Studies on the mechanism of hormonal induction of alkaline phosphatase in human cell cultures. I. Effects of puromycin and actinomycin D.

Increased alkaline phosphatase activity is induced in certain epithelial cell cultures by hormones with adrenal glucocorticoid activity or their analogues such as prednisolone (Delta(I)-hydrocortisone). Enzyme induction occurs in two distinct phases. During the first 12 hr after the addition of prednisolone, there is a small increase in alkaline phosphatase levels. After 15 to 24 hr, the enzyme activity shows a sudden, marked linear rise, reaching a maximum at 60 to 80 hr. Puromycin blocks enzyme induction immediately, even when added during the period of rapid increase of enzyme. Actinomycin D blocks induction when added no later than 8 hr after the addition of prednisolone. On the other hand, Actinomycin D added during the phase of rapid enzyme induction has no effect for at least 12 hr. These findings suggest that de novo protein synthesis is involved in prednisolone induction of alkaline phosphatase and that the RNA messenger for this enzyme is relatively stable.

Studies on cell communication with enucleated human fibroblasts.

Metabolic cooperation, the correction of the mutant phenotype in cells deficient in hypoxanthine phosphoribosyltransferase (HPRT-) by intimate contact with normal cells (HPRT+), represents a form of cell communication that is easily studied with radioautography. In the present study it was found that the formation of cell junctions needed for communication does not require protein synthesis nor is it under the immediate control of the cell nucleus. Enucleated normal cells efficiently communicate with HPRT- mutant cells. The effectiveness of enucleated cells as donors in metabolic cooperation provides evidence that it is the transfer of small molecules, nucleotide, or nucleotide derivatives that is responsible for correction of the mutant phenotype. Karyoplasts (nuclei with small amounts of cytoplasm surrounded by a plasma membrane) are unable to efficiently communicate with intact cells. The utilization of [3H]hypoxanthine by communicating mixtures of HPRT+ and HPRT- human cells is not significantly different than in the normal cells alone. Metabolic cooperation, as studied involves a redistribution of purine-containing compounds among communicating cells.

Hormonal induction of increased zinc uptake in mammalian cell cultures: requirement for RNA and protein synthesis.

The zinc content of HeLa S(3) cells is markedly increased after growth in medium containing adrenal glucocorticoid hormones. Studies with inhibitors indicate that the synthesis of RNA and protein is required for enhanced zinc uptake. When protein synthesis is blocked in the presence of the steroid, an intermediate, presumably messenger RNA, which specifies enhanced zinc uptake accumulates and is expressed when the inhibition of protein synthesis is removed.

E2 transacylase-deficient (type II) maple syrup urine disease. Aberrant splicing of E2 mRNA caused by internal intronic deletions and association with thiamine-responsive phenotype.

Maple syrup urine disease (MSUD) or branched-chain alpha-ketoaciduria is an autosomally inherited disorder in the catabolism of branched-chain amino acids leucine, isoleucine, and valine. The disease is characterized by severe ketoacidosis, mental retardation, and neurological impairments. MSUD can be classified into genetic subtypes according to the genes of the branched-chain alpha-ketoacid dehydrogenase (BCKD) complex which are affected in patients. We describe here four intronic deletions and an intronic nucleotide substitution in the E2 transacylase gene of type II MSUD, in which the E2 subunit of the BCKD complex is deficient. These new E2 mutations comprise an internal 3.2-kb deletion in intron 4 (causing a 17-bp insertion in mRNA), an internal 12-bp (ttaccttgttac) deletion in intron 4 (creating a 10-bp insertion), a 10-bp (catttctaG) deletion in intron 10/ exon 11 junction (leading to a 21-bp deletion), a 2-bp deletion in the exon 5/intron 5 junction (ATgt--> A-t) (resulting in the skipping of exon 5), and a G to A transition at nucleotide -7 of intron 9 (causing a 6-bp insertion). These intronic mutations were initially detected by secondary alterations in the mutant E2 mRNA, as a result of aberrant splicing. The 3.2-kb deletion in intron 4 was determined by the amplification of the entire intron from both a normal subject (11.2 kb) and a homozygous patient (8 kb) by long PCR, followed by subcloning and sequencing of regions flanking the deletion. Similar methods were used to identify and characterize the other intronic alterations. Our results depict heretofore undescribed splicing errors caused by the deletion of internal intronic segments, and provide an approach for detecting this class of novel and rare human mutation. The association of the thiamine-responsive phenotype with a subset of the type II MSUD patients studied is also discussed.

Familial hyperlysinemia with lysine-ketoglutarate reductase insufficiency.

Fibroblasts grown in tissue culture from the skin of normal subjects have lysine-ketoglutarate reductase activity (lysine: alpha-ketoglutarate: triphosphopyridine nucleotide (TPNH) oxidoreductase (epsilon-N-[L-glutaryl-2]-L-lysine forming)). The activity of the enzyme is considerably reduced in the skin fibroblasts grown from three siblings with hyperlysinemia. The high concentrations of lysine in the blood of these patients, the previous demonstration in the intact subject of a reduction in the ability to degrade lysine, and the present demonstration of diminished lysine-ketoglutarate reductase activity, accurately define the metabolic defect and establish the saccharopine (epsilon-N-[L-glutaryl-2]-L-lysine) pathway as the major degradative pathway for lysine in the human.

Disparate enzyme activity in erythocytes and leukocytes. A variant of hypoxanthine phosphoribosyl-transferase deficiency with an unstable enzyme.

A family is reported in which each of two sisters has a son with no detectable hypoxanthine phosphoribosyltransferase (HPRT) (EC 2. 4. 2. 8) in his erythrocytes, a finding considered pathognomonic of Lesch-Nyhan disease. However, neither has the stigmata of the disease. One boy is neurologically normal, and the other is moderately retarded. There was only a slight increase in urinary uric acid, but the amounts of hypoxanthine and xanthine, and their ratios, were similar to those found in Lesch-Nyhan disease, strongly indicating that excesses of these last two oxypurines are not responsible for the symptomatology in that disease. In contrast to the nondetectable HPRT activity in the red blood cells, leukocyte lysates from the two boys have 10-15% of normal activity, possibly reflecting continuing synthesis of an unstable enzyme. This hypothesis is supported by the demonstration that at 4 degrees C HPRT activity was rapidly lost in the propositus while the activity increased in control subjects. The mother's cells were intermediate between the two. The intact and disrupted leukocytes of the hemizygote, in the absence of added phosphoribosyl converted as much hypoxanthine to inosinate as the normal cell, and appropriate tests indicated that under these circumstances enzyme concentration is not rate limiting whereas the concentration of the cosubstrate, phosphoribosyl pyrophosphate, is. The capacity for normal function in the intact mutant cell is more representative of in vivo conditions than the lysate, which may explain the important modification of clinical symptomatology, the relatively mild hyperuricosuria, and the presence of mosaicism in the circulating blood cells of the heterozygotes. A similar explanation may apply to other genetic diseases in which incomplete but severe enzyme deficiencies are found in clinically normal individuals. An associated deficiency in glucose-6-phosphate dehydrogenase in this family permitted confirmation of previous observations on linkage with hypoxanthine phosphoribosyltransferase.

Hypoxanthine phosphoribosyltransferase activity in intact fibroblasts from patients with X-linked hyperuricemia.

Discordance between clinical phenotype and the level of a mutant enzyme activity may reflect differences between enzyme function in vivo and that measured by the customary enzyme assays on cell extracts. In the present study, the conversion of hypoxanthine to phosphorylated products was measured in intact skin fibroblasts and in cell extracts from seven patients with mutant hypoxanthine-guanine phosphoribosyltransferase (HPRT) and six control subjects. The patient's phenotypes ranged from asymptomatic hyperuricemia to the Lesch-Nyhan syndrome. Although there was a general correlation between the HPRT activity in cell extracts assayed by the usual methods and the function of the purine salvage pathway in patients, as reflected by urinary oxypurine excretion, there were notable exceptions. A more accurate appraisal of the functioning of the pathway at the cellular level is achieved by measuring the conversion of substrate to product in the intact cell at physiological concentrations of substrates, activators, and product and metabolite inhibitors, and in a physiological ionic environment. In one of the seven patients, the standard enzyme assay indicated normal function, whereas measurements in the intact cell exposed severe dysfunction of the salvage system. In another, the standard assay suggested a severe deficiency not evident in the intact cell or in the patient.

Maple syrup urine disease in Mennonites. Evidence that the Y393N mutation in E1 alpha impedes assembly of the E1 component of branched-chain alpha-keto acid dehydrogenase complex.

Maple Syrup Urine Disease (MSUD) in Mennonites is associated with homozygosity for a T to A transversion in the E1 alpha gene of the branched-chain alpha-keto acid dehydrogenase complex. This causes a tyrosine to asparagine substitution at position 393 (Y393N). To assess the functional significance of this missense mutation, we have carried out transfection studies using E1 alpha-deficient MSUD lymphoblasts (Lo) as a host. The level of E1 beta subunit is also greatly reduced in Lo cells. Efficient episomal expression in lymphoblasts was achieved using the EBO vector. The inserts employed were chimeric bovine-human cDNAs which encode mitochondrial import competent E1 alpha subunit precursors. Transfection with normal E1 alpha cDNA into Lo cells restored decarboxylation activity of intact cells. Western blotting showed that both E1 alpha and E1 beta subunits were markedly increased. Introduction of Y393N mutant E1 alpha cDNA failed to produce any measurable decarboxylation activity. Mutant E1 alpha subunit was expressed at a normal level, however, the E1 beta subunit was undetectable. These results provide the first evidence that Y393N mutation is the cause of MSUD. Moreover, this mutation impedes the assembly of E1 alpha with E1 beta into a stable alpha 2 beta 2 structure, resulting in the degradation of the free E1 beta subunit.

Molecular and biochemical basis of intermediate maple syrup urine disease. Occurrence of homozygous G245R and F364C mutations at the E1 alpha locus of Hispanic-Mexican patients.

Maple syrup urine disease (MSUD) is caused by a deficiency of the mitochondrial branched-chain alpha-keta acid dehydrogenase (BCKAD) complex. The multienzyme complex comprises five enzyme components, including the E1 decarboxylase with a heterotetrameric (alpha 2 beta 2) structure. Four unrelated Hispanic-Mexican MSUD patients with the intermediate clinical phenotype were diagnosed 7 to 22 mo after birth during evaluation for developmental delay. Three of the four patients were found homozygous for G to A transition at base 895 (exon 7) of the E1 alpha locus, which changes Gly-245 to Arg (G245R) in that subunit. The remaining patient was homozygous for T to G transversion at base 1,253 in the E1 alpha gene, which converts Phe-364 to Cys (F364C) in the gene product. Transfection studies in E1 alpha-deficient lymphoblasts indicate that both G245R and F364C mutant E1 alpha subunits were unable to significantly reconstitute BCKAD activity. Western blotting showed that both mutant E1 alpha subunits in transfected cells failed to efficiently rescue the normal E1 beta through assembly. The putative assembly defect was confirmed by pulse-chase labeling of E1 subunits in a chaperone-augmented bacterial overexpression system. The kinetics of initial assembly of the G245R E1 alpha subunit with the normal E1 beta was shown to be slower than the normal E1 alpha. No detectable assembly of the F364C E1 alpha with normal E1 beta was observed during the 2 h chase. Small amounts of recombinant mutant E1 proteins were produced after 15 h induction with isopropyl thiogalactoside and exhibited very low or no E1 activity. Our study establishes that G245R and F364C mutations in the E1 alpha subunit disrupt both the E1 heterotetrameric assembly and function of the BCKAD complex. Moreover, the results suggest that the G245R mutant E1 alpha allele may be important in the Hispanic-Mexican population.

Carbon isotope fractionation by anoxygenic phototrophic bacteria in euxinic Lake Cadagno.

Anoxygenic phototrophic bacteria utilize ancient metabolic pathways to link sulfur and iron metabolism to the reduction of CO2 . In meromictic Lake Cadagno, Switzerland, both purple sulfur (PSB) and green sulfur anoxygenic phototrophic bacteria (GSB) dominate the chemocline community and drive the sulfur cycle. PSB and GSB fix carbon utilizing different enzymatic pathways and these fractionate C-isotopes to different extents. Here, these differences in C-isotope fractionation are used to constrain the relative input of various anoxygenic phototrophs to the bulk community C-isotope signal in the chemocline. We sought to determine whether a distinct isotopic signature of GSB and PSB in the chemocline persists in the settling fraction and in the sediment. To answer these questions, we also sought investigated C-isotope fractionation in the water column, settling material, and sediment of Lake Cadagno, compared these values to C-isotope fractionation of isolated anoxygenic phototroph cultures, and took a mass balance approach to investigate relative contributions to the bulk fractionation signature. We found a large C-isotope fractionation between dissolved inorganic carbon (DIC) and particulate organic carbon (POC) in the Lake Cadagno chemocline. This large fractionation between the DIC and POC was also found in culture experiments carried out with anoxygenic phototrophic bacteria isolated from the lake. In the Lake Cadagno chemocline, anoxygenic phototrophic bacteria controlled the bulk C-isotope fractionation, but the influence of GSB and PSB differed with season. Furthermore, the contribution of PSB and GSB to bulk C-isotope fractionation in the chemocline could be traced in the settling fraction and in the sediment. Taken together with other studies, such as lipid biomarker analyzes and investigations of other stratified lakes, these results offer a firmer understanding of diagenetic influences on bacterial biomass.

Modular synthesis of 4-aminocarbonyl substituted 1,8-naphthalimides and application in single molecule fluorescence detection.

Robust methodology to install amide, carbamate, urea and sulfonamide functionality to the 1,8-naphthalimide scaffold has been developed and exemplified. New benzamidonaphthalimide 6, synthesised using this approach, was found to be sensitive to base whereupon fluorescence emission strongly increases (>10-fold) and red-shifts (>4000 cm-1). The optical properties of deprotonated 6 allow for single molecule fluorescence detection, the first example of such behaviour from this class of fluorophore.

The reduction of artificial electron acceptors at sub-zero temperatures by chloroplasts suspended in fluid media.

1. Chloroplasts can be suspended in aqueous/organic mixtures which are liquid at sub-zero temperatures with a good retention of the ability to reduce artificial electron acceptors. The reduction of ferricyanide and 2,6-dichlorophenolindophenol at temperatures above 0 degrees C is about 50% inhibited by 50% (v/v) ethylene glycol. Higher concentrations cause more extensive inhibition. 2. Different solvents were compared on the basis of their ability to cause a given depression of the freezing point of an aqueous solution. Ethylene glycol caused less inhibition of electron transport than glycerol, which in turn was found to be superior to methanol. 3. The reduction of oxidised 2,3,5,6-tetramethyl-p-phenylenediamine could be measured at -25 degrees C in 40% (v/v) ethylene glycol. Using an acceptor with a high extinction coefficient, methyl purple (a derivative of 2,6-dichlorophenolindophenol) it was possible to observe electron flow at temperatures as low as -40 degrees C in 50% (v/v) ethylene glycol. 4. From studies of the effects of the inhibitors 3(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone it is suggested that electron flow from the donor side of Photosystem II to the acceptor side of Photosystem I can occur at temperatures at least as low as -25 degrees C. The ultimate electron donor is presumably water but it was not possible to demonstrate this directly.

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors.

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced. 2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the "resting" oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis. 3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a3 (k1obs = 7.5 s-1 for the oxygen-pulsed oxidase). 4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the "oxygenated" compound, but there were small differences between the two spectra.

Effect of growth temperature on membrane dynamics in a thermophilic cyanobacterium: a spin label study.

A strain of Synechococcus sp. was grown at its optimal growth temperature (58 degrees C) and at 38 degrees C, in order to investigate possible adaptations of membrane-related properties to growth temperature. Light-induced electron transport in thylakoid membranes from both types of cells showed linear Arrhenius plots with the same activation energy (48 kJ/mol). Membranes from cells grown at 58 degrees C had a higher temperature optimum (53 degrees C) than those from cells grown at 38 degrees C (41 degrees C). Growth at 38 degrees C caused an increase in the proportion of unsaturated fatty acids compared to growth at 58 degrees C. The fluidity of the membranes was investigated by measuring the temperature dependence of the parameters derived from electron spin resonance spectra of the spin-labels 5-doxyldecane, 5-doxylstearate and 16-doxylstearate. Only small differences between the dynamic properties of the membranes from cells grown at different temperatures could be detected. This suggests that the observed change in fatty acid composition of the membranes following the change in growth temperature does not serve to maintain a constant viscosity at the growth temperature.

Transient and long-term differential modulations of branched-chain alpha-keto acid decarboxylase activity in hypophysectomized rats.

Hypophysectomy caused a marked but transient increase in branched-chain alpha-keto acid decarboxylase activities in rat liver mitochondria, peaking at about nine days post-surgery. The magnitude of increase is different for each of the three branched-chain alpha-keto acids. The activities then fall to a new steady state in three weeks with alpha-ketoisovalerate decarboxylase activity within the normal range, alpha-keto-beta-methylvalerate decarboxylase activity at twice normal, and alpha-ketoisocaproate decarboxylase activity decreased to a level too low for accurate measurements.

Differential processing of human and rat E1 alpha precursors of the branched-chain alpha-keto acid dehydrogenase complex caused by an N-terminal proline in the rat sequence.

The N-terminal sequences of the E1 alpha, E1 beta and E2 subunits of the human branched-chain alpha-keto acid dehydrogenase complex have been determined by microsequencing. The N-terminal of human E1 beta and E2 subunits (Val and Gly, respectively) are identical to those of the corresponding rat and bovine subunits. However, the N-terminus of the human E1 alpha subunit (Ser) is identical to bovine, but differs from the rat E1 alpha (Phe) subunit. Comparison of the N-terminal sequences of human and rat E1 alpha subunits shows that the serine residue at the +1 position in the human sequence is replaced by a proline residue in the rat sequence. The presence of the proline residue apparently causes a 5'-shift by one residue in the cleavage site by the mitochondrial processing peptidase in the rat sequence, when compared to the human sequence. The results provide evidence that the mitochondrial processing peptidase cannot cleave an X-Pro bond, similar to trypsin, chymotrypsin and microsomal signal peptidases.

The complete cDNA sequence for dihydrolipoyl transacylase (E2) of human branched-chain alpha-keto acid dehydrogenase complex.

We have determined the complete nucleotide sequence for the cDNA encoding human dihydrolipoyl transacylase (E2) using the rapid amplification of cDNA ends (RACE) procedure. The full-length E2 cDNA is 3535 nucleotides in length. The coding region spans 1446 bp and the 3'-noncoding region spans 2074 bp. The latter contains three Alu repetitive sequences and two transcription termination sites.

Coordinate regulation of the messenger ribonucleic acids encoding the alpha- and beta-subunits of human chorionic gonadotropin in HeLa cells and butyrate-resistant variants.

Short term exposure of HeLa S3 cells to sodium butyrate induces accumulation of the mRNAs encoding both the alpha- and beta-subunits of hCG. Both mRNAs accumulate with the same kinetics and reach maximal levels with the same concentration of butyrate, suggesting that the levels of alpha and hCG beta mRNAs are coordinately regulated. In addition, induction of both mRNAs is tightly coupled with a similar increase in secreted levels of hCG subunit protein, further suggesting that regulation of CG biosynthesis in HeLa cells occurs before translation. To determine whether HeLa cells which have overcome the growth inhibitory properties of butyrate show uniformly high levels of alpha and hCG beta mRNAs, we isolated clonal variants by stepwise selection with increasing concentrations of butyrate. Of 69 isolated variants, at least 2 (AO and B2) display different patterns of CG gene expression. In AO cells, alpha-subunit mRNA was not detectable, while in B2 cells, the level of alpha-subunit mRNA was similar to that of wild-type HeLa S3 cells. Conversely, hCG beta mRNA levels in both variant cell lines approximated levels found in butyrate-treated HeLa S3 cultures, suggesting that the mRNAs for alpha and hCG beta are regulated independently. Analysis of genomic DNA by blot hybridization indicated that the alpha-subunit gene was present as a single unrearranged copy in HeLa S3 cells and in both variants, indicating that differences in alpha-subunit gene expression are not associated with major genomic alterations, but probably reflect more subtle changes.

Structure of the gene encoding the entire mature E1 alpha subunit of human branched-chain alpha-keto acid dehydrogenase complex.

We report the isolation of a 22-kb human genomic clone (G7) that contains 8 exons encoding a partial mitochondrial presequence, the entire mature peptide and the complete 3' untranslated region of the E1 alpha mRNA of human branched-chain alpha-keto acid dehydrogenase complex. Based on this gene structure, exon 9 contains the Tyr393----Asn mutation previously identified in the E1 alpha subunit of Mennonite and other maple syrup urine disease (MSUD) patients. Moreover, the homozygous mutation appears to cause skipping of exon 6 in the mutant E1 alpha transcript. The information on the gene structure for the entire mature E1 alpha subunit will facilitate investigations into the molecular basis of MSUD involving this subunit.

Molecular cloning of the mature E1b-beta subunit of human branched-chain alpha-keto acid dehydrogenase complex.

We have isolated a cDNA encoding the E1b-beta subunit of the human branched-chain alpha-keto acid dehydrogenase complex. The human E1b-beta cDNA is 1401 base pairs in length. It encodes the entire mature E1b-beta subunit consisting of 342 amino acid residues, and a mitochondrial targeting presequence of 31 residues. The calculated molecular mass of the mature human E1b-beta subunit is 37,851 Da, and the calculated isoelectric point is pH 5.18. A hydropathy plot shows that the human E1b-beta subunit is highly hydrophobic. Northern blot analysis shows that the human E1b-beta mRNA is approximately 1.4 kb in size. It is present at the normal level in fibroblasts from two unrelated maple syrup urine disease patients.