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.     

Plant regeneration from stem cortex protoplasts of Brassica napus

Cell layer strips composed of the epidermis and 7–9 layers of subepidermal cells were isolated from the 3–4 terminal internodes of Brassica napus cv Westar plants at the early flowering stage. The strips were precultured for one day in modified liquid MS [11] medium and subsequently incubated for 17–18 h in a 0.4 M mannitol solution containing 1% Macerozyme and 1% Cellulase Onozuka R-10. Protoplast yield was 2–2.8×10⁶ per 1.0g of tissue. Protoplasts were cultured at 1×10⁵/ml in three different media: S₁ [13], B [12] and L[8]. The first cell divisions occurred after 2–8 days of culture at frequencies of 20–54%. The highest growth rate of colonies was obtained in L medium containing 0.4 M sucrose and 2% Ficoll. After 4 weeks, green calli, 1–2 mm in diameter were transferred onto B₅ [2] medium with 3 mgl^-1 zeatin, 1% sucrose, 0.1 M mannitol and 0.5% agarose for shoot regeneration. Up to 20% of the calli regenerated shoots which subsequently were rooted and established in soil in the greenhouse.     

Inhibition of N2 fixation by white clover (Trifolium repens L.) at low concentrations of NO inf3 sup− in flowing solution culture

The impact of sustained low external concentrations of NO ₃ ^– (0, 10, 100 and 1000 mmol m^–3) on plant growth and the relative acquisition of N through N₂ fixation and NO ₃ ^– uptake by established, nodulated white clover (Trifolium repens L. cv. Blanca) was studied over 28 days in flowing solution culture. Nitrogen fixation was measured by N difference and ¹⁵N dilution methods. Plants supplied with NO ₃ ^– achieved higher relative growth rates (% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGabmiEayaara% aaaa!3702!\[\bar x\]=0.091 d^–1) compared with control plants dependent on N₂ fixation (0.073 d^–1). Nitrate plants showed progressive increases in shoot: root d.w. ratios from 4 to 6.5–7.6 between days 0–28, compared with 5.1 on day 28 for control plants. Increases in both nodule d.w. and numbers per plant were inhibited after day seven at all concentrations of NO ₃ ^– . The severity of inhibition of N₂ fixation increased with increasing NO ₃ ^– concentration and with time. The total amounts of N₂ fixed per plant between days 0–7 after supplying 10, 100 and 1000 mmol m^–3 NO ₃ ^– , respectively, were 37–39, 28–30 and 0–13%, of the total N acquired. Between days 7–28 the proportional contributions of N₂ fixation to total N acquisition declined to 3, 0.5 and 0%, respectively, in these treatments. The corresponding mean specific rates of N₂ fixation between days 0–7 were, respectively, 5.4, 3.2, and 2.0 mmol N d^–1 g^–1 nodule d.w., compared with 7.9 mmol N d^–1 g^–1 nodule d.w. for zero NO ₃ ^– plants. There was no evidence of a transitory increase in N₂ fixation following the addition of NO ₃ ^– , even at the lowest supply concentration.     

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.     

Stimulation of potato tuber respiration by cold stress is associated with an increased capacity of both plant uncoupling mitochondrial protein (PUMP) and alternative oxidase

The CO₂ evolution of intact potato tubers (Solanum tuberosum, L., var. Bintje) was analyzed during a 10-day period of their warm (25 ± 2°C) or cold (5 ± 1°C) storage, to evaluate cold-stress effects on expression and activities of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX). CO₂ evolution rates were analyzed at 20°C, to reflect their possible capacities. The 20°C CO₂ production declined from 13 to 8 mg kg^–1 h^–1 after 2 days of warm storage and then (after 3 to 7 days) decreased from 8 to 6.5 mg kg^–1 h^–1. In contrast, 20°C CO₂ evolution did not change after the first day of cold storage, increased up to 14.5 mg kg^–1 h^–1 after 2 days, and decreased to about 12 mg kg^–1 h^–1 after 3 to 7 days of cold storage. Cold storage increased PUMP expression as detected by Western blots and led to elevated capacities of both PUMP (44%) and CN-resistant AOX (10 times), but not the cytochrome pathway. Since we found that cold storage led to about the same mitochondrial respiration of 40 nmol O₂ min^–1 mg^–1 attributable to each of the respective proteins, we conclude that both AOX and PUMP equally contribute to adaptation of potato tubers to cold.     

The use of quantitative cytochemical analyses in rapid prenatal detection and somatic cell genetic studies of metabolic diseases

Synopsis The biochemical analysis of enzyme activities in cultured human cells has become an increasingly important tool in the (prenatal) diagnosis of inborn errors of metabolism and in fundamental studies of somatic cell genetics and metabolic interaction of normal and mutant human cells. In all these fields the use of microchemical analyses offers new possibilities in the assay of enzymes in small numbers or even single cultured cells.When cell cultivation is carried out in dishes with a thin plastic foil at the bottom, groups of a few hundred cells and single cells can be sampled after quick freezing and freeze-drying of the culture. Incubation of this material in (sub)microlitre volumes ofp-nitrophenyl or methylumbelliferyl substrates, followed by extinction or fluorescence measurements in microdroplets using a microspectrofluorometer enables lysosomal enzyme activities in the range of 10^–11–10^–14 mol/hr/cell to be detected.Application of these techniques in assays of -1,4-glucosidase and -galactosidase on groups of 100–300 freeze-dried cultured amniotic fluid cells have enabled the prenatal diagnosis of glycogenosis-II and Fabry's disease respectively within 10–12 days after amniocentesis in the 14–15th week of pregnancy. This is a considerable reduction in the time interval hitherto required, 4–6 weeks, for the prenatal diagnosis of metabolic diseases.Examples of application of enzyme assays on single cultured cells are presented for studies on the interaction between enzyme-deficient and normal cultured cells and for genetic complementation studies after hybridization of two human cell strains. By using Lowry's principle of NADP(H) cycling, glucose-6-phosphate dehydrogenase (G-6-PD) activity could be reliably measured in single binuclear hybrid cells after fusion of normal and G-6-PD deficient human cell strains. With the same technique we were also able to detect two cell populations (normal and G-6-PD-deficient) within one culture of a heterozygous carrier for G-6-PD deficiency without the need of elaborate cell cloning techniques.Finally, a microfluorometric assay procedure was developed to enable certain lysosomal enzyme activities to be measured in single (hybrid) human cells. An example of this application is presented for -galactosidase activity measurements in single cells from a mixed culture of normal fibroblasts and cells from a patient with GM1-gangliosidosis (-galactosidase-deficient). In none of our experiments was any evidence obtained for the metabolic correction of enzyme-deficient cells by the uptake of enzyme from normal cells.     

Levels of glutamate,aspartate, GABA,and taurine in different regions of the cerebellum after X-irradiation-induced neuronal loss

The levels of glutamate (Glu), aspartate (Asp), -amino-n-butyric acid (GABA), and taurine (Tau) were determined in the cortex, molecular layer, and deep nuclei of cerebella of adult rats exposed to X-irradiation at 12–15 days following birth (to prevent the acquisition of late-forming granule cells; 12–15x group) and 8–15 days following birth (to prevent the acquisition of granule and stellate cells; 8–15x group). Also, the levels of the four amino acids were measured in the crude synaptosomal fraction (P₂) isolated from the whole cerebella of the control, 12–15x, and 8–15x groups. The level of Glu was significantly decreased by (1) 6–20% in the cerebellar cortex; (2) 15–20% in the molecular layer; and (3) 25–50% in the P₂ fraction of the X-irradiated groups relative to control values. The content of Glu in the deep nuclei was not changed by X-irradiation treatment. Regional levels of Asp were unchanged by X-irradiation, while its level in P₂ decreased by 15–30% after treatment. The levels of GABA and Tau in the molecular layer, deep nuclei, or P₂ were not changed in the experimental groups. However, there was a 15% increase in the levels of GABA and Tau in the cerebellar cortex of the 8–15x group relative to control values. The data support the proposed role of glutamate as the excitatory transmitter released from the cerebellar granule cells but are inconclusive regarding a transmitter role for either Tau or GABA from cerebellar stellate cells.     

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.     

Seasonal variation in the microbial contamination of game carcasses in an Austrian hunting area

In 2003, 50 game carcasses (ungulates) originating from one Austrian hunting ground were subject to visual examination for (fecal) contamination of the body cavities and microbiological testing of the body cavities in order to assess variations in microbial surface contamination in the season June–August compared to October–December. No carcass tested positive for the bacterial pathogens Salmonella or Listeria. Bacterial surface counts in October–December (median values: total aerobic count: 4.12 log₁₀ colony-forming-units (cfu)/cm²; Enterobacteriaceae: 2.48 log₁₀ cfu/cm²) were significantly lower than those in June–August (median values: total aerobic count: 5.65 log₁₀ cfu/cm²; Enterobacteriaceae: 3.45 log₁₀ cfu/cm²). The cooling regime (0.4 °C, 62% relative humidity) allowed no microbial growth for 96 h but was associated with weight loss of the carcasses. All carcasses had undergone a precooling phase of 8–12 h, with temperatures of 17.8±1.2 °C in the season June–August and 9.8±1.2 °C in October–December. This temperature difference was identified as the most probable effector for the observed seasonal variation. The results demonstrate the need for a continuous cool chain after evisceration of game carcasses.     

Comparison of cyclopropene, 1-methylcyclopropene, and 3,3-dimethylcyclopropene as ethylene antagonists in plants

A comparison has been made of cyclopropene (CP), 1-methylcyclopropene (1-MCP), and 3,3-dimethyl-cyclopropene (3,3-DMCP) in their ability to protect plants against ethylene. In bananas, both CP and 1-MCP are effective around 0.5 nL L^–1, and 3,3-DMCP was effective at 1 L L^–1. Bananas treated with CP and 1-MCP again become sensitive to ethylene at 12 days and those treated with 3,3-DMCP at 7 days. Mature green tomatoes are protected by 5–7 nL L^–1 of 1-MPC for 8 days at 25°C and tomatoes treated with 3,3-DMCP at 5–10 L L^–1 are protected for 5 days. Carnation flowers are protected with CP or 1-MCP after exposure to 0.5 nL L^–1 for 24 hours and by 1 L L^–1 of 3,3-DMCP. The display life of Campanula flowers is increased from 3.3 to 5.4 days by 10 L L^–1 of 3,3-DMCP and to 9 days by 20 nL L^–1 of 1-MCP. Ethylene inhibition of pea seedlings is reduced by treatment with 1-MCP at 10 L L^–1 of ethylene but as ethylene is increased to 3000 L L^–1 growth inhibition increases. 3,3-DMCP treatment causes very little reduction of the ethylene effect even at very low concentrations.     

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.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)₃AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l^–1 thidiazuron, 0.5 mg l^–1 -naphthaleneacetic, 10 mg l^–1 kanamycin (Km), and 250 mg l^–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m^–2 s^–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.     

Temperature conditions enhancing resting egg production of the euryhaline rotifer Brachionus plicatilis O. F. Müller (Kamiura strain)

We examined the effect of low temperature treatment(12°C), followed by transfer to highertemperature (25°C), on resting egg formation ofthe rotifer Brachionus plicatilis, Kamiurastrain. This strain has been mass cultured as livefeed at Kamiura Station (Japan Sea FarmingAssociation) for 9 years at 20°C without theappearance of sexual reproductive stages.After preculture in 20 l of 27 seawater at 12°C for 0, 10, 20, and 30 days,rotifers were inoculated into 0.5 l mass cultures andcultured at 25°C for 7–9 days. The inoculationdensities were changed from 20 to 400 ind. ml^–1,depending on mixis rate. Condensed and frozen Nannochloropsis oculata was fed to rotifers at thefeeding rate of 0.14 µg (dry weight)rotifer^–1day^–1. The control was cultured at12°C for the entire 36 day experiment. No mixisappeared and no resting eggs were produced when thelow temperature treatment was 0 or 10 days. However,mixis rates reached 50-60% after 20 or 30 days ofexposure to 12°C. The number of resting eggsproduced in these treatments reached 25,500 about 13 times higher than the control. Our resultssuggest that low temperature stimulated mictic femaleproduction and the transfer to the high temperatureaccelerated resting egg formation. This method may beuseful for producing resting eggs of rotifer strainsthat lack sexual reproduction in the common culturecondition at larval rearing facilities.     

The intraperitoneal delivery of radiolabeled monoclonal antibodies: studies on the regional delivery advantage

Summary The i.p. delivery of murine monoclonal antibody was compared with i.v. delivery in normal mice and rats, in normal nude mice and in those with i.p. human ovarian carcinoma xenografts. In normal rats, all classes of antibodies and antibody fragments evaluated were cleared from the peritoneal cavity at comparable rates. The regional delivery (Rd¹) advantage to the peritoneal cavity following i.p. delivery was thus most dependent on the rate of clearance of the antibody or fragment from the blood stream. Determining the exact i.p. delivery advantage was problematic due to the difficulty in reliably obtaining peritoneal fluid later than 9–10 h after i.p. injection in normal animals. During the first 9 h following i.p. injection, the Rd(0–9/0–9) was, for a murine IgG2ak Fab>F(ab)₂>IgG (at 13.6>10>7.9). Two murine IgMs evaluated differed in Rd(0–9) at 27.1 and 9.2 respectively. When blood levels were extrapolated to infinity, these Rd (0–9/) values were considerably lower with the Fab having the highest Rd at 4.67. The i.p. Rd advantage was almost solely due to the i.p. antibody levels seen in the first 24 h after injection, as after that time, blood levels become comparable to those seen following i.v. injection. Normal tissues obtained at sacrifice 5–7 days after i.p. injection. Normal tissues obtained at sacrifice 5–7 days after i.p. or i.v. injection in rats showed comparable levels of radioantibody activity, whether the injection was i.p. or i.v. (except for higher diaphragmatic levels following i.p. delivery). In nude mice with i.p. human-derived ovarian tumors, intact IgG clearance from the peritoneal cavity to the blood was considerably slower than in normal animals, and early i.p. tumor uptake of specific antibody was significantly higher than that following i.v. antibody delivery. With higher early tumor uptake and lower systemic exposure, early tumor/nontumor ratios were significantly greater than those for i.v. delivery, though not beyond 48 h after i.p. injection. This study demonstrates the pharmacokinetic rationale for i.p. monoclonal antibody delivery, especially for agents cleared rapidly from the blood, such as antibody fragments. In addition, definite i.p. delivery benefit for antibody specific to i.p. tumors in the i.p. ovarian cancer system was shown soon after injection. These data regarding i.p. antibody delivery should be useful in rationally planning diagnostic and therapeutic studies involving the i.p. delivery of unmodified and immunoconjugated monoclonal antibodies.Rd is area under the curve (AUC) for peritoneal fluid activity/AUC for blood radioactivity. Rd (0–9/0–9) is the Rd measured from 0 to 9 h for both peritoneal fluid and blood. Rd (0–9/) is the conservative estimate of Rd with i.p. fluid AUC measured to 9 h, with blood levels extrapolated to infinity. Rd₂ is Rd/(AUC i.p. fluid (0–9)/AUC blood (0–9)) after i.v. injection.     

Lipolytic and adenyl-cyclase-stimulating activity of glucagon1–6: comparison with glucagon derivatives chemically modified in the 7–29 sequence

Glucagon_1–6 has a maximum lipolytic activity (L_max) in the rat adipocyte which is 66% of that of glucagon. The N-guanidyl derivative, modified at Lys₁₂ , has about the same L_max as glucagon_1–6. Modifying the carboxyl groups of glucagon with glycinamide or removing the COON-terminal residues with cyanogen bromide reduces L_max to less than 25% of the level of glucagon. The potency of each of these analogs (A₅₀) in M is as follows: glucagon 6×10^–3; glucagon^1–6 2 ×10^–2; N-guanidyl glucagon 9×10^–3; glycinamide glucagon 10^–2; cyanogen bromide peptide of glucagon 2 ×10^–1. The ability of all of the glucagon analogs to stimulate adenyl cyclase was somewhat less than their tipolytic activities with the exception of the glycin-amide derivative and the cyanogen bromide peptide, which were slightly more active in stimulating adenyl cyclase than in lipolysis. Glucagon_1–6 is much more potent in stimulating adipocyte than liver adenyl cyclase.     

β-Adrenoreceptors of multiple affinities in a clonal capillary endothelial cell line and its functional implication

-Adrenoreceptor has been studied in a clonal capillary endothelial cell line established from the vascular bed of the bovine adrenal medulla. [³H]Dihydroalprenolol ([³H]DHA) binding to the isolated plasma membranes from these cells has demonstrated the presence of -adrenoreceptors with two different affinities. the dissociation constants (K_d) have been found to be 0.27±0.09×10^–9 M and 2.96±0.31×10^–9 M, respectively with the corresponding B_max of 5.1±0.05 and 70.0±0.2 pmol/mg protein, respectively. Inhibition of [³H]DHA binding to the -receptor by atenolol (a ₁-antagonist) and ICI 118,551 (a ₁-antagonist) has suggested that the IC_50cor (=K_i) for atenolol and ICI 118,551 for high affinity site are 0.08±0.03×10^–12 M and 0.25±0.08×10^–12 M, respectively. This, therefore, indicates that both atenolol and ICI 118,551 are able to displace the bound ligand effectively but the ₁-selective antagonist atenolol is 3 times more potent than its ₂ counterpart, ICI 118,551. Displacement of [³H]DHA binding to the endothelial cell plasma membrane by the agonists isoproterenol, epinephrine and norephinephrine has established a relative order of K_i for these agents as isoproterenol (0.56±0.19×10^–9 M)–9 M)>-norepinephrine (0.71±0.24×10^–9 M) for the high affinity site. The corresponding values for the low affinity site, however, are 4.62±0.64×10^–9 M, 6.21±0.86×10^–9 M and 5.90±0.82×10^–9 M, respectively for the same agonists. Increased intracellular cAMP accompanied with cellular proliferation in the presence of isoproterenol has suggested not only the coupling of -adrenoreceptors to the adenylate cyclase system but also its involvement in endothelial cell proliferation.Abbreviations DHA Dihydroalprenolol - cAMP 3:5 cyclic adenosine monophosphate - DTT dithiothreitol - MEM minimal essential medium - 8Br-cAMP 8-bromo-adenosine 3:5 cyclic monophosphate     

Effect of water stress on subsequent uptake of chloride by wheat plants

Summary Wheat plants that were grown for 30 days in flowing nutrient solution were transferred to CaSO₄ solution, and water stress was developed by increasing the evaporative demand on the tops and decreasing the amount of the root system in the solution. The stress was maintained for 3 or 9 h. Uptake of³⁶Cl by the plants was measured immediately after removal of the stress and at intervals up to 36 h later.The water potential of the leaves ranged from –5 bar in the control to –12 bar in stressed plants. Stressed plants transpired less water after removal of the stress than did unstressed plants.Chloride uptake immediately after the removal of water stress was unaffected by the stress, but when measurements were made 36 h later previously stressed plants absorbed only 2 M h^–1 chloride compared to 7 M h^–1 for unstressed plants.     

Fine root mass in relation to soil N supply in a cool temperate forest

Soil inorganic nitrogen supply and fine root mass in the top layers of mineral soil (0–5 and 5–10cm) were investigated at upper and lower sites of a cool temperate forest where Fagus crenata and Quercus crispula dominate. At both sites, soil inorganic nitrogen supply was greatest in the 0–5cm layer. The predominant forms of soil inorganic nitrogen supply were NH₄⁺-N at the upper site and NO₃^–-N at the lower site. Fine roots were concentrated in the 0–5cm layer at the upper site, but not at the lower site. The form of supplied soil inorganic nitrogen supply can be important in determining the vertical distribution of fine roots.     

Pattern of cadmium accumulation and essential cations during growth of cadmium-tolerant fungi

The present study evaluates the growth response of two strains of filamentous fungi; a Fusarium sp. and Alternaria tenuis, grown on both solid and liquid Czapek Dox medium amended with different concentrations of CdCl₂. Colony extension and the mycelial dry weight of both fungi were significantly inhibited by high concentrations of cadmium. Extended lag phases and low growth rates resulted from cadmium administration. Cadmium drastically affected fungal morphogenesis by the production of stunted sterile thick mycelial filaments of the Fusarium sp. and chains of uncharacterized swellings instead of conidia in A. tenuis. Experiments showed that cadmium accumulation by the Fusarium sp. grown in liquid medium was a concentration dependent, and over the incubation time it displayed a plateau pattern. The cells grown on medium containing 0.25 mmol l^–1 CdCl₂ accumulated up to 89 ± 12 mol Cd (gm dw)^–1 after two days, falling to 29 ± 10 mol Cd (gm dw)^–1 after five days. At 0.5 mmol l^–1 CdCl₂ treatment the maximum cellular cadmium content was 132 ± 14 mol (gm dw)^–1, attained after 3 days, and decreased to 98 ± 9 mol (gm dw)^–1 at the end of the incubation time. There was a simultaneous marked drop in cadmium content and pH of the growth medium during the first few days. The presence of cadmium markedly altered the cellular essential cations; K⁺ and Mg²⁺ being decreased while Na⁺ increased during the growth period. Such findings resulted a reverse pattern of cellular Na⁺/K⁺ ratio for cells grown on cadmium-containing medium in respect to the control treatment. The results are discussed in relation to a further dimension of cadmium effects that might reflect its toxicity, as well as the implication of cadmium extrusion for tolerance during fungal growth.     

Is there a “dopaminergic glial cell”?

Intracellular cAMP increased 9-fold in cerebral hemisphere primary cultures after incubation with dopamine (10^–4M). The effect was dose- and time-dependent (10^–6 M-10^–4M; 2–10 minutes). It was mimicked, to some extent, by the partial agonist apomorphine (10^–5 M-10^–4 M) and antagonized by fluphenazine (10^–5 M-10^–4 M). The elevation of cAMP caused by dopamine was incompletely antagonized by propanolol (10^–5 M-10^–4 M), obviating an interaction with -adrenergic receptors. A -adrenergic effect was antagonized by propranolol but only slightly by fluphenazine. The effect of dopamine on cAMP-level was more pronounced in a subpopulation of the hemisphere culture, i.e. in astroglial cultures from the striatum, 12-fold compared with controls at 10^–4 M. No dopamine stimulated formation of cAMP was found in primary cultures from brain-stem. The results demonstrated some heterogeneity among astroglial cells. The cultures used contained mainly astroglial-like cells, as judged from immnohistochemical localization mainly astroglial-like cells, as judged from immunohistochemical localization of the glial specific proteins S 100 and GFA (-albumin). No mature neurons or oligodendroglial cells have so far been demonstrated in the cultures.