Light-cytokinin interaction in shoot formation in cultured cotyledon explants of radiata pine

Previous studies utilizing cotyledon explants of radiata pine ( Pinus radiata D. Don) revealed that cytokinin was required for shoot formation. This was confirmed and extended in the present study, which also showed that light was required. As little as three days of exposure to 16 h photoperiod light at a photon fluence rate of ca 80 μmol m⁻² s⁻¹ was sufficient to give some meristematic tissue formation. Longer exposure to light increased this formation. While cytokinin must be present during the first three days in culture for shoot formation, the exposure of the cotyledons to light could be delayed at least until day 10, but after 21 days in darkness, transfer to light did not permit shoot formation. Anatomical examination of the cotyledons confirmed the morphogenetic interactions of light and cytokinin in shoot formation. The data suggest that cytokinin is directly involved in the induction of shoot initiation and both light and cytokinin are required for the development of meristematic tissue and subsequent shoot formation.     

Boron uptake by sunflower, squash and cultured tobacco cells

Boron uptake was studied in sunflower ( Helianthus annuus cv. Ha301), squash ( Cucurbita pepo cv. Early prolific straight neck) and cultured tobacco ( Nicotiana tobacum L. cv. TXD Monsanto cell line) cells with the use of stable B isotopes and inductively coupled plasma mass spectrometry. Boron uptake increased linearly with increasing B concentrations in the uptake medium, did not exhibit multiphasic kinetics and was not saturable over a wide concentration range. The addition of respiratory inhibitors to the uptake solution or exposure to low (2°C) or high (42°C) temperatures did not inhibit B uptake. The majority of the B within the plants, including recently absorbed B, was present in a nonexchangeable form and could not be removed by repeated rinsing with deionized water or exchange with B isotope. These results demonstrate that in these species B uptake is a passive, nonmetabolic process and that the formation of nonexchangeable B-complexes within the cytoplasm and cell wall is a key factor in determining the uptake of B by plants.     

Resistance to methyl mercaptopurine riboside in cultured hamster cells: Preliminary characterization of resistant cells and conditions affecting their selection in quantitative mutation studies

Cells resistant to high concentrations of methyl mercaptopurine riboside (MMPR), an analogue of adenosine, were found at a frequency of about 6 × 10⁻⁶ per viable cell in untreated cultures of V79 Chinese hamster cells. Resistant cells were selected less efficiently if purines were added to the MMPR-medium, but high cell densities had little effect upon selection. 6 independently-isolated spontaneous MMPR-resistant sublines were characterized by their resistance to the toxicity of different purines, rate of purine excretion, incorporation of radioactive adenosine, electrophoresis of cell extracts, and expression of resistance in hybrids to an MMPR-sensitive line. 5 of these sublines showed recessive expression of MMPR-resistance in hybrids and had characteristics consistent with loss of adenosine kinase activity, while the remaining subline was much less resistant to MMPR and showed semi-dominant expression of resistance without loss of adenosine kinase activity.Cells with high resistance to MMPR were not found in a “tetraphoid” (hybrid) V79 line or in freshly-isolated human cell cultures, but occurred at a comparable frequency to V79 in another commonly-used aneuploid hamster line, CHO-K1. The frequency of MMPR-resistant cells in V79 cultures was increased to a similar extent by treatment with γ-rays or with ethyl methanesulphonate providing a suitable post-treatment interval was allowed for the expression of resistance. A genetic interpretation of these data is given in which it is proposed that resistance most usually arises through mutation of an autosomally-linked gene of which one copy has been inactivated or lost in V79 and in CHO-K1 cells.In comparison to published data on the selection of “mutants” resistant to 6-thiguanine, it is argued that MMPR could be as useful a selective agent as thioguanine and may select a different range of types of mutagenic event.     

Thymidine-kinase activity of cultured cells from individuals with inherited galactokinase deficiency

Cells of a person homozygous for galactokinase deficiency and of her heterozygous parents were found to be deficient in the enzyme thymidine kinase. The decrease in thymidine-kinase activity may be the result of a qualitative alteration in the enzyme molecule. This is reflected in the apparent alteration in the sensitivity of the enzyme to trifluorothymidine. It is suggested that this relationship between the galactokinase and thymidine kinase is not fortuitous but a reflection of their interdependence as found previously in the Chinese hamster.     

Riboflavin deficiency in cultured rat hepatoma cells: A model for studying the hepatic effects of riboflavin deficiency

Summary The acyl-CoA dehydrogenases are a family of mitochondrial flavoenzymes required for fatty acid beta-oxidation and branched-chain amino acid degradation. The hepatic activity of these enzymes, particularly the short-chain acyl-coenzyme A (CoA) dehydrogenase, is markedly decreased in riboflavin deficient rats. We now report that the in vivo effects of riboflavin deficiency on the beta-oxidation enzymes of this group are reproduced in FAO rat hepatoma cells cultured in riboflavin-deficient medium. Although it has been long known that hepatic short-chain acyl-CoA dehydrogenase activity is the most severely affected of the straight-chain specific enzymes in riboflavin deficiency, the mechanism by which its activity is decreased has not been reported. We have used this new cell culture system to characterize further this mechanism. Whole cell extracts from riboflavin-deficient and control cells were subjected to analysis by denaturing polyacrylamide gel electrophoresis. The contents of the gels were then electroblotted onto nitrocellulose filters and probed with short-chain acyl-CoA dehydrogenase-specific antiserum. The relative abundance of enzyme antigen was estimated autoradiographically. Our findings indicate that short-chain acyl-CoA dehydrogenase activity changes in parallel with its antigen, suggesting that riboflavin deprivation does not affect the activity of individual enzyme molecules. Further, no evidence of extramitochondrial enzyme precursor was found on the blots, making unlikely a significant block in the mitochondrial uptake process. These findings suggest that changes in short-chain acyl-CoA dehydrogenase activity in riboflavin deficiency result from either increased synthesis or decreased degradation of the enzyme. This work was supported by grants from the VA Medical Research Service, the Diabetes Association of Greater Cleveland, and the National Institutes of Health (HD25299), Bethesda, MD. Portions of the work presented here were presented at the 71st meeting of the Endocrine Society, Seattle, WA.     

Selenite or selenomethionine interaction with methylmercury on uptake and toxicity showing a weak selenite protection: studies on cultured K-562 cells

Selenium and methylmercuric chloride (MMC) interactions regarding cellular uptake and selenium protection on MMC toxicity have been studied. Human K-562 cells were pretreated or simultaneously treated with either selenite (5 or 50 μM) or selenomethionine (10 or 50 μM) together with (3.5 or 5 μM) MMC. Cells simultaneously treated with selenite or selenomethionine and 3.5 μM MMC showed a decreased mercury concentration with increased selenium dose especially seen in the selenite combinations. The simultaneous selenite and MMC 3.5 μM combinations showed growth curves with an increasing number of viable cells with increased selenite dose. All combinations with 5 μM MMC were toxic to the cells. Interactions between selenite or selenomethionine and MMC regarding cellular uptake of mercury and selenium were observed and indications of selenite protection against MMC toxicity in human K-562 cells were noticed.     

Regulation of secretion and synthesis of gastrointestinal hormones: studies with cultured gut endocrine cells

Small number of endocrine cells are diffusely distributed in the gut mucosa. Studies on their secretory mechanisms have been further complicated by numerous neural, paracrine, and endocrine factors affecting their response. Recent technical development for isolation and culture of gut endocrine cells has circumvented these problems and enabled to study their receptors, signal transduction mechanism, and biosynthesis of gut hormones. In this review, current progress made in the cellular physiology of gut endocrine cells is summarized.     

Cellular and molecular aspects of thiamin uptake by human liver cells: studies with cultured HepG2 cells

The liver is an important site for thiamin metabolism, utilization, and storage. Little is known about the mechanism of thiamin uptake by the human liver. In this study, we examined cellular and molecular aspects of the human liver thiamin uptake process using the human-derived liver HepG2 cells as a model system. Our studies showed that the initial rate of thiamin uptake to be: (1) Na(+)-independent and occurs with no detectable metabolic alterations in the transported substrate, (2) highly pH-dependent with diminished uptake upon decreasing incubation buffer pH from 8.0 to 5.0, (3) higher following cell acidification compared to unacidified control cells, (4) saturable as a function of concentration with an apparent K(m) of 7.7+/-1.6 microM, (5) inhibited by the thiamin structural analogues oxythiamin and amprolium but not by the unrelated organic cations tetraethylammonium (TEA) and N-methylnicotinamide (NMN), and (6) inhibited in a concentration-dependent manner by the membrane transport inhibitor amiloride. Both of the recently cloned human thiamin transporters, i.e., SLC19A2 and SLC19A3, were found to be expressed in liver HepG2 cells with the former being the predominant form. High promoter activity of the predominant form, i.e., SLC19A2, was detected in HepG2 cells, and the minimal region of the SLC19A2 promoter required for its basal activity in these cells was found to be encoded in a sequence between -356 and -36 and has multiple putative cis-regulatory elements. Mutation of a number of these putative cis-elements diminished promoter activity of the SLC19A2 minimal region. These results show the involvement of a specialized carrier-mediated mechanism for thiamin uptake by human liver HepG2 cells. In addition, SLC19A2 was found to be the predominant thiamin uptake carrier expressed in these cells and its promoter displays a high level of activity in them.     

A simple method for the quantitative evaluation of functional effects of xenobiotics with cultured cells

We present a simple, noninvasive, nondestructive all-purpose method for the quantitative evaluation of functional effects of xenobiotics with cultured cells and the work station for its routine, easy implementation. At present 1 to 150 cells growing in one to six dishes can be studied in parallel or otherwise at time intervals ranging from 10 s to 6 h or more, over periods of time ranging from a few tens of minutes to 3–4 days. Any aspect of cell physiological behavior can be studied (differentiation-dedifferentiation, migration, division, degeneration, death) without preliminary staining and/or fixation provided it results in optically visible changes.     

The interaction of 125I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method

The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when 125I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, 125I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.     

The characterization of lipoproteins in the high density fraction obtained from patients with familial lecithin:cholesterol acyltransferase deficiency and their interaction with cultured human fibroblasts

Lipoproteins of density 1.063--1.21 g/ml were isolated from the plasma of three sisters of Irish origin with familial LCAT deficiency. Fractionation of the lipoproteins on the basis of particle size by chromatography on Sephacryl S-300 permitted partial separation of two major and at least three other minor components which differed in their lipid:protein ratio and their apolipoprotein content. One of the major components was a small spherical lipoprotein whose sole apolipoprotein was apoA-I; the second major component contained predominantly apoA-I, together with apoE, and in addition, an apolipoprotein of molecular weight 46,000 that was not cleaved by reduction of disulfide bonds, and which was identified as apoA-IV. This apoprotein has not previously been detected in the lipoproteins of LCAT-deficient patients. A second apoE-containing lipoprotein, which contained apoA-I and apoE in a ratio of approximately 2:1, was also present as a minor component, together with two or more minor components whose apoproteins were comprised of apoA-I and apoC. The apoE-containing lipoproteins competed efficiently with 125I-labeled LDL for binding to high affinity LDL-receptor sites on the surface of cultured human skin fibroblasts. The ability to bind to the LDL-receptor was directly proportional to the apoE content of the lipoproteins, even when other apoproteins, with the exception of apoB, were present in relatively large proportions. ApoE-containing 125I-labeled lipoproteins from an LCAT-deficient subject were also taken up and degraded by the cultured cells.     

Boron accumulation and partitioning in wheat cultivars with contrasting tolerance to boron deficiency

Two pot experiments at the Plant Environment Laboratory (PEL), Reading, UK investigated sterility, boron (B) accumulation and B partitioning of wheat cultivars grown with limited B in the growing medium. The first experiment evaluated nine cultivars of spring wheat with diverse field responses to low available soil B, supplied with or without 20 μM B. A second experiment examined the response of a susceptible (SW-41) and a tolerant (Fang-60) cultivar to B-deficiency. These cultivars were supplied with either 20 μM B from sowing to flag leaf emergence and no added B thereafter, or 20 μM B from sowing to maturity. When B was not supplied in the nutrient solution, the number of grains ranged from 4 per ear (cv. BL-1135) to 32 per ear (cv. BL-1249) and sterility of competent florets ranged from 39% to 93%. Boron concentration in the flag leaf at anthesis did not differ greatly when the growing medium contained limited B, but differences between cultivars were evident when B was unlimited. Tolerance of B-deficiency was not related to the B concentration in the flag leaf. Some cultivars produced viable pollen and set grains while others failed to do so at similar B concentrations in the flag leaf. The two contrasting cultivars did not differ much in their pattern of B partitioning when B supply was restricted from flag leaf emergence onwards. Similarly, little evidence was found that the tolerant cultivars translocated B from their leaves, roots or stems when the supply in the growing medium was restricted. The proportion of total B partitioned in different organs was the same irrespective of B supply and cultivar. On average, leaves contained 68% of the total B content in the whole plant compared to 16% in the roots, 10% in the ears and only 6% in the stems. Tolerant or susceptible cultivars of wheat could not be distinguished based on the B concentration and B content of the flag leaf. This revised version was published online in June 2006 with corrections to the Cover Date.