A -glucuronidase deficiency mucopolysaccharidosis: studies in cultured fibroblasts

Cytochrome P-450 cannot be detected spectrophotometrically in testis mitochondria of untreated rats because of the high cytochrome a₃ to Cytochrome P-450 ratio. Injection of human chorionic gonadotrophin (HCG) causes a large increase in mitochondrial cytochrome P-450. After 14 days injection, mitochondrial cytochrome P-450 levels are increased 15- to 30-fold (from 0.007 to 0.134 nmoles/mg protein) over control levels. Levels of cytochrome a + a₃ are not altered by this treatment. Mitochondrial cytochrome P-450 can also be demonstrated by injection of HCG into rats which were hypophysectomized 24 days previously. During hypophysectorny the mitochondrial cytochromes c + c_i, a + a₃ and mitochondrial protein decay with halflives of 14, 16, and 15.5 days, respectively. HCG treatment for 8 days increases mitochondrial cytochrome P-450 (from < 0.003 to 0.24 nmoles/mg protein) without altering the levels of the other mitochondrial cytochromes. The control of cytochrome P-450 levels in the mitochondria by HCG suggests that the level of this key component of cholesterol side-chain cleavage enzyme may be of importance in the regulation of steroidogenesis in the testis.     

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 in sunflower,squash and cultured tobacco cells: Studies with stable isotope and ICP-MS

A direct correlation was found between fractional losses of added N and the change in δ¹⁵N‰ during 19 years in an experiment with annual additions of N at three rates to a Scots pine (Pinus sylvestris L.) forest in northern Sweden. This confirms that processes leading to losses of N discriminate against¹⁵N, and opens possibilities to conduct retrospective studies of the N balance in forests.     

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.     

The interaction of dianhydrogalactitol with DNA in cultured Yoshida sarcoma cells

Using alkaline sucrose gradient sedimentation centrifugation it was found that treatment of Yoshida sarcoma cells in culture for 1 h with increasing concentrations of dianhydrogalactitol (DAG) enhanced the sedimentation rate of DNA in a dose-dependent manner. There was no difference between the amount of protein which co-sedimented with DNA released from treated and untreated cells. When DNA was extracted from the cells using a p-amino-salicylate-phenol mixture, the protein content of DNA seemed not to be affected by DAG. The possibility that DAG could form interstrand cross-linking in cellular DNA was suggested from renaturation studies. The appearance of a fast sedimenting DNA in the alkaline sucrose gradient and the evidence for a cross-linked DNA detected by renaturation technique, only appeared later than 6 h after treatment. A similar delayed effect on the depression in the rate of DNA synthesis was also observed. These data suggest that the inhibition of DNA synthesis may be related to the delayed formation of DNA interstrand cross-linked.     

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.     

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.     

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.     

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 between microtubules and intermediate filaments in cultured cells treated with taxol and nocodazole

Using double-label immunofluorescence and electron microscopy we studied the interaction between microtubules (MT) and intermediate filaments (IF) in MO cells treated with various combinations of taxol and nocodazole. With taxol, the organized MT of cultured cells are replaced by free MT and MT bundles. This rearrangement of MT is followed by a rearrangement of the IF. As in untreated cells a close association between these two filamentous systems is observed. In cells pretreated with nocodazole followed by addition of taxol, to induce the bundles of free MT, the preexisting IF coils disappear and IF associate with the MT. From these experiments we conclude that an interaction between MT and IF exists independent of the normal organisation of the MT system. The redistribution of IF always follows the redistribution of MT. The data show that MT determine the spatial distribution of IF which most probably involves some kind of physicochemical link.     

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.