First Hour Of Incubation Research Articles

Transforming Growth Factor-β1 Release from a Porous Electrostatic Spray Deposition–Derived Calcium Phosphate Coating

This study evaluated the utilization of a porous coating, derived with electrostatic spray deposition (ESD), as a carrier material for transforming growth factor-beta1 (TGF-beta1). A porous beta-tricalcium phosphate coating was deposited with ESD, and 10 ng of (125) I-labeled TGF-beta1 was loaded on the substrates. A burst release during the first hour of incubation of >90% was observed, in either culture medium or phosphate-buffered saline (PBS). Ninety-nine percent of the growth factor was released after 10 days of incubation. All samples were able to inhibit epithelial cell growth, indicating that the growth factor had remained bioactive after release. Thereafter, osteoblast-like cells were seeded upon substrates with or without 10 ng of TGF-beta1. While proliferation of osteoblast-like cells was increased on TGF-beta1-loaded substrates, differentiation was inhibited or delayed. In conclusion, a porous ESD-derived calcium phosphate coating can be used as a carrier material for TGF-beta1, when a burst release is desired.

Resveratrol induces apoptotic cell death in rat H4IIE hepatoma cells but necrosis in C6 glioma cells

Resveratrol ( trans-3,5,4′,-trihydroxystilbene) is assumed to possess cancer-preventive and cancer-therapeutic properties. The aim of this project was to analyze cellular effects of resveratrol in metabolically active H4IIE rat hepatoma cells in comparison to metabolically poorly active C6 rat glioma cells. Resveratrol is rapidly taken up by both cell types and acts as a potent intracellular antioxidant. On the other hand, resveratrol in higher concentrations is relatively toxic to both cell lines as measured by the neutral red accumulation assay. In H4IIE cells, resveratrol concentrations rapidly decline to very low levels during the first hours of incubation due to formation of resveratrol glucuronides. The first resveratrol effect found at 3 h after the start of resveratrol treatment was the induction of mild DNA damage as detected by the comet assay. Cell death was caused via induction of apoptosis as detected by caspase activation, oligonucleosomal DNA fragmentation and formation of apoptotic nuclei. Following DNA damage, resveratrol led to an activation of caspases 2 and 8/10 at 6 h and consequently of caspase 3 at 12 h, but failed to activate caspase 9. In contrast to H4IIE cells, resveratrol is not metabolised in C6 glioma cells and accumulates to concentrations which are assumed to drive the cell into necrosis. This suggests that the mode of cell death caused by resveratrol and the usefulness of resveratrol for cancer prevention and treatment critically depends on the metabolic capacity of the tumor cell to be eradicated.

Cytotoxicity and cell signalling induced by continuous mild hyperthermia in freshly isolated mouse hepatocytes

An increasing body of data has been demonstrating that mammalian cells have elaborate networks of molecular signalling in counteracting heat shock and in developing adaptation to oxidative stress to avoid cell death. However, the precise mechanisms linking heat shock, oxidative stress and cell survival/cell death mechanisms are not yet clearly understood. The purpose of this study was thus to study the time course of hyperthermia-induced oxidative stress and cellular signalling through the activation of heat shock factor 1 (HSF1) and heat shock protein 70 (HSP70), using freshly isolated mouse hepatocytes. The results accomplished in this work demonstrated that mild continuous hyperthermia (41°) leads to oxidative stress and loss of cellular viability in a time-dependent manner, with significant effects already observed at the first hour of incubation. These toxic effects developed concomitantly with activation of HSF1 and emerged before the formation of HSP70 levels. Thus, although cell signalling was triggered through the transcriptional activation of HSP70 via HSF1, this putative protective process did not modify the trend of hepatotoxic effects mediated by this type of hyperthermic challenging.

Hypoxia interferes with ABA metabolism and increases ABA sensitivity in embryos of dormant barley grains

Two mechanisms have been suggested as being responsible for dormancy in barley grain: (i) ABA in the embryo, and (ii) limitation of oxygen supply to the embryo by oxygen fixation as a result of the oxidation of phenolic compounds in the glumellae. The aim of the present work was to investigate whether hypoxia imposed by the glumellae interferes with ABA metabolism in the embryo, thus resulting in dormancy. In dormant and non-dormant grains incubated at 20 degrees C and in non-dormant grains incubated at 30 degrees C (i.e. when dormancy is not expressed), ABA content in the embryo decreased dramatically during the first 5 h of incubation before germination was detected. By contrast, germination of dormant grains was less than 2% within 48 h at 30 degrees C and embryo ABA content increased during the first hours of incubation and then remained 2-4 times higher than in embryos from grains in which dormancy was not expressed. Removal of the glumellae allowed germination of dormant grains at 30 degrees C and the embryos did not display the initial increase in ABA content. Incubation of de-hulled grains under 5% oxygen to mimic the effect of glumellae, restored the initial increase ABA in content and completely inhibited germination. Incubation of embryos isolated from dormant grains, in the presence of a wide range of ABA concentrations and under various oxygen tensions, revealed that hypoxia increased embryo sensitivity to ABA by 2-fold. This effect was more pronounced at 30 degrees C than at 20 degrees C. Furthermore, when embryos from dormant grains were incubated at 30 degrees C in the presence of 10 microM ABA, their endogenous ABA content remained constant after 48 h of incubation under air, while it increased dramatically in embryos incubated under hypoxia, indicating that the apparent increase in embryo ABA responsiveness induced by hypoxia was, in part, mediated by an inability of the embryo to inactivate ABA. Taken together these results suggest that hypoxia, either imposed artificially or by the glumellae, increases embryo sensitivity to ABA and interferes with ABA metabolism.

Photodynamic Effect of Curcumin on NPC/CNE2 Cells

Nasopharyngeal carcinoma (NPC) is highly prevalent in Southern China. Radiotherapy is the primary treatment of NPC, but the rate of tumor recurrence is significant. Photodynamic therapy (PDT) and the use of natural compounds become one of the new approaches in the investigation of NPC treatment. PDT is an alternate method of cancer treatment while curcumin (CUR) is a compound derived from the traditional Chinese medicinal (TCM) herbs. The purpose of the study focuses on the photodynamic effect of CUR on one of the NPC cell lines, NPC/CNE2. Cytotoxicity and photocytotoxicity of CUR were evaluated by 3-(4,5-dimthyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Uptake kinetics of CUR in NPC/CNE2 was examined by flow cytometry. The mode of cell death induced by CUR was studied by fluorescence microscopy. Summarizing the results, CUR showed dark cytotoxicity as well as photocytotoxic effects on NPC/CNE2 cells. LC50 of CUR in the dark was about 16 microM. The cytotoxicity of CUR was enhanced by the irradiation of visible light and blue filtered light (maximum transmittance at 300 approximately 400 nm) with light doses of 300 kJ/m2 and 60 kJ/m2 respectively. NPC/CNE2 was found to rapidly take up CUR in the first hour of incubation, and the uptake kinetics steadily increased to a plateau level after 20 hr of incubation. Cell shrinkage and membrane bledding appeared under the observation of fluorescence microscopy. Such evidences proved that CUR might induce apoptosis on NPC/CNE2 cells. The preliminary study confirmed that CUR demonstrated dark cytotoxicity and photocytotoxicty to NPC/CNE2. The mode of action is likely to be induced by apoptotic pathway. CUR may be developed as a potential photosensitizer as well as a chemotherapeutic agent in clinical application.

Induction of toll-like receptors by Propionibacterium acnes

The bacterium Propionibacterium acnes is involved in the induction and maintenance of the inflammatory phase of acne. Recent studies have found that keratinocytes express toll-like receptors (TLRs) implicated in immediate immunity. No studies have, to date, been carried out on the action of P. acnes upon TLR activation in keratinocytes. Focusing on the inflammatory phase of acne, to clarify the role of P. acnes in immediate immunity by inducing expression of TLR-2 and TLR-4 by keratinocytes. We also studied how the secretion and expression of matrix metalloproteinase (MMP)-9 is induced by P. acnes. The work was carried out on two levels: in vivo with the study of the expression of TLR-2 and TLR-4 proteins in biopsies of acne lesions and in vitro on cultured keratinocyte monolayers to study the modulating effects of P. acnes on the expression of TLR-2 and TLR-4 and also on the expression and secretion of MMP-9. Our findings reveal that in vivo TLR-2 and TLR-4 expression is increased in the epidermis of acne lesions. In vitro, an increase in TLR-2 and TLR-4 expression by human keratinocytes occurred in the first hours of incubation with bacterial fractions as well as an increase of the expression and secretion by the keratinocytes of MMP-9, which plays a role in inflammation. This work demonstrates that P. acnes induces TLR expression and that this mechanism could play an essential role in acne-linked inflammation. These receptors could be involved notably in acute acne.

Rat hepatocyte invasion by Listeria monocytogenes and analysis of TNF-alpha role in apoptosis

Listeria monocytogenes, etiological agent of severe human foodborne infection, uses sophisticated mechanisms of entry into host cytoplasm and manipulation of the cellular cytoskeleton, resulting in cell death. The host cells and bacteria interaction may result in cytokine production as Tumor Necrosis Factor (TNF) alpha. Hepatocytes have potential to produce pro-inflammatory cytokines as TNF-alpha when invaded by bacteria. In the present work we showed the behavior of hepatocytes invaded by L. monocytogenes by microscopic analysis, determination of TNF-alpha production by bioassay and analysis of the apoptosis through TUNEL technique. The presence of bacterium, in ratios that ranged from 5 to 50,000 bacteria per cell, induced the rupture of cellular monolayers. We observed the presence of internalized bacteria in the first hour of incubation by electronic microscopy. The levels of TNF-alpha increased from first hour of incubation to sixth hour, ranging from 0 to 3749 pg/mL. After seven and eight hours of incubation non-significant TNF-alpha levels decrease occurred, indicating possible saturation of cellular receptors. Thus, the quantity of TNF-alpha produced by hepatocytes was dependent of the incubation time, as well as of the proportion between bacteria and cells. The apoptosis rate increased in direct form with the incubation time (1 h to 8 + 24 h), ranging from 0 to 43%, as well as with the bacteria : cells ratio. These results show the ability of hepatocyte invasion by non-hemolytic L. monocytogenes, and the main consequences of this phenomenon were the release of TNF-alpha by hepatocytes and the induction of apoptosis. We speculate that hepatocytes use apoptosis induced by TNF-alpha for release bacteria to extracellular medium. This phenomenon may facilitate the bacteria destruction by the immune system.

Binding and transfer of an oligodeoxynucleotide containing the translation initiation site of the BCL2 mRNA into K562 cells

A study was conducted of the interaction of an octadecameric oligodeoxynucleotide (dON) containing the BCL2 mRNA translation start with K562 cells. Both in solution and upon lipofection, the binding of dON used at a low concentration (≤30 nM) at 37°C involved two steps: saturating surface binding with the cell membrane and internalization. Three phases were revealed in the dynamics of internalization: the extent and rate of internalization increased during the first hour of incubation; decreased during the second hour; and then increased again, which was assumed to reflect the priming of new dON-binding sites. The binding constant and the number of binding sites were estimated at 10°C (the conditions preventing internalization) by consecutive dissociation of dON-cell complexes formed in 1 h at 37°C. Incubation of dON with cells led to the priming of new high-affinity binding sites and an increase of the binding constant to a level characteristic of high-affinity ligand-receptor interactions (109 M−1). High-affinity receptor-mediated binding preceded internalization of dON. Lipofection increased the binding constant and the number of binding sites severalfold but had virtually no effect on the temporal pattern and the extent of dON internalization.

The effect of a nitric oxide donor on the synthesis of cGMP in Hymenolepis diminuta: a radiometric study

The formation of cGMP in homogenates of the adult rat-tapeworm Hymenolepis diminuta was followed with a radiometric assay during 3 h after stimulation with the nitric oxide donor sodium nitroprusside (SNP) and in the presence of isobutylmethylxanthine (IBMX). The level of cGMP was stable in worms incubated with IBMX during the first hour. After 3 h of incubation, the level of cGMP had declined by 27%. Addition of SNP stimulated the formation of cGMP during the first hour of incubation. After 3 h of incubation, a two-fold decline in cGMP formation was observed. The rate of nitric oxide (NO) release by the worm was determined by a spectrophotometric assay for the accumulation of nitrites and nitrates, the stable degradation products of NO, using the Griess reaction. The results are discussed from the perspective of the current concept on the role of the nitrergic mechanisms in the flatworm nervous system.

Characterization of events associated with apoptosis/anoikis induced by snake venom metalloproteinase BaP1 on human endothelial cells

Human endothelial EA.hy926 cells were incubated with BaP1, a hemorrhagic metalloproteinase purified from Bothrops asper snake venom. Since the first hour of incubation with the proteinase, cells started showing DNA fragmentation, detected by a terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL)-based photometric enzyme-linked immunosorbent assay (ELISA). At later times, DNA fragments were predominantly located outside the cells, evidencing plasma membrane rupture. DNA fragmentation was completely abolished by Batimastat, a potent inhibitor of metalloproteinase enzymatic activity. Apoptosis induced by BaP1 on endothelial cells was independent of two Bcl-2 family members (anti-apototic Bcl-xL and pro-apoptotic Bax), that did not show any changes in their expression during a 24 h-treatment period. Interestingly, IkappaBalpha, an inhibitor of NFkappaB, decreased after 24 h of treatment, suggesting further activation of the transcription factor. When some elements of the apoptotic extrinsic pathway were assessed, it was observed that procaspase-8 completely disappeared after 24 h of treatment with BaP1, probably indicating its activation by a death receptor, whereas caspase-8 inhibitor, cellular FLICE-inhibitory protein (cFLIP(L)), increased its expression since the first hours of BaP1 incubation. In conclusion, treatment of human endothelial cells with BaP1 induces apoptosis/anoikis, independently of Bcl-2 family members Bax and Bcl-xL and associated with caspase-8 activation and cFLIP(L) up-regulation. Apoptosis was completely dependent on BaP1 enzymatic activity. Similarities between this and other endothelial cell anoikis-related systems suggest that BaP1 and other snake venom metalloproteinases may be useful experimental tools in the study of death-related events that occur when adherent cells loose contact with extracellular matrix.

Multidimensional long-term time-lapse microscopy of in vitro peripheral nerve regeneration.

In order to test the effectiveness of a new advanced time-lapse microscopy imaging and image processing and analysis system, and to do quantitative and qualitative temporal analyses of in vitro peripheral nerve regeneration, long-term time-lapse imaging of cultures of mouse dorsal root ganglia (DRGs) was performed. DRGs were placed in a Petri dish, covered with collagen gel, their attached peripheral nerves were cut in the middle, creating a gap, and the dish was filled with culture medium. Six preparations were kept on the time-lapse imaging system, which provides a suitable incubation environment and enables to capture images from multiple coordinates at x,y,z axes at desired time intervals for 13 days. In general, the time-lapse imaging system proved quite stable and efficient, although some improvements are certainly required. Two main components of peripheral nerve regeneration, outgrowth of axons and activities of resident cells, were examined. Axons started to grow during the first hour of incubation with a 16.5 microm/h rate and showed the slowest rates (0.7 microm/h) on days 8 and 9, after which they resumed higher speeds again. The first cell came out of the proximal end of the cut nerve on the second day and it was a Schwann cell (SC), which was the prominent cell type in the preparations throughout the experiment. SCs were higher in number (83.15% of all cells) but slower in migration (3.4 vs. 7.3 microm/h, P < 0.001) than other cells. Other observed characteristics of axonal outgrowth and cellular activity and interactions between axons and the cells are discussed.

Uptake, Distribution and Release of 14C-Triclosan in Human Gingival Fibroblasts

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is an antibacterial agent included in dentifrices and mouth rinses. Previously, we reported that triclosan reduces the production of the inflammatory mediators in gingival fibroblasts. The aim of this study was to investigate the uptake, distribution, and release of (14)C-triclosan in gingival fibroblasts. Time-course studies showed that the uptake of (14)C-triclosan in cytoplasmic and nuclear fraction started within the first minute of incubation, increased gradually, and reached constant levels after 1 h in the nuclear fraction and slightly increased in the cytoplasmic fraction between 3 and 24 h. The distribution of (14)C-triclosan in the cytoplasmic and the nuclear fractions was, on an average, 84 and 16%, respectively. Autoradiographic results based on transmission electron microscopy confirmed the distribution of (14)C-triclosan in the cytoplasm and nucleus of the cell. The release of (14)C-triclosan showed that the radioactivity of the agent in the medium gradually increased during the first hour of incubation and then reached steady-state levels. After repeated washing of preloaded fibroblasts, the level of (14)C-triclosan in the cytoplasmic fraction decreased by 77% whereas the level in the nuclear fraction remained unchanged. Our results demonstrate that triclosan is distributed in the cytoplasm and remains associated with the nucleus of gingival fibroblasts, suggesting that the agent may affect the intracellular signal pathways involved in the production of inflammatory mediators.

Photodynamic activities of sulfonamide derivatives of porphycene on nasopharyngeal carcinoma cells.

Two sulfonamide derivatives of porphycene, namely PS6 and PS6A, were synthesized, and their photodynamic efficacies on the nasopharyngeal carcinoma (NPC) cell line NPC/CNE-2 were evaluated. By comparing the 50% lethal concentrations (LC(50)) of these photosensitizers, we found that PS6A with a cationic ammonium group on the side chain exhibited potent photocytotoxicity on the NPC cell line. At a light dose of 1 J/cm(2), the LC(50) values of PS6 and PS6A for NPC cells were 11.6 and 1.92 microM, respectively. CNE-2 was found to rapidly take up PS6A in the first hour of incubation, and the uptake kinetics steadily increased to a plateau level after 18 h of incubation. The uptake of PS6A was temperature dependent. Over 99% of CNE-2 cells were sensitized by PS6A 24 h after drug treatment. Collapse of the mitochondrial membrane potential was also observed in PS6A photodynamic therapy (PDT)-treated CNE-2 cells 1.5 h after PDT. Confocal microscopy revealed that PS6A was predominantly localized in the mitochondria, lysosomes and Golgi bodies of NPC cells. Significant genotoxicity was not observed in CNE-2 cells. In functional studies, the in vitro formation of a capillary-like network of human umbilical vein endothelial cells in Matrigel was greatly inhibited by PS6A PDT in a dose-dependent manner. In conclusion, PS6A mediates both in vitro antitumor and antiangiogenic activities. PS6A might be a candidate for photodynamic treatment of NPCs.

Folate-liposome-mediated antisense oligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo.

The objective of this study was to investigate the use of folate-targeted liposomes for the delivery of encapsulated oligonucleotides to folate receptor (FR)-positive tumor cells in vitro and in vivo. This project involved the synthesis and biological evaluation of many folate-PEG-lipid conjugates, where the chemical form of the folate moiety (pteroate) and the length of the PEG linker chain were varied widely. Folate-targeted oligonucleotide-containing liposomes were prepared using conventional methods, and the extent of cell uptake was evaluated using, among others, the FR positive KB cell line. Oligonucleotide-loaded folate-targeted liposomes were found to rapidly associate with the KB cells, and saturation was typically reached within the first hour of incubation at 37 degrees C. Nearly 100,000 liposomes per cell were bound or internalized at saturation. Importantly, cell association was blocked by a large excess folic acid, thus reflecting the FR-specific nature of the cell interaction. Full targeting potential was achieved with PEG linkers as low as 1000 in molecular weight, and pteroates bearing glycine or gamma-aminobutyryl residues juxtaposed to the pteroic acid moiety were also effective for targeting, provided that a terminal cysteine moiety was present at the distal end of the PEG chain for added hydrophilicity. When tested in vivo, folate-targeted liposomes were found to deliver approximately 1.8-fold more oligonucleotide to the livers of nude mice (relative to the nontargeted PEG-containing formulations); however, no improvement in KB tumor uptake was observed. We conclude from these results that folate liposomes can effectively deliver oligonucleotides into folate receptor-bearing cells in vitro, but additional barriers exist in vivo that prevent or decrease effective tumor uptake and retention.

Effects of angiotensin II and natriuretic peptides of the eel on prolactin and growth hormone release in the tilapia, Oreochromis mossambicus

The effects of angiotensin II (ANG II) and natriuretic peptides (NPs) of the eel (ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; and VNP, ventricular natriuretic peptide) on prolactin (PRL 188 and PRL 177) and growth hormone (GH) release from the organ-cultured tilapia pituitary were examined. Eel ANG II at concentrations greater than 1 nM stimulated the release of PRL 188 and PRL 177 in a dose-related manner during the first hour of incubation. Significant stimulation by 100 nM ANG II on PRL 177 release was observed until 4 h of incubation, and on PRL 188 release until 12 h. No effect of ANG II was seen on GH release. None of the NPs altered the release of PRLs at any time point. On the other hand, eel VNP at concentrations greater than 1 nM stimulated GH release in a dose-related manner after 4 h, and significant stimulation was observed until 48 h. Eel CNP was less effective than eel VNP; significant stimulation of GH release was observed at 1 and 10 nM during 24–48 h of incubation. No significant effect of eel ANP on GH release was seen at any concentration. ANG II had no effect on GH release at any time point. There was no change in mRNA levels of PRLs or GH in the pituitaries incubated with ANG II for 8 h or those incubated with the NPs for 48 h. These results indicate rapid and short-lasting stimulation by ANG II on PRL release and slow and long-lasting stimulation by VNP and CNP on GH release from the tilapia pituitary.

Cryopreservation of Rat Precision-cut Liver Slices is Associated with Major Metabolic Stress and Ionic Perturbations

Background/Aims: Cryopreserved precision-cut liver slices (PCLS) in culture exhibit a rapid decline in ATP level and anabolic processes by unknown mechanisms. The aim of this study is to elucidate the key events explaining the alterations occurring in cryopreserved PCLS. Methods: Glucose metabolism, mitochondrial activities, ionic homeostasis and caspase-3 activity were assessed in fresh or cryopreserved (rapid freezing-thawing conditions) rat PCLS within the first hour of incubation in Williams’ medium E at 37°C. Results: Despite a similar glycolytic activity under both conditions, only fresh PCLS were able to gradually recover their ATP and potassium content. Glycogen content dropped more rapidly in cryopreserved than in control PCLS. Only cryopreserved PCLS exhibited a decline in O₂ consumption and a lower ATP/ADP ratio from 30 min of incubation with a loss of coupling of oxygen consumption to ATP synthesis and mitochondrial calcium transport after 1 hour of incubation. Caspase-3 activation was already present in cryopreserved PCLS after a few minutes of incubation. Conclusions: The lack of restoration of potassium/sodium exchange might be a primary event in the metabolic alterations occurring in cryopreserved PCLS after thawing, that contributes to further mitochondrial alterations, caspase activation and compromises ATP-dependent anabolic pathways.

Broad-spectrum bactericidal activity of Ag(2)O-doped bioactive glass.

Bioactive glass has found extensive application as an orthopedic and dental graft material and most recently also as a tissue engineering scaffold. Here we report an initial investigation of the in vitro antibacterial properties of AgBG, a novel bioactive glass composition doped with Ag(2)O. The bacteriostatic and bactericidal properties of this new material and of two other bioactive glass compositions, 45S5 Bioglass and BG, have been studied by using Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus as test microorganisms. Concentrations of AgBG in the range of 0.05 to 0.20 mg of AgBG per ml of culture medium were found to inhibit the growth of these bacteria. Not only was AgBG bacteriostatic, but it also elicited a rapid bactericidal action. A complete bactericidal effect was elicited within the first hours of incubation at AgBG concentrations of 10 mg ml(-1). 45S5 Bioglass and BG had no effect on bacterial growth or viability. The antibacterial action of AgBG is attributed exclusively to the leaching of Ag(+) ions from the glass matrix. Analytical measurements rule out any contribution to AgBG-mediated bacterial killing by changes in pH or ionic strength or the dissolution of other ionic species from the biomaterials. Our observations of the dissolution profiles of Ag(+) from AgBG in the presence and absence of bacteria are consistent with silver accumulation by the bacteria.

Oleic, linoleic and linolenic acids enhance receptor-mediated uptake of low density lipoproteins in Hep-G2 cells

In the present study, the binding, internalization and degradation of low-density lipoprotein (LDL) was investigated in Hep-G2 cells treated with 18:0, 18:1, 18:2 and 18:3. In non-treated control cells, the surface binding (heparin-releasable) of 125I-LDL progressed in a saturable manner reaching equilibrium within 2 h, amounting 24.0 ± 1.1, 29.5 ± 1.3 and 31.4 ± 2.8 (ng/mg cell protein) at 1, 2 and 4 h, respectively. The cells rapidly internalized 125I-LDL reaching a plateau at 2 h (72.4 ± 6.3/1 h, 96.7 ± 4.3/2 h and 100.8 ± 4.6 ng/mg protein/4 h, respectively). The degradation of internalized LDL progressed slowly during the first hour of incubation reflecting the time required to an uptake and delivery of LDL to the cellular lysosomes. The levels of degraded LDL discharged into the medium then increased rapidly in a linear manner after the initial lag period, amounting 16.8 ± 1.2, 51.8 ± 7.0 and 118.2 ± 5.7 ng/mg protein at 1, 2 and 4 h, respectively. The treatment of cells with of 1.0 mM of fatty acids for 4 h resulted in a significant increase in the surface binding of 125I-LDL compared to the control (34.9 ± 3.0), but it was significantly lower in cells exposed to 18:0 (48.2 ± 2.0) than to 18:1 (56.8 ± 5.1), 18:2 (56.0 ± 3.5) and 18:3 (57.8 ± 6.0 ng/mg protein/4 h) ( P < 0.05). The levels of degraded LDL in cells remained nearly the same regardless of fatty acid treatments, but degraded LDL levels in the medium were much higher in cells exposed to 18:1 (167.6 ± 10.1), 18:2 (159.8 ± 7.7) and 18:3 (165.1 ± 14.7) than to 18:0 (142.1 ± 8.4) and the control (121.2 ± 3.4 ng/mg protein/4 h) ( P < 0.05). The present finding that 18:1 is equally effective in enhancing the receptor-mediated LDL uptake and its degradation as those of 18:2 and 18:3 suggests that the major action of 18:1 in lowering LDL-cholesterol levels also involves an increased clearance of LDL via hepatic LDL-receptors.

Surge ammonium uptake of the cultured seaweed, Kappaphycus alvarezii (Doty) Doty (Rhodophyta: Gigartinales)

The possibility that the cultured seaweed Kappaphycus alvarezii may demonstrate surge ammonium uptake was studied using the perturbation method. Cultivars of the green morphotype of K. alvarezii were incubated at three different initial NH 4 + concentrations (i.e., 10, 20, 30 μM) at the same light intensity. Additional incubations were conducted at 30 μM initial NH 4 + concentration in the dark. K. alvarezii were also placed in a 30 μM NH 4 +-enriched seawater medium for 1 h before incubation at 30 μM initial NH 4 + concentration. Levels of enrichment and time had significant effects on ammonium uptake rates. Surge ammonium uptake tended to be higher with higher initial ammonium concentration. Mean uptake rates at 10 μM NH 4 + initial concentration were significantly lower compared to other treatments. Higher variability in uptake was noted during the first hour of incubation. Ammonium uptake in the dark was not significantly different from that in light (at 30 μM NH 4 + initial concentration). Surge uptake was higher for incubations without additional enrichment than when the alga was additionally exposed to enriched medium before the experiment. On average, surge uptake ( V S) occurred within the first 30 min and ranged from 15 to 35 μmol NH 4 + g −1 DW h −1. Internally controlled uptake ( V I) ranged from 5 to 15 μmol NH 4 + g −1 DW h −1 and occurred from 30 min to 6 h after exposure to the nutrient. Externally controlled uptake ( V E) was less than 5 μmol NH 4 + g −1 DW h −1 and occurred from 6 to 8 h after exposure when the medium was significantly depleted of ammonium (as in 10 and 20 μM NH 4 + initial concentrations). K. alvarezii from Danajon Reef, central Philippines thus exhibits surge ammonium uptake and some form of nitrogen limitation. Such a strategy is needed for surviving in environments with low or erratic nutrient supplies.

The early increase of S-adenosylmethionine decarboxylase activity is essential for the normal germination and tube growth in tomato ( Lycopersicon esculentum Mill.) pollen

The effect of polyamine biosynthesis inhibitors on pollen germination and tube growth and putrescine (Put), spermidine (Spd) and spermine (Spm) levels in germinating tomato ( Lycopersicon esculentum Mill.) pollen was investigated. Pollen germination was initiated 20 min after incubation at 25 °C and completed within 2 h. The activity of arginine decarboxylase (ADC), an enzyme catalyzing Put synthesis, exhibited a marked increase during the initial 2 h and remained high thereafter. The activity of S-adenosylmethionine decarboxylase (SAMDC), an essential enzyme in the synthesis of Spd and Spm, increased transiently during the first hour of incubation. Consequently, the germinating pollen showed elevated ratios of Spd+Spm to Put during the first hour in all of the free, conjugated and bound forms. The addition to the germination medium of either methylglyoxal-bis(guanylhydrazone) (MGBG) or cyclohexylamine, inhibitors of SAMDC and spermidine synthase, respectively, caused a marked decrease of Spd and Spm contents, together with ratios of Spd+Spm to Put, and resulted in inhibition of pollen germination and tube growth. Simultaneous addition of Spd or Spm reversed the adverse effect of MGBG. However, Put failed to reverse the inhibitory effect of d-arginine, a competitive inhibitor of ADC. MGBG treatment 2 h after incubation was still inhibitory to tube growth, which was partially reversed by Spd. The results suggest that the increased activity of SAMDC and the resultant elevation of Spd and Spm contents during the early pollen germination phase are essential for the occurrence of normal germination and tube growth in tomato pollen.