Stimulation of guanylate cyclase and RNA polymerase II activities in HeLa cells and fibroblasts by biotin

HeLa cells cultured in a biotin-deficient medium showed reduced rates of protein synthesis, DNA synthesis and growth. Continuous synthesis is required for the increase in DNA synthesis observed upon addition of biotin to cells cultured in biotin-deficient medium. The addition of biotin to the biotin-deficient culture medium increased the activity of guanylate cyclase in both HeLa cells and fibroblasts. Both cell types cultured in biotin deficient medium showed reduced activity of RNA Polymerase II. The exogenous addition of biotin to the biotin-deficient cell cultures also resulted in increased activity of RNA Polymerase II in HeLa cells and fibroblasts. The maximal response was observed in 4 hours. Significant increase in enzyme activity was observed at 10^–8 M biotin in the culture medium. The growth promoting effect of biotin seems to involve stimulations of cellular guanylate cyclase and RNA Polymerase II activity.     

A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.     

Biotin uptake, utilization, and efflux in normal and biotin-deficient rat hepatocytes.

Biotin uptake, utilization, and efflux were studied in normal and biotin-deficient cultured rat hepatocytes. Biotin-deficient cells accumulate about 16-fold more biotin than do normal cells when incubated with a physiological concentration of biotin for 24 h. This difference is due to the greater amount of protein-bound biotin relative to free biotin in biotin-deficient hepatocytes, and is attributable to the presence of more apocarboxylases in deficient cells. The rate of biotin uptake and the rate of activation of the carboxylases, acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and beta-methylcrotonyl-CoA carboxylase, are proportional to the concentration of exogenous biotin. Increases in carboxylase activities are proportional to the concentration of biotin only at exogenous biotin concentrations of less than 410 nM. Concentrations of 410 nM or more biotin increase carboxylase activities to normal or near normal. Biocytin inhibits biotin uptake at very high concentrations, whereas desthiobiotin and lipoic acid have no effect. Biocytin in the medium results in carboxylase activation either intracellularly or extracellularly by conversion to biotin by biotinidase. Investigation of the efflux of biotin from normal and biotin-deficient cells preincubated with the vitamin showed greater retention of biotin by biotin-deficient cells than by normal cells over 24 h. Retention of free biotin is similar in biotin-deficient and normal cells. The greater amount of biotin retained by biotin-deficient cells is accounted for by the greater amount of bound biotin in these cells. These results suggest that the free and bound biotin pools are independently regulated. The ready loss of free biotin from these cells has implications for the treatment of inherited, biotin-responsive carboxylase deficiencies.     

Biotin uptake in cultured hepatocytes from normal and biotin-deficient rats

Biotin uptake was studied in isolated cultured hepatocytes of normal and biotin-deficient rats. Biotin uptake was temperature-dependent with respect to physical, but not to chemical, processes, proportional to the exogenous biotin concentration in the medium, independent of pH and sodium ion concentrations of the medium, and uneffected by the presence of structural analogues of biotin or metabolic inhibitors in both normal and biotin-deficient hepatocytes. These results suggest that biotin uptake occurs by a passive, nonmediated, non-energy-dependent mechanism in rat hepatocytes.     

Biotin-deficient growth of Bacillus polymyxa

When Bacillus polymyxa, a wild-type biotin auxotroph, is grown in biotin-deficient medium, a retardation of cell division and consequential cell elongation are the initial detectable consequences of limited biotin. Subsequent events in biotin-deficient cells include, in chronological order: inhibition of net ribonucleic acid (RNA) synthesis and a simultaneous arithmetical accumulation of protein; loss of net RNA, deoxyribonucleic acid, and protein synthesis; morphological aberration, death, and lysis. Incorporation studies employing ³²P-phosphate and ¹⁴CO₂ demonstrate an initial selective inhibition of net ribosomal RNA synthesis over that of ribosomal protein or total protein. Biotin could not be replaced by various extracts from which biotin had been removed, nor could osmotic stabilizers be found which could prevent lysis of the culture.     

Interruption of the adipose conversion of 3T3 cells by biotin deficiency: differentiation without triglyceride accumulation

During the adipose conversion of 3T3 cells in medium supplemented with 10% serum, the accumulation of triglyceride depends upon a small molecular weight component in the serum. When this is removed by exhaustive dialysis of the serum, the cells undergo some changes that are part of the adipose conversion, but very little triglyceride accumulates. After the addition of either a serum dialysate or commercial biotin, cellular lipid begins to accumulate rapidly. The dialyzable factor in the serum has numerous chemical properties of biotin.When cells begin the adipose conversion in the absence of biotin, they are unable to increase their rate of acetate incorporation into triglyceride, but they do undergo the same change in shape to spherical, the same increase in activity of cytoplasmic (NAD-linked) glycerophosphate dehydrogenase (300 fold) and the same increase in lipoprotein lipase (>50 fold) as in the presence of biotin. On the other hand, in the biotin-deficient state the activity of glycerophosphate acyltransferase and of malic enzyme rises to only a small fraction of the control level. After the addition of biotin to the biotin-deficient cultures, the activity of both enzymes increases to the control level within 24–48 hr.These results are consistent with the concept that the glycerophosphate dehydrogenase is a primary enzyme in the adipose conversion: its response (measured as the amount of extractable activity) does not depend upon the rate of cellular fatty acid synthesis. Lipoprotein lipase is similarly independent. On the other hand, both malic enzyme and glycerophosphate acyltransferase may be considered as secondary enzymes in the sense that their response during the adipose conversion is linked to the supply of fatty acid.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

Growth and G1 arrest of sarcoma virus trnasformed cells in serum free media.

Rous-sarcoma transformed BHK cells can be continuously cultured in a medium containing Eagle's Minimal Essential Medium, iron and biotin. The rate of cell multiplication increased when serine, or serine plus other non-essential amino-acids were added to the medium. With biotin deleted from the medium there is a reduction in DNA synthesis and most cells are blocked in G1.     

The biotin requirement of human fibroblasts in culture

We have examined the effect of biotin deficiency on the growth, viability, biotin content, and the activities of biotin-dependent and biotin-independent enzymes of human fibroblasts. There was a significant decrease in viability of the biotin-deficient cells even when the medium contained serum lipids. Propionyl CoA carboxylase activity reflected the decreased biotin content of the cells whereas alkaline phosphatase activity was not altered. The inclusion of avidin bound biotin in the growth medium resulted in an increase in biotin content as well as propionyl CoA carboxylase activity over that seen when free biotin was included in the medium. The cells appeared to bind and internalize the avidin-biotin complex by adsorptive pinocytosis. These findings are similar to those demonstrated using HeLa cells.     

Mammalian pyruvate carboxylase: effect of biotin on the synthesis and translocation of apo-enzyme into 3T3-L adipocyte mitochondria

The effect of biotin on the induction (and possible requirement for uptake into mitochondria) of apopyruvate carboxylase has been examined in 3T3-L adipocytes. Cells fed biotin-sufficient medium contained only holoenzyme in mitochondria and no apoenzyme was detected. The amount of apoenzyme elaborated in biotin-deficient 3T3-L adipocytes was comparable to the holopyruvate carboxylase protein found in cells maintained on biotin-sufficient medium. Like the holoenzyme, the apoenzyme was detected exclusively in the mitochondrial fraction of 3T3-L adipocytes. This indicates that the synthesis of apopyruvate carboxylase and its translocation into mitochondria occur independently of the cofactor, biotin.     

Optimisation of culture conditions with respect to biotin requirement for the production of recombinant avidin in Pichia pastoris

Due to its very high affinity to biotin, avidin is one of the most widely exploited proteins in modern biotechnological and biomedical applications. Since biotin is an essential vitamin for the growth of many microorganisms, we examined the effect of biotin deficiency on growth for a recombinant Pichia pastoris strain expressing and secreting a recombinant glycosylated avidin. The results showed that biotin deficiency lowers growth rate and biomass yield for P. pastoris. Substitution of biotin in the medium by the two structurally unrelated compounds, aspartic acid and oleic acid, which do not bind to recombinant avidin was analyzed quantitatively. These two compounds had a growth promoting effect in biotin-deficient medium, but did not replace biotin completely. Indeed, in chemostat culture, wash-out occurred after about six liquid residence times and recombinant avidin productivity was lowered. However, addition of low amounts of biotin (20 microg L(-1) of biotin for a cell density of 8 g L(-1)) resulted in stable chemostat cultures on methanol with the production of recombinant biotin-free avidin. The specific avidin production rate was 22 microg g(-1) h(-1) at a dilution rate of 0.06 h(-1).     

The uncoupling of macromolecular synthesis from cell division in SV3T3 cells by glucocorticoids: The imposition of a G2 block

Through a receptor-mediated process glucocorticosteroids block cell division by 20–45 hours in SV40-transformed 3T3 (SV3T3) mouse fibroblasts growing in a low calf serum (0.30% v/v) medium containing biotin. However, the rate of DNA synthesis, determined at various times after dexamethasone addition by the incorporation of radioactive thymidine into acid-insoluble material, is not inhibited by this steroid as late as 66 hours. A modest decrease is observable by 91 hours. There is also no reduction in the uptake of exogenous thymidine into acid-soluble cellular pools. Similarly, RNA synthesis and the uptake of radioactive uridine are not affected by the glucocorticoid up to 69 hours. Measurements of the amounts of cellular DNA (by the fluorescent dye, 4′,6-diamidino-2-phenylindole) and protein revealed that both macromolecules are present in elevated quantities in steroid-treated cells. (The constancy of the protein content in the nonproliferative stage suggests that protein synthesis and degradation are occurring at equal rates.) If the steroid is removed and fresh 10% calf serum medium added, cell division commences (even if nearly 90% of protein synthesis is inhibited by cycloheximide) as early as 45 minutes later such that by 2 hours the viable cell count increases by as much as 70%. Since the growth curve after recovery resembles a step function, it appears that the cells are partially synchronized by the glucocorticoid. These results demonstrate that the glucocorticoid cytostatic effect in SV3T3 cells is the result of a block not in G₁, as previously thought, but in G₂.     

Synthesis of Mono- and Sesquiterpenoids

The synthesis of isocitrate lyase in Candida tropicalis, the growth of which was stimulated by exogenously added biotin, was released from repression by glucose under biotin-deficient conditions. Biotin deficiency reduced remarkably the levels of biotin-enzymes, pyruvate carboxylase and acetyl-Co A carboxylase, in the glucose-utilizing cells of this yeast. A marked increase in intracellular level of pyruvate was observed in the biotin-deficient cells. Acetyl-CoA-donating compounds, such as pyruvate, acetate and alkanes, stimulated the formation of isocitrate lyase in the yeast regardless of the presence or absence of biotin. On the other hand, malate and succinate did not affect the enzyme synthesis. The isocitrate lyase synthesis under biotin-sufficient conditions was repressed by not only glucose but also glucosamine and 2-deoxyglucose. This repression by glucose was not eliminated by cAMP. The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was also observed in C. albicans and C. guilliermondii growing on glucose.     

Apparent coordination of the biosynthesis of lipids in cultured cells: its relationship to the regulation of the membrane sterol:phospholipid ratio and cell cycling

The coordination of the syntheses of the several cellular lipid classes with one another and with cell cycle control were investigated in proliferating L6 myoblasts and fibroblasts (WI-38 and CEF). Cells cultured in lipid-depleted medium containing one of two inhibitors of hydroxymethylglutaryl-CoA reductase, 25-hydroxycholesterol or compactin, display a rapid, dose-dependent inhibition of cholesterol synthesis. Inhibition of the syntheses of each of the other lipid classes is first apparent after the rate of sterol synthesis is depressed severalfold. 24 h after the addition of the inhibitor, the syntheses of DNA, RNA, and protein also decline. The inhibition of sterol synthesis leads to a threefold reduction in the sterol:phospholipid ratio that parallels the development of proliferative and G1 cell cycle arrests and alterations in cellular morphology. All of these responses are reversed upon reinitiation of cholesterol synthesis or addition of exogenous cholesterol. A comparison of the timing of these responses with respect to the development of the G1 arrest indicates that the primary factor limiting cell cycling is the availability of cholesterol provided either from an exogenous source or by de novo synthesis. The G1 arrest appears to be responsible for the general inhibition of macromolecular synthesis in proliferating cells treated with 25-hydroxycholesterol. In contrast, the apparent coordinated inhibition of lipid synthesis is not a consequence of the G1 arrest but may in fact give rise to it. Sequential inhibition of lipid syntheses is also observed in cycling cells when the synthesis of choline-containing lipids is blocked by choline deprivation and is observed in association with G1 arrests caused by confluence or differentiation. In the nonproliferating cells, the syntheses of lipid and protein do not appear coupled.     

Lipid requirement for cell cycling : The effect of selective inhibition of lipid synthesis

Tissue culture cells require lipid which must be provided exogenously or synthesized via endogenous pathways. The exogenous supplies can be largely removed by growing cells in medium containing delipidized serum. Pathways for synthesis of lipid can then be blocked at three steps: (1) fatty acids by removal of biotin, an essential coenzyme; (2) phosphatidylcholine and sphingomyelin by deleting choline from the growth medium; and (3) cholesterol by inhibiting HMG-CoA reductase with 25-hydroxycholesterol. Sustained proliferation is prevented when lipid synthesis is blocked at any one of these steps. Cell proliferation resumes upon restoring synthesis with biotin, choline, or mevalonate (the product of the HMG-CoA reductase reaction) or by providing the lipid end products oleic acid or cholesterol. Using a combined cytophotometric-autoradiographic analysis to determine cell cycle distributions we have demonstrated that prereplicative (G1) cell cycle arrests develop in parallel with the proliferative inhibition. Each of the G1 arrests can be reversed by restoring the synthetic pathways or their lipid products. These observations suggest a causal relationship between the supply of lipids and passage through G1.     

Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae.