Activation of the cAMP-generating system by vasoactive intestinal polypeptide (VIP) in the human laryngeal malignant cell line HEp-2

In the presence of 3-isobutyi-l-methylxanthine, VIP produced a dose-related (3×10^–9–10^–7 M) increase (g-fold) in cAMP production in isolated HEp-2 cells incubated at 15°C in KRP buffer. Among the peptides structurally related to VIP, including secretin (10^–7 M), pancreatic glucagon (10^–6 M), PHI, somatostatin-14 (10^–6 M), hpGRF (10^–8–4×10^–M), GIP (2×10^–7 M), only PHI (3×10^–7 M and above) is able to activate the cAMP-generating system in HEp-2 cells, but at 102 times lower potency. Under the same conditions, histamine (10^–3 M) was also ineffective, while PGE 2 (10^–7–10^–4 M) increased (0-fold) basal cAMP levels in HEp-2 cells. The VIP effect is related to the interaction os the peptide on VIP recognition sites (12SI-VIP-binding capacity ), coupled to the membrane-bound adenylate cyclase . The results indicate that the transformed laryngeal cell line HEp-2 possessesa receptor-cAMP system preferentially activated by VIP (relative potencies: VIP > PHI other peptides of the secretin family), and suggest that this neuropeptide could modulate biological functions in normal laryngeal epithelia in man.     

Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l^–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11^* cells which had been engineered to express a humanised antibody, CAMPATH^*-1H, were routinely grown using serum-containing medium. From a seeding density of 10⁵ cells ml^–1, cells grown in static culture with serum reached a maximal cell density of 6.5×10⁵ cells ml^–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l^–1 after 11 days in culture. CHO 3D11^* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10⁵ cells ml^–1 reached a maximal viable cell density of 2.16×10⁶ cells ml^–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l^–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids     

Effects of griseofulvin on mitosis in PtK1 cells

The concentration dependent effects of griseofulvin (GF) on mitosis in PtK₁ cells were studied using a combination of time lapse cinematography and polarization and electron microscopy. Low concentrations of GF (4×10^–5 M) allowed a substantial number of cells to enter and complete an apparently normal mitosis. At higher concentrations of GF (1×10^–4 M and 2.5×10^–4 M) all cells entering mitosis were arrested. Typical c-mitotic chromosome arrays were observed at 1×10^–4 M GF with microtubules present but no spindle formed. At 2.5×10^–4 M GF chromosomes did not orient toward a common center to form a c-mitotic figure, but instead remained in a loosely clustered grouping at the center of the cell. Electron microscopy showed microtubules to be absent but revealed an irregularly shaped electron dense cloud around the centrioles. Quantitative polarization microscopy of metaphase cells perfused with GF showed rapid loss of spindle birefringence after exposure to the drug. Coinciding with loss of birefringence the spindle shrank rapidly with a pronounced shortening of pole to pole distance.     

Induction of germ-tube formation by Candida albicans in amino acid liquid synthetic medium at 25°C

Candida albicans (3153A) was found to exhibit extensive germ-tube and mycelial development at 25°C when transferred from amino acid synthetic medium at pH 6 to medium of pH 7. Significant germ-tube formation was detectable after approximately 8 h and in all experimental treatments, the peaks of maximal germination occurred at approximately 40–44 h. Such a transition was not only dependent on the initial pH of the medium but also on the glucose concentration and inoculum size. The optimum initial glucose concentration and inoculum size for maximal germ-tube development was 1.25% and 2×10⁶ cells ml^–1 respectively and above or below these values the extent of germ-tube formation was greatly reduced.     

A low-cost chemically defined protein free medium for a recombinant CHO cell line producing prothrombin

A chemically defined protein free medium, DF6S, was developed for the cultivation of a recombinant Chinese hamster ovary cell line (CHO2DS) producing human prothrombin in suspension batch culture. DF6S was formulated by optimizing DME/F12 with amino acids and supplementing the optimized DME/F12 with aurintricarboxylic acid, ethanolamine, ferric sulfate, Pluronic F68, putrescine and sodium pyruvate. From a seeding density of 2.3 × 10⁵ cells ml^–1, CHO2DS cells grown in suspension in DF6S medium reached a maximal cell density of 1.92 × 10⁶ cells ml^–1 with an accumulated prothrombin concentration of 16.7 mg l^–1 after 6 days in culture.     

Transgenic plants of Agrostis alba obtained by electroporation-mediated direct gene transfer into protoplasts

A system for genetic transformation of Agrostis alba plants by electroporation-mediated DNA transfer to protoplasts is described. The npt II gene was used as a selectable marker. Selection with 20 mg/1 G418 (geneticin) yielded a total of over 50 resistant cell colonies from three independent experiments. Overall frequency of resistant colony formation was 1–3 × 10^–6 based on the number of protoplasts plated and 1–2 × 10^–5 based on the number of cell colonies recovered. Subsequent subcultures led to the development of plants with an apparently normal morphology. DNA analysis (PCR and Southern hybridization) and enzymatic analysis showed that the G418 resistant plants carried the transgene and expressed it. This is the first successful genetic transformation of an economically important temperate grass, Agrostis.     

The basal permeability to water of human red blood cells evaluated by a nuclear magnetic resonance technique

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes by a doping nuclear magnetic resonance technique. In order to estimate the basal permeability the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzene sulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 0.7×10^–3 cm s^–1 at 10°C, 1.2×10^–3 cm s^–1 at 15°C, 1.4×10^–3 cm s^–1 at 20°C, 1.8×10^–3 cm s^–1 at 25°C, 2.1×10^–3 cm s^–1 at 30°C and 3.5×10^–3 cm s^–1 at 37°C. The mean value of the activation energy of water diffusion (E_a,d) was 25 kJ/mol for control and 43.7 kJ/mol for PCMBS-inhibited erythrocytes. The values of P and E_a,d obtained after induction of maximal inhibition of water diffusion by PCMBS can be taken as references for the basal permeability to water of the human red blood cell membrane.     

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in -minimal essential medium containing either vehicle, genistein (10^–7–10^–5 M) or daidzein (10^–7–10^–5 M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [³H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein (10^–5 M) or daidzein (10^–5 M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10^–7 M) or cycloheximide (10^–6 M) in the absence or presence of isoflavones. Moreover, when genistein (10^–7–10^–5 M) or daidzein (10^–6 and 10^–5 M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10^–7 M). In addition, [³H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10^–6 and 10^–5 M) or daidzein (10^–5 M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.     

Glucagon control of cyclic AMP accumulation in isolated intact rat liver parenchymal cells in vitro

Cyclic AMP levels were measured in suspensions of isolated rat liver parenchymal cells during incubation in vitro. Glucagon caused a rapid elevation of cyclic AMP content. With 1.4·10⁻⁶ M (5 μg/ml) of the hormone the levels increased about 10-fold during the first minute, thereafter the elevation was less rapid. Maximal values were reached at 5–10 min. Theophylline slightly increased the basal cyclic AMP levels, and markedly augmented the response to glucagon. Teh major part of the cyclic AMP was located within cells, but a siginificant fraction was present in the incubation medium, and the relative amount present extracellularly increased with incubation time. Significant elevation of the cyclic AMP levels was produced by glucagon 1.4·10⁻¹⁰M, and half-maximal stimulation occured at about 2·10⁻⁹ M. The initial rate of cyclic AMP accumulation was such more rapid in the parenchymal cells than in liver slices, and the maximal levels obtained were about 3 times higher (comparisons based on the finding that 1 mg liver tissue contains about 10⁵ parenchymal cells). It is concluded that preparations of parenchymal liver cells are useful in the study of glucagon actions on liver tissue.     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

Localization of carbonic anhydrase in the rat lung

Summary The localization of carbonic anhydrase in the rat lung has been demonstrated, at light and electron microscopic levels, by the cobalt bicarbonate histochemical method of Hansson. Focal deposits of the cobalt sulfide reaction product were found not only in the capillary endothelium of the alveolar walls, but also in the small and large alveolar cells. The histochemical reaction was abolished by two potent inhibitors, acetazolamide (10^–5 to 10^–6 M) and KCNO (5×10^–3 to 10×10^–3 M). Physiological assay with Maren's method indicated that values for carbonic anhydrase activity in rat lung are 4.4±0.8 UA/mg of protein, 25.0±5.5 UA/mg of nitrogen, and 369±86 UA/g of wet weight. In addition, it was calculated that after fixation in glutaraldehyde-formaldehyde-picric acid about 9% activity is retained.     

Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function

The effect of zinc compounds on osteoclast-like cell formation in rat marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing hormone (1, 25-dihydroxyvitamin D₃ and parathyroid hormone [1–34]). Osteoclast-like cell formation was estimated by staining for tartrateresistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D₃ (10^–8 M) or parathyroid hormone (PTH; 10^–8 M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10^–7 to 10^–5 M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10^–6 M) did not have an effect on PTH (10^–8 M)-induced osteoclast-like cell formation in the presence of EGTA (5 × 10^–4 M), dibucaine (10^–5 M) or staurosporine (10^–9 M). Moreover, when osteoclasts isolated from rat femoraldiaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10^–7 to 10^–5 M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and -glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.     

Lysis by interleukin 2-stimulated tumor-infiltrating lymphocytes of autologous and allogeneic tumor target cells

Summary Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70–75% large granular lymphocytes with 10–13 times greater NK activity) at a flow rate of 38–44 ml/min using a fixed rotor speed of 3000 rpm at 27° C. However, the mean cell recovery was <1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19⁺ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19⁺ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80±15% (n=10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1–4% of total MNC, but their subsequent expansion was at least 10–22-fold during 8–14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 × 10⁹ normal MNC, obtained by leukapheresis, yielded 5 × 10⁸ A-LAK cells with a total of 5.7 × 10⁵ lytic units of cytotoxicity against K562 and a total of 3.3 × 10⁵ lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer.     

Effects of epinephrine, glucagon and vasoactive intestinal polypeptide on chloride secretion by teleost opercular membrane

Summary The effects of epinephrine, glucagon and vasoactive intestinal polypeptide on chloride secretion by chloride cell-containing isolated opercular membranes from the seawater-adapted euryhaline teleost, the tilapiaSarotherodon mossambicus, have been examined. Epinephrine inhibits chloride secretion, measured as the short-circuit current (I _sc), via -receptors, in a dose-dependent fashion. The minimum effective dose is 10^–9 M, ED₅₀ equals 2×10^–7 M and maximal inhibition at 10^–5 M is nearly 80%. Inhibition of phosphodiesterase by isobutylmethylxanthine (IBMX; 10^–4 M), does not alterI _sc in untreated tissues, but it completely reverses the epinephrine inhibition ofI _sc, suggesting that hormones which modulate cAMP in chloride cells may alter chloride secretion. Glucagon and vasoactive intestinal polypeptide also stimulateI _sc in epinephrine-inhibited tissues, an effect potentiated by IBMX. The effect of glucagon is dose-dependent with a minimum effective dose of 10^–9 M, ED₅₀ equal to 8×10^–8 M and a maximum stimulation of 72% at 10^–5 M.Analysis of the effects of epinephrine and IBMX onI _sc and tissue conductance suggests that these agents act antagonistically on a nonconductive transport mechanism. It is proposed that IBMX and hormones which increase intracellular cAMP levels stimulate chloride secretion in epinephrine-inhibited tissues by stimulating a neutral sodium chloride cellular entry-step mechanism.Abbreviations ED ₅₀ effective dose causing half-maximal inhibition or stimulation - IBMX isobutylmethylxanthine - VIP vasoactive intestinal polypeptide     

Growth and uptake kinetics of a facultatively oligotrophic bacterium at low nutrient concentrations

In oligotrophic waters, not only community structure but also physiological properties of heterotrophic bacteria are influenced by the concentration of organic matter.The relationship between growth rate of two facultatively oligotrophic strains ofAeromonas sp. No. 6 andFlavobacterium sp. M1 was studied in comparison with that of two eutrophic strains ofEscherichia coli 7020 andFlavobacterium sp. M2. These strains had two or three different substrate constants (Ks values) depending on substrate concentrations: Ks values for the two former were remarkably lower than those for the two latter. For instance, Ks value forAeromonas sp. No. 6 was about 8.9M when substrate concentration was greater than 53M and about 1.1M when substrate concentration was less man 53M. InE. coli the Ks value was about 260M at greater than 5600M and about 47M at less than 5600M substrate concentration.Uptake kinetics ofAeromonas sp. grown in a medium containing 2.7 mM glutamate (H-cell) and 0.11M glutamate (L-cell) have been determined for the intact cells. H-cell had two distinct values of Km for glutamate assimilation and respiration, and L-cell had three distinct values of Km for glutamate assimilation and respiration: In H-cell Km of assimilation was 2.8×10^–7 M and 1.5×10^–4 M, and Km of respiration was 2.3×10^–7 M and 1.7×10^–4 M; in L-cell Km of assimilation was 7.4×10^–8 M, 8.3×10^–6 M, and 1.3×10^–4 M, and Km of respiration was 2.5×10^–7, 8.9×10^–6M, and 1.7×10^–4 M. More than 60% of glutamate taken up by the H- and L-cells was respired when the substrate concentration was less than 10^–6 M, although at greater than 10^–6 M, 50% and 30% of glutamate was respired by H-cells and L-cells, respectively. These results suggest that the facultatively oligotrophic bacteria grow with high efficiency in environments with extremely low nutrient concentration, such as oligotrophic waters of lakes and ocean, as compared with in their growth in conditions of high nutrient concentraton, such as nutrient broth.     

Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.     

The uptake and incorporation of leucine and cystine by the mycelial and yeastlike phases of Blastomyces dermatitidis

Uptake and incorporation of L-leucine-C¹⁴ and L-cystine-S³⁵ was studied in the mycelial [MP] and yeastlike [YP] phases of the dimorphic fungal pathogen,Blastomyces dermatitidis. Both amino acids entered the cells of the two morphological forms ofB. dermatitidis by a permease-like system at low external concentrations of substrate. At high substrate levels, the amino acids entered the cells by a simple diffusion-like process in addition to the permease-like system. Michaelis-Menten constants [K_m] for L-leucine was found to be 1.1×10^–5 M and 4.4×10^–5 M for the MP and YP phases, respectively. The K_m for L-cystine was found to be 1.0×10^–5 M for the MP and 0.5×10^–5 M for the YP. A requirement for energy supplied by metabolic activity was demonstrated by the inhibition of uptake and incorporation of the amino acids by cells incubated with either 2,4-dinitrophenol or sodium azide. Amino acid uptake was broadly tolerant of hydrogen ion concentration, but definite optima were demonstrated at pH 7.0 to 7.5.     

Effect of β-alanyl-L-histidinato zinc on protein components in osteoblastic MC3T3-El cells: Increase in osteocalcin,insulin-like growth factor-I and transforming growth factor-β

The effect of -alanyl-L-histidinato zinc (AHZ) on protein components in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 3 days at 37°C in CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further 3 or 6 days. The homgenate of cells was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of AHZ (10^–7 to 10^–5 M) caused an appreciable increase of many protein components in cells. Especially, the 67 killo-dalton (kDa) and 44 kDa proteins which are the major components from control cells were clearly increased by the presence of AHZ. Furthermore, the concentrations of osteocalcin, insulin-like growth factor-I and transforming growth factor- in the culture medium secreted from osteoblastic cells were markedly increased by the presence of AHZ (10^–6 and 10^–5 M). The effect of AHZ was a greater than that of zinc sulfate (10^–6 and 10^–5 M). The present findings suggest that AHZ can increase many proteins which are involved in the stimulation of bone formation and cell proliferation in osteoblastic cells.     

Examining the relationship between bacteria and heterotrophic nanoflagellates in Funka Bay (Japan) using the size-fractionation method

The chemical and biological conditions, and the bacteria-heterotrophic nanoflagellate (HNF) relationship were investigated in the vicinity of Funka Bay, southwest of Hokkaido, Japan during early spring 1999. At the time of sampling, chlorophyll a concentration, bacteria, phycoerythrin rich-cyanobacteria, and HNF abundance were in the following ranges: 0.3–3.6 g l^–1, 2.5–5.6 × 10⁵ cells ml^–1, 0.6–1.2 × 10³ cells ml^–1, and 2.2–4.2 × 10³ cells ml^–1, respectively. Dissolved inorganic nitrogen, phosphate and silicate concentrations were in the ranges: 8.7–12.2 M, 0.9–2.0 M, and 21.6–25.5 M, respectively. Primary production ranged from 6.4 to 76.3 mg C m^–3 d^–1. Using water samples from regions of different productivity levels (in and outside bay), the bacteria - HNF relationship was uncoupled experimentally by the size-fractionation technique. Higher primary production (19.9 mg C m^–3 d^–1) in the bay supported higher bacterial growth rate (0.029 h^–1). However, outside the bay both primary production (6.4 mg C m^–3 d^–1) and bacterial growth rate (0.007 h^–1) were lower. The HNF growth rates and grazing rates were similar for both but by comparing both HNF grazing capacity and bacterial production, there was net decrease in bacterial abundance outside the bay and net increase inside the bay. The microbial parameters (rates and abundance) and the amount of carbon flow estimated through the phytoplankton – dissolved organic matter (DOM) – bacteria loop were different between the coastal station and the open ocean station. However HNF grazing and growth rates was similar for both stations.     

Effect of β-alanyl-L-histidinato zinc on differentiation of osteoblastic MC3T3-El cells: Increases in alkaline phosphatase activity and protein concentration

The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10^–7–10^–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10^–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10^–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10^–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.