein kinase C (PKC) inhibitor Go6976, the novel PKC inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89, and the protein tyrosine kinase (PTK) inhibitor herbimycin A. Among these, the blocking effect of the novel PKC (PKC delta isoenzyme) inhibitor Rottlerin was the most pronounced. Studies employing reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Sophy beta-glucan stimulated the expression of IL-8 mRNA in the U937 cells, and that this induction was inhibited by Rottlerin. Sophy beta-glucan also blocked the stimulator cell induction of DNA synthesis and IFN-gamma production in the responder cells in a one-way mixed lymphocyte reaction (MLR) using allogenic PBMCs. Interestingly, immunoglobulin G (IgG), but not IgM to Sophy beta-glucan was detected in the sera derived from normal adult donors and from the umbilical cord blood of neonates. Taken together, these findings strongly suggest that the Sophy beta-glucan may have unique immune regulatory or enhancing properties that could be exploited by the health food, medical and pharmaceutical industries.

Phosphorylation of HuR by Chk2 Regulates SIRT1 Expression

The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.

Overexpression of a truncated TrkB isoform increases the proliferation of neural progenitors

The truncated isoform of TrkB, TrkB.T1, has been shown to be expressed in the neurogenic region of rodent brain. TrkB.T1 lacks tyrosine kinase activity and it may modify the action of the full-length TrkB. We show here that the full-length TrkB and TrkB.T1 are expressed at the same relative expression levels in mouse neural progenitor cell cultures. The number of neurosphere-forming progenitors was reduced and apoptosis increased in neurospheres generated from mice overexpressing TrkB.T1 when compared with wild-type neurospheres. The proliferation of the transgenic neural progenitors was increased, as indicated by the larger average diameter of spheres (140% of control), the increased cell growth in an MTT assay (137% of control) and the faster rate of 3H-thymidine incorporation (128% of control) in the transgenic cell cultures than in controls. The proliferation of neural progenitors was also increased after lentivirus-mediated TrkB.T1 overexpression. A significant increase in 3H-thymidine incorporation (119% of control) and the average diameter of spheres (112% of control) in the TrkB.T1-transduced neurospheres compared with neurospheres transduced with the control vectors confirmed the role of TrkB.T1 in proliferation of neural progenitor. When induced to differentiate, progenitors overexpressing TrkB.T1 generated two to three times more neurons than did wild-type cells. The increase in the number of neurons correlated with an increase in the number of apoptotic cells (two-fold) at these time points. The data indicate that changes in the relative expression levels of different TrkB isoforms influence the replicative capacity of neural progenitors.

Discriminating between effects of metals and natural variables in terrestrial bacterial communities

This study aimed to assess effects of metals on bacterial communities in grassland soil and to discriminate these effects from natural variability in soil properties. Changes in gross parameters of bacterial communities were investigated by the determination of 14C-leucine and 3H-thymidine incorporation rates, CO 2 evolution, biomass indicators and N mineralization rates. 14C-leucine incorporation rate and CO 2 evolution showed correlations with all metals tested as well as with organic matter content. 3H-thymidine incorporation rates, biomass indicators and N mineralization rates did not strongly correlate with any physical or chemical parameters. Further, community-level physiological profiles (CLPP) and polymerase chain reaction (PCR)-amplified denaturing gradient gel electrophoresis (DGGE) of 16S rDNA were performed. Monte-Carlo permutation testing was performed to analyse CLPP and DGGE data to allow for a stringent discrimination between sources of effects. CLPP changes correlated with the Pb concentration and pH in the soil. DGGE changes correlated with Pb and Cu concentrations and organic matter content. Pollution-induced community tolerance (PICT) was not found for any of the metals assessed. A causal relation between the effects observed on bacterial communities and the presence of metals was not established with PICT. The negative outcome of PICT can probably be attributed to indirect effects or to methodological problems. We concluded that the observed shifts in CLPP and DGGE patterns are a strong indication of metals effects on bacterial communities in this grassland. These effects could only be filtered from the total variation by multivariate analyses.

Anti- Ki-67 peptide nucleic acid affects the proliferation and apoptosis of human renal carcinoma cells in vitro

We treated in vitro human renal carcinoma cells (cell line 786-0) with the lipid-delivered peptide nucleic acids (PNAs) against Ki-67 gene. Corresponding control groups were treated with the antisense oligonucleotides (ASOs) of the same nucleobase sequence, and with mismatched PNAs. In cells treated by anti- Ki-67 PNAs, the Ki-67 expression rate, Ki-67 protein level, cell growth and the DNA synthesis-indicative 3H-thymidine incorporation rate were lower than in the ASO-treated groups, and reduced significantly compared to untreated controls, whereas the rate of apoptosis was markedly increased by PNA treatment. We conclude that anti- Ki-67 PNA has more strong (than ASO) and dose-dependent effects on the proliferation and apoptosis of human renal carcinoma cells. Our results indicate that the strategy of using PNA against the Ki-67 gene might be a promising approach in renal carcinoma therapy.

Expression and activity of the CDK inhibitor p57Kip2 in chondrocytes undergoing hypertrophic differentiation.

Growth plates of p57-null mice exhibit several abnormalities, including loss of collagen type X (CollX) expression. The phenotypic consequences of p57 expression were assessed in an in vitro model of hypertrophic differentiation. Adenoviral p57 expression was not sufficient for CollX expression but did augment induction of CollX by BMP-2. During hypertrophic differentiation, chondrocytes pass from an actively proliferative state to a postmitotic, hypertrophic phenotype. The induction of growth arrest is a central feature of this phenotypic transition. Mice lacking the cyclin dependent-kinase inhibitor p57Kip2 exhibit several developmental abnormalities including chondrodysplasia. Although growth plate chondrocytes in p57-null mice undergo growth arrest, they do not express collagen type X, a specific marker of the hypertrophic phenotype. This study was carried out to investigate the link between p57 expression and the induction of collagen type X in chondrocytes and to determine whether p57 overexpression is sufficient for the induction of hypertrophic differentiation. Neonatal rat epiphyseal or growth plate chondrocytes were maintained in an aggregate culture model, in defined, serum-free medium. Protein and mRNA levels were monitored by Western and Northern blot analyses, respectively. Proliferative activity was assessed by fluorescent measurement of total DNA and by 3H-thymidine incorporation rates. An adenoviral vector was used to assess the phenotypic consequences of p57 expression. During in vitro hypertrophic differentiation, levels of p57 mRNA and protein were constant despite changes in chondrocyte proliferative activity and the induction of hypertrophic-specific genes in response to bone morphogenetic protein (BMP)-2. Adenoviral p57 overexpression induced growth arrest in prehypertrophic epiphyseal chondrocytes in a dose-dependent manner but was not sufficient for the induction of collagen type X, either alone or when coexpressed with the related CDKI p21Cip1. Similar results were obtained with more mature tibial growth plate chondrocytes. p57 overexpression did augment collagen type X induction by BMP-2. These data indicate that p57-mediated growth arrest is not sufficient for expression of the hypertrophic phenotype, but rather it occurs in parallel with other aspects of the differentiation pathway. Our findings also suggest a contributing role for p57 in the regulation of collagen type X expression in differentiating chondrocytes.

Time-course of cadmium-induced acute hepatotoxicity in the rat liver: the role of apoptosis

Exposure to toxic metals and pollutants is a major environmental problem. Cadmium is a metal causing acute hepatic injury but the mechanism of this phenomenon is poorly understood. In the present study, we investigated the mechanism and time-course of cadmium-induced liver injury in rats, with emphasis being placed on apoptosis in parenchymal and nonparenchymal liver cells. Cadmium (3.5 mg/kg body weight) was injected intraperitoneally and the rats were killed 0, 9, 12, 16, 24, 48 and 60 h later. The extent of liver injury was evaluated for necrosis, apoptosis, peliosis, mitoses and inflammatory infiltration in hematoxylin-eosin-stained liver sections, and by assaying serum enzyme activities. The number of cells that died via apoptosis was quantified by TUNEL assay. The identification of nonparenchymal liver cells and activated Kupffer cells was performed histochemically. Liver regeneration was evaluated by assaying the activity of liver thymidine kinase and by the rate of 3H-thymidine incorporation into DNA. Both cadmium-induced necrotic cell death and parenchymal cell apoptosis showed a biphasic elevation at 12 and 48 h and peaked at 48 and 12 h, respectively. Nonparenchymal cell apoptosis peaked at 48 h. Peliosis hepatis, another characteristic form of liver injury, was first observed at 16 h and, at all time points, closely correlated with the apoptotic index of nonparenchymal liver cells, where the lesion was also maximial at 48 h. Kupffer cell activation and neutrophil infiltration were minimal for all time points examined. Based on thymidine kinase activity, liver regeneration was found to discern a classic biphasic peak pattern at 12 and 48 h. It was very interesting to observe that cadmium-induced liver injury did not involve inflammation at any time point. Apoptosis seems to be a major mechanism for the removal of damaged cells, and constitutes the major type of cell death in nonparenchymal liver cells. Apoptosis of nonparenchymal cells is the basis of the pathogenesis of peliosis hepatis. The first peaks of necrosis and parenchymal cell apoptosis seem to evolve as a result of direct cadmium effects whereas the latter ones result from ischemia.

Mesoscale variability in bacterial activity in the Northeast Pacific Ocean off Oregon, USA

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 25:21-30 (2001) - doi:10.3354/ame025021 Mesoscale variability in bacterial activity in the Northeast Pacific Ocean off Oregon, USA Evelyn B. Sherr*, Barry F. Sherr, Timothy J. Cowles College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, Oregon 97330-5503, USA *E-mail: sherre@oce.orst.edu ABSTRACT: Variation in bacterial abundance and activity was assessed by sampling the upper 35 to 80 m of the water column during 2 to 5 d periods at 3 sites: eutrophic-mesotrophic midshelf, mesotrophic-oligotrophic slope, and oligotrophic gyre edge, off the Oregon coast in late summer 1997 and 1998. Bacterial abundances varied 10-fold, from 0.2 to 2.3 x 106 cells ml-1, and leucine incorporation rates varied 160-fold, from 1.5 to 240 pM h-1. During the strong El Niño event in 1997, bacterial abundances were similar at all 3 sites, but midshelf 3H-leucine incorporation rates were ~6-fold higher than rates at the slope and gyre sites. In 1998, after relaxation of the El Niño, bacterial abundances were lower, and average 3H-leucine incorporation rates were only 2.5 times higher at the midshelf site than at the slope and gyre sites. There was a close correlation between estimates of bacterial cell production rate based on 3H-leucine and on 3H-thymidine incorporation rates for the midshelf and slope sites, but no relation between the 2 estimates for the gyre site. During both years, bacterial abundance varied inversely with depth, salinity, and macronutrients and positively with temperature. Bacterial activity varied positively with chlorophyll concentration, temperature, and bacterial biomass. Rates of bacterial 3H-leucine incorporation were most strongly related to chlorophyll concentrations at the midshelf site and less related at sites farther offshore. There was no significant relation of bacterial parameters with the concentration of dissolved organic carbon. Our results showed dynamic mesoscale variability, on scales of 10s of meters to 10s of kilometers, and on scales of hours to days in rates of bacterial activity, which was positively related to phytoplankton concentration as a proxy for trophic state of the water mass. We also found inter-annual differences in distribution of bacterial abundance and activity in this region, which appeared to be related to climatic variability. KEY WORDS: Marine bacteria · Bacterial activity · Mesoscale · El Niño · Northeast Pacific Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 25, No. 1. Online publication date: August 10, 2001 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2001 Inter-Research.

Experimental cryptorchidism inhibited growth of the rat ventral prostate.

Adult Sprague-Dawley male rats, weighing about 350 g, were rendered cryptorchid by suturing the testes to the lateral abdominal wall. Twenty-eight days later, cryptorchidism resulted in a significant decline in testis weight and suppressed spermatogenesis. The ventral prostate was significantly smaller in cryptorchid rats. There was no significant difference in serum testosterone levels between the normal and cryptorchid rats. Charcoal-stripped aqueous extracts of the testis from intact and cryptorchid animals were tested on primary cultures of rat prostatic stromal cells. Cultures treated with extract from the intact testis had a significantly increased cell proliferation as assessed by cell count and by the rate of 3H-thymidine incorporation. Additionally, extracts of seminiferous tubules significantly increased prostate stromal cell proliferation compared to extracts of testicular interstitial components. Furthermore, this proliferative effect of testicular extracts is specific to the prostate as extract of both normal and cryptorchid testis stimulated proliferation of rat footsole fibroblasts in culture, but only extracts from intact testis stimulated proliferation of prostate stromal cells. These observations demonstrate that the testis produces nonandrogenic substances that can promote growth of prostatic stromal cells and that these substances were eliminated in the cryptorchid testis.

Hyaluronic acid stimulates human fibroblast proliferation within a collagen matrix.

Human dermal fibroblasts suspended in a collagen matrix exhibit a 4-day delay in cell division, while the same cells in monolayer divided by day 1. The initial rates of 3H-thymidine incorporation by cells in monolayer or suspended in collagen were not significantly different. When suspended in collagen, there was a threefold increase in the proportion of cells in a tetraploidal (4N) DNA state compared to the same cells in monolayer. Flow cytometry analysis and 3H-thymidine incorporation studies identified the delay of cell division as a consequence of a block in the G2/M of the cell cycle and not an inhibition of DNA synthesis. The inclusion of 150 microg/ml of hyaluronic acid (HA) in the manufacture of fibroblast populated collagen lattices (FPCL) caused a stimulation of cell division, as determined by cell counting; increased the expression of tubulin, as determined by Western blot analysis; and reduced the proportion of cells in a 4N state, as determined by flow cytometry. HA added to the same cells growing in monolayer produced a minimal increase in the rate of cell division or DNA synthesis. HA supplementation of FPCLs stimulated cell division as well as tubulin concentrations, but it did not enhance lattice contraction. The introduction of tubulin isolated from pig brain or purchased tubulin into fibroblasts by electroporation prior to their transfer into collagen lattices promoted cell division in the first 24 hours and enhanced FPCL contraction. It is proposed that tubulin protein, the building blocks of microtubules, is limited in human fibroblasts residing within a collagen matrix. When human fibroblasts are suspended in collagen, one effect of added HA may be to stimulate the synthesis of tubulin which assists cells through the cell cycle.

The regulation of the scleral growth associated with deprivation myopia in chicks.

The mechanism of axial elongation caused by experimental or clinical myopia is still unknown. We sought to explore the changes of scleral chondrocytes during myopia formation through the cell biology model. White Leghorn chicks were used for this study. The right eye was covered with a translucent goggle after hatching, and the left eye was left uncovered for control. The chicks were maintained on 12 hours light-dark cycle for two weeks, then sacrificed every other day and the eyeballs removed for study. Our results in the primary culture of scleral chondrocytes showed that the densities of chondrocytes on myopic eyes were significantly higher than those of the controlled non-myopic eyes, and 3H-thymidine incorporation rate also increased with the increasing of the concentration of fetal bovine serum. The PCNA index of chondrocytes in myopic eyes was also higher than that of the controlled non-myopic eyes. Thus, axial elongation of experimental myopia in the chick is the result of active tissue remodeling rather than passive scleral stretching alone.

Bacterial carbon production in the northern Baltic:a comparison of thymidine incorporation and FDC based methods

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 153:59-66 (1997) - doi:10.3354/meps153059 Bacterial carbon production in the northern Baltic: a comparison of thymidine incorporation and FDC based methods Tuomi P Bacterial production in the open Gulf of Finland was estimated by 3H-thymidine incorporation rate (TTI) and by the frequency of dividing cells (FDC), counted by transmission electron microscopy (TEM). To relate TTI and FDC to bacterial growth rate, batch cultures with the natural bacterial community from the study area were used. Conversion factors between 0.2 and 7.2 x 1018 cells mol-1 TdR, or 0.3 and 24.3 x 1017 µm3 mol-1 TdR, were obtained for TTI, depending on the season and calculation model used (integrative, cumulative, or modified derivative). From FDC and growth rate (µ) based on bacterial cell numbers, the following equation was derived: µ = 0.002FDC - 0.001. Bacterial carbon production in the study area, estimated using the FDC method, was 20 to 80% higher than TTI-based estimates. Virus abundances were followed in the batch cultures and in the field study. Viral lysis may have been a significant cause of bacterial mortality in the field, but it remains to be shown whether viral-induced mortality was already included in the empirical conversion factor. Bacteria · 3H-thymidine incorporation rate · Frequency of dividing cells · Baltic Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 153. Publication date: July 10, 1997 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 1997 Inter-Research.

Regulation of annual variation in heterotrophic bacterial production in the Schelde estuary (SW Netherlands)

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 12:223-232 (1997) - doi:10.3354/ame012223 Regulation of annual variation in heterotrophic bacterial production in the Schelde estuary (SW Netherlands) Goosen NK, van Rijswijk P, Kromkamp J, Peene J Heterotrophic bacterioplankton production (3H-thymidine incorporation rate) and abundance in the surface water of the Schelde estuary (The Netherlands) were studied during an annual cycle in 1991 along the salinity gradient (0.8 to 33 psu). Bacterial production and numbers increased from the lower to the upper estuary. This spatial variability of bacterial production was positively correlated with concentrations of dissolved and particulate organic carbon and negatively correlated with the oxygen concentration. On an annual scale the ratio gross bacterial production:net primary production was larger than 1 in the whole estuary and increased from the lower to the upper estuary. This indicates that bacteria processed more carbon than was put into the system by phytoplankton primary production. A comparable situation is found in other estuaries and shows the high degree of heterotrophy of these systems. Despite the high degree of heterotrophy, temporal variation in bacterial production in the lower Schelde estuary was characterised by a spring and summer bloom and was correlated with phytoplankton primary production. This apparent contradiction is explained by rapid uptake of labile algal exudates by bacteria and a subsequent increase of bacterial production rate during and after the phytoplankton bloom. This idea is corroborated by the absence of an increase in dissolved organic carbon during phytoplankton blooms in the lower estuary. Due to an excess of allochthonous organic carbon and nutrients in the upper estuary, temperature was the most important factor in explaining the variability of bacterial production in this region. Heterotrophic bacterial production · Organic carbon · Primary production · Estuary Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 12, No. 3. Publication date: May 29, 1997 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1997 Inter-Research.

Apoptosis in mesangial cells induced by ionizing radiation and cytotoxic drugs

Mesangial proliferation contributes to the pathogenesis of many forms of glomerulonephritis. To evaluate the role of apoptosis on the pharmacologic effects of cytotoxic drugs and ionizing radiation, we studied their effects on cultured rat mesangial cells (MC), whose apoptotic response to these drugs is unknown. Mesangial cells were cultured with or without stimuli to induce apoptosis and were harvested at 24 and 48 hours. MC morphology was examined by light microscopy, in situ end labeling technique using terminal deoxy-transferase (TUNEL) and by electrophoresis of extracted total cellular DNA. MCs exposed to cytotoxic drugs or irradiation demonstrated statistically significant increases in apoptotic cells identified by light microscopy. DNA fragmentation of apoptotic cells was also visualized as characteristic staining by the TUNEL method and statistically significant increases in apoptotic cell number in cells exposed to cytotoxic drugs and irradiation were noted compared to control cultures. In general, the number of TUNEL positive cells was greater than that of morphologically apoptotic cells. DNA extracted from these cells also showed the characteristic ladder pattern of internucleosomal chromatin cleavage of 180 bp fragments on agarose gel electrophoresis. To further analyze whether MC apoptosis induced by these drugs alters the cell cycle, 3H-thymidine incorporation rates were measured in both the cell culture monolayer and in those cells shed into the supernatant when cultured with or without cyclophosphamide (N = 5). 3H-thymidine incorporation corrected for total cellular DNA showed a similar pattern in both control and cyclophosphamide treated groups, suggesting that cyclophosphamide did not alter the mesangial cell cycle. Considering that the dosage of the cytotoxic drugs utilized in these experiments in nearly the therapeutic plasma level in humans, these results suggest that cytotoxic drugs used to treat glomerular disease can induce apoptotic mesangial cell death and may operate in part via this mechanism.