Isotope Incorporation Studies Research Articles

Free Piperazic Acid as a Precursor to Nonribosomal Peptides.

Piperazic acid (Piz) is a nonproteinogenic amino acid possessing a rare nitrogen-nitrogen bond. However, little is known about how Piz is incorporated into nonribosomal peptides, including whether adenylation domains specific to Piz exist. In this study, we show that free piperazic acid is directly adenylated and then incorporated into the incarnatapeptin nonribosomal peptides through isotopic incorporation studies. We also use in vitro reconstitution to demonstrate adenylation of free piperazic acid with a three-domain nonribosomal peptide synthetase from the incarnatapeptin gene cluster. We furthermore use bioinformatics and site-directed mutagenesis to outline consensus sequences for the adenylation of piperazic acid, which can now be used for the prediction of gene clusters linked to piperazic-acid-containing peptides. Finally, we discover a fusion protein of a piperazate synthase and an adenylation domain, highlighting the close biosynthetic relationship of piperazic acid formation and its adenylation. Altogether, our work demonstrates the evolution of biosynthetic systems for the activation of free piperazic acid through adenylation, a pathway we suggest is likely to be employed in the majority of pathways to piperazic-acid-containing peptides.

Biosynthesis of Cylindrospermopsin in Cyanobacteria: Characterization of CyrJ the Sulfotransferase.

7-Deoxy-desulfo-cylindrospermopsin was purified at small-scale from the supernatant of a culture of the cyanobacterium Oscillatoria sp. PCC 10702. This metabolite was obtained in a pure form using a three-step chromatographic procedure, and its identity was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS quantification showed that this metabolite was excreted in the culture medium of Oscillatoria sp. PCC 10702. Isotopic incorporation studies using [2-13C,15N]glycine, a cylindrospermopsin precursor, and Oscillatoria sp. PCC 10702 cells showed that glycine was incorporated into 7-deoxy-desulfo-cylindrospermopsin, 7-deoxy-cylindrospermopsin, 7-epi-cylindrospermopsin, and cylindrospermopsin. The isotopic incorporation rate was consistent with the following metabolic flux: 7-deoxy-desulfo-cylindrospermopsin → 7-deoxy-cylindrospermopsin → 7-epi-cylindrospermopsin and cylindrospermopsin. We have cloned the cyrJ gene into an expression vector and overproduced the putative sulfotransferase CyrJ in Escherichia coli. The purified protein CyrJ catalyzed, in vitro, the transfer of a sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to 7-deoxy-desulfo-cylindrospermopsin to give 7-deoxy-cylindrospermopsin. Kinetic analysis afforded the following apparent constants: KMapp. (PAPS) = 0.12 μM, Vmaxapp. = 20 nM/min, KMapp. (7-deoxy-desulfo-cylindrospermopsin) = 0.12 μM, and KIapp. (7-deoxy-desulfo-cylindrospermopsin) = 4.1 μM. Preliminary data suggested that CyrJ catalyzed the reaction through a ternary-complex kinetic mechanism. All these data confirmed that CyrJ catalyzed a sulfotransfer during the penultimate step of the biosynthesis of cylindrospermopsin.

Novel copper-containing membrane monooxygenases (CuMMOs) encoded by alkane-utilizing Betaproteobacteria.

Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short-chain alkanes and alkenes. In a metagenome constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with <59% derived protein sequence identity to genes from known bacteria. Stable isotope probing experiments combined with a specific xmoA qPCR assay demonstrated that the bacteria possessing these genes were incapable of methane assimilation, but did grow on ethane and propane. Single-cell amplified genomes (SAGs) from propane-enriched samples were screened with the specific PCR assay to identify bacteria possessing the target gene cluster. Multiple SAGs of Betaproteobacteria belonging to the genera Rhodoferax and Polaromonas possessed homologues of the metagenomic xmoCABD gene cluster. Unexpectedly, each of these two genera also possessed other xmoCABD paralogs, representing two additional lineages in phylogenetic analyses. Metabolic reconstructions from SAGs predicted that neither bacterium encoded enzymes with the potential to support catabolic methane or ammonia oxidation, but that both were capable of higher n-alkane degradation. The involvement of the encoded CuMMOs in alkane oxidation was further suggested by reverse transcription PCR analyses, which detected elevated transcription of the xmoA genes upon enrichment of water samples with propane as the sole energy source. Enrichments, isotope incorporation studies, genome reconstructions, and gene expression studies therefore all agreed that the unknown xmoCABD operons did not encode methane or ammonia monooxygenases, but rather n-alkane monooxygenases. This study broadens the known diversity of CuMMOs and identifies these enzymes in non-nitrifying Betaproteobacteria.

Half-Sandwich Iridium Complexes for Homogeneous Water-Oxidation Catalysis

Iridium half-sandwich complexes of the types Cp*Ir(N-C)X, [Cp*Ir(N-N)X]X, and [CpIr(N-N)X]X are catalyst precursors for the homogeneous oxidation of water to dioxygen. Kinetic studies with cerium(IV) ammonium nitrate as primary oxidant show that oxygen evolution is rapid and continues over many hours. In addition, [Cp*Ir(H(2)O)(3)]SO(4) and [(Cp*Ir)(2)(μ-OH)(3)]OH can show even higher turnover frequencies (up to 20 min(-1) at pH 0.89). Aqueous electrochemical studies on the cationic complexes having chelate ligands show catalytic oxidation at pH > 7; conversely, at low pH, there are no oxidation waves up to 1.5 V vs NHE for the complexes. H(2)(18)O isotope incorporation studies demonstrate that water is the source of oxygen atoms during cerium(IV)-driven catalysis. DFT calculations and kinetic experiments, including kinetic-isotope-effect studies, suggest a mechanism for homogeneous iridium-catalyzed water oxidation and contribute to the determination of the rate-determining step. The kinetic experiments also help distinguish the active homogeneous catalyst from heterogeneous nanoparticulate iridium dioxide.

Should we use one‐, or multi‐compartment models to describe 13C incorporation into animal tissues?

Understanding rates of isotopic incorporation and discrimination factors between tissues and diet is an important focus of ecologists seeking to use stable isotopes to track temporal changes in diet. We used a diet-shift experiment to measure differences among tissues in (13)C incorporation rates in house sparrows (Passer domesticus). We predicted faster incorporation rates in splanchnic than in structural tissues. We also assessed whether isotopic incorporation data were better supported by the one-compartment models most commonly used by ecologists or by multi-compartment models. We found large differences in the residence time of (13)C among tissues and, as predicted, splanchnic tissues had faster rates of isotopic incorporation and thus shorter retention times than structural tissues. We found that one-compartment models supported isotopic incorporation data better in breath, excreta, red blood cells, bone collagen, and claw tissues. However, data in plasma, intestine, liver, pectoralis muscle, gizzard, and intestine tissues supported two-compartment models. More importantly, the inferences that we derived from the two types of models differed. Two-compartment models estimated longer (13)C residence times, and smaller tissue to diet differences in isotopic composition, than one-compartment models. Our study highlights the importance of considering both one- and multi-compartment models when interpreting laboratory and field isotopic incorporation studies. It also emphasizes the opportunities that measuring several tissues with contrasting isotopic residence times offer to elucidate animal diets at different time scales.

Biosynthesis and Structures of Cyclomarins and Cyclomarazines, Prenylated Cyclic Peptides of Marine Actinobacterial Origin

Two new diketopiperazine dipeptides, cyclomarazines A and B, were isolated and characterized along with the new cyclic heptapeptide cyclomarin D from the marine bacterium Salinispora arenicola CNS-205. These structurally related cyclic peptides each contain modified amino acid residues, including derivatives of N-(1,1-dimethylallyl)-tryptophan and delta-hydroxyleucine, which are common in the di- and heptapeptide series. Stable isotope incorporation studies in Streptomyces sp. CNB-982, which was first reported to produce the cyclomarin anti-inflammatory agents, illuminated the biosynthetic building blocks associated with the major metabolite cyclomarin A, signifying that this marine microbial peptide is nonribosomally derived largely from nonproteinogenic amino acid residues. DNA sequence analysis of the 5.8 Mb S. arenicola circular genome and PCR-targeted gene inactivation experiments identified the 47 kb cyclomarin/cyclomarazine biosynthetic gene cluster (cym) harboring 23 open reading frames. The cym locus is dominated by the 23 358 bp cymA, which encodes a 7-module nonribosomal peptide synthetase (NRPS) responsible for assembly of the full-length cyclomarin heptapeptides as well as the truncated cyclomarazine dipeptides. The unprecedented biosynthetic feature of the megasynthetase CymA to synthesize differently sized peptides in vivo may be triggered by the level of beta oxidation of the priming tryptophan residue, which is oxidized in the cyclomarin series and unoxidized in the cyclomarazines. Biosynthesis of the N-(1,1-dimethyl-2,3-epoxypropyl)-beta-hydroxytryptophan residue of cyclomarin A was further illuminated through gene inactivation experiments, which suggest that the tryptophan residue is reverse prenylated by CymD prior to release of the cyclic peptide from the CymA megasynthetase, whereas the cytochrome P450 CymV installs the epoxide group on the isoprene of cyclomarin C post-NRPS assembly. Last, the novel amino acid residue 2-amino-3,5-dimethylhex-4-enoic acid in the cyclomarin series was shown by bioinformatics and stable isotope experiments to derive from a new pathway involving condensation of isobutyraldehyde and pyruvate followed by S-adenosylmethionine methylation. Assembly of this unsaturated, branched amino acid is unexpectedly related to the degradation of the environmental pollutant 3-(3-hydroxyphenyl)propionic acid.

Homeostatic Proliferation of Chronic Lymphocytic Leukemia.

Slow accumulation of chronic lymphocytic leukemia cells in vivo was considered due to defective apoptosis rather than proliferation. However, recent data, based on isotope incorporation studies, suggested significant continuous proliferation of CLL cells in vivo and similar observations has been made for human memory B cells. As previous gene expression studies revealed close relation of CLL cells to memory B cells we asked, whether the mechanisms driving homeostatic proliferation of human memory B cells were stimulating CLL cells as well. Comparison of CLL samples (n=20) and memory B cells of healthy donors showed high expression levels of Toll-like receptor (TLR) 7 and TLR9 and of IL2 and IL15 cytokine receptors compared to naive B cells. Proliferation of CLL cells compared to their normal counterparts was analyzed after FACS sorting and CFSE-labeling.Naive B cells did neither respond to TLR7 ligand resiquimod, TLR9 ligand CpG2006, IL2 and IL15 nor to their combination. In contrast, CLL cells and memory B cells showed comparable patterns of proliferation. Remarkably, under these conditions, terminal differentiation to plasma cells was observed for memory B cells only, while proliferation of CLL ceased after the 4th division without differentiation into Ig-secreting cells. To estimate in vivo turnover rates, B cells of healthy donors and CLL cells were stained for intracellular Ki67, that identifies recently divided cells, and were analyzed by flow cytometry. Memory B cells showed high in vivo turnover rates (2–5% Ki67+) while naive B cells where largely quiescent - results that are in line with previous isotope incorporation studies with healthy donors. Turnover rates of CLL ex vivo ranged from 0.025–1.4% Ki67+ cells, which in absolute numbers (per mL of blood) was comparable to memory B cells. These results are consistent with a concept of homeostatic proliferation of both memory B cells and CLL cells, the latter being arrested without terminal differentiation, which might account for their accumulation in vivo.

Impaired gastric acid and pancreatic enzyme secretion in patients with Crohn's disease may be a consequenece of a poor nutritional state.

Impaired pancreatic function has been reported in Crohn's disease, the cause of which is uncertain. This study investigated the effect of malnutrition, and subsequent re-feeding, on digestive function and protein synthesis in Crohn's disease patients. Gastric acid and pancreatic secretion studies were performed on malnourished Crohn's patients before, and after a period of intensive nutritional support. Whole body, as well as pancreatic enzyme protein synthesis was investigated by [14C]leucine isotope incorporation studies. Results were evaluated in comparison to normal healthy volunteers. The mean body mass index (BMI) of the Crohn's patients was 14.14 kg/m2. The Crohn's patients had reduction in the secretion of gastric acid (7.36 versus 25.53 mEq/h; P < 0.01), and the pancreatic enzymes, amylase (759.6 versus 2305 U/h; P < 0.01), lipase (33.01 versus 118.6 U/h; P < 0.01) and trypsin (97.43 versus 341.4 U/h; P < 0.01). Resting energy expenditure (REE), expressed in relation to body mass, was greater in the malnourished Crohn's disease patients (38.25 versus 25.36 kcal/kg/d; P = 0.01). Total body protein synthesis was reduced (2.82 versus 4.39 g protein/kg/d; P < 0.05), with significant impairment in the synthesis of pancreatic enzymes, and reduction of zymogen stores. Following re-feeding, the BMI of the Crohn's patients improved to 16.80 +/- 0.66 kg/m2. Pancreatic enzyme synthesis improved, with significant increase in pancreatic enzyme stores and secretion, to levels similar to control values. Gastric acid secretion also improved, although still lower than the control value. Malnutrition may play a significant role in the impairment of gastric acid and pancreatic secretion in Crohn's disease patients.

The mechanism of the reaction catalyzed by ADP-beta-L-glycero-D-manno-heptose 6-epimerase.

ADP-l-glycero-d-manno-heptose 6-epimerase (AGME, RfaD) is a bacterial enzyme that is involved in lipopolysaccharide biosynthesis and interconverts ADP-beta-l-glycero-d-manno-heptose (ADP-l,d-Hep) with ADP-beta-d-glycero-d-manno-heptose (ADP-d,d-Hep). AGME is known to require a tightly bound NADP+ cofactor for activity and presumably employs a mechanism involving transient oxidation of the substrate. Four mechanistic possibilities are considered that involve transient oxidation at either C-7' ', C-6' ', or C-4' ' of the heptose nucleotide. In this contribution, the use of solvent isotope incorporation studies and alternate substrates provides strong evidence for a mechanism involving nonstereospecific oxidation/reduction directly at C-6' '. It was found that the epimerization proceeds without any detectable incorporation of solvent-derived deuterium or 18O-isotope into the product. This argues against mechanisms involving either proton transfers at carbon or dehydration/rehydration events. In addition, the deoxygenated analogues, 7' '-deoxy-ADP-l,d-Hep and 4' '-deoxy-ADP-l,d-Hep, were both found to serve as substrates for the enzyme, indicating that oxidation at either C-7' ' or C-4' ' is not required for catalysis.

Investigations of Terpenoid Biosynthesis by the Dorid Nudibranch Cadlina luteomarginata.

Stable isotope incorporation studies with [1,2-(13)C(2)]acetate, [1,2-(13)C(2),(18)O(1)]acetate, and [2-(13)C]mevalonolactone have been used to investigate the biosynthesis of the terpenoids albicanyl acetate (1), cadlinaldehyde (2), and luteone (3) by the dorid nudibranch Cadlina luteomarginata. The results have shown that all three terpenoids are synthesized de novo by C. luteomarginata and the incorporation patterns are consistent with the biogenetic proposal that the new cadlinalane and luteane carbon skeletons are formed by degradation of a sesterterpenoid precursor. This represents the first demonstration of sesterterpenoid biosynthesis by a marine mollusc. Quantitative analysis has shown that only a small turnover of metabolites takes place during the feeding experiments but that the newly formed molecules have extremely high levels of incorporation of labeled precursors.

Site-directed mutagenesis and oxygen isotope incorporation studies of the nucleophilic aspartate of haloalkane dehalogenase.

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds. The X-ray structure suggests that Asp124, which is located close to an internal cavity, carries out a nucleophilic attack on the C alpha of the substrate, releasing the halogen. To study the mechanism of hydrolysis, this aspartate residue was mutated to alanine, glycine, or glutamate. The mutant enzymes showed no activity toward 1,2-dichloroethane and 1,2-dibromoethane. Incubation of purified wild-type dehalogenase with 1,2-dichloroethane in the presence of H2(18)O resulted in the incorporation of 18O in 2-chloroethanol and in the carboxylate group of Asp124. This shows that the reaction proceeds by covalent catalysis with the formation of an alkyl-enzyme intermediate that is hydrolyzed by attack of solvent water on the carbonyl carbon of Asp124. On the basis of amino acid sequence similarity between haloalkane dehalogenase and epoxide hydrolases, it is proposed that a conserved aspartate residue is also involved in covalent catalysis by the latter enzymes.

The galactose binding lectin from the beet armyworm, Spodoptera exigua: Distribution and site of synthesis

The Spodoptera exigua galactose binding lectin, extracted from hemolymph by affinity chromatography, was comprised of large molecular weight aggregates (100–700 kDa). SDS-PAGE of this lectin preparation revealed it to contain 2 subunits of 33.2 and 34.4 kDa in equimolar concentrations. These subunits had similar amino acid profiles, possessed identical N-terminal sequences and reacted equally to a bank of antilectin monoclonal antibodies. By staining Western blots with various lectin conjugate probes, we demonstrated that the 34.4 kDa subunit contains complex mannose residues, suggesting that this subunit is the glycosylated form of the 33.2 kDa subunit. The N-terminal sequence of the S. exigua lectin was distinct from other invertebrate galactose binding lectins. Light microscopy in combination with immunoelectron microscopy was used to localize the S. exigua lectin in the granules of the granulocyte class of hemocytes. Degranulation of these cells resulted in the release of the lectin. Isotope incorporation studies followed by immunoprecipitation with a S. exigua monoclonal antibody demonstrated that the fat body was the major site of lectin synthesis. Similar studies with hemocyte monolayers did not result in the production of detectable levels of 35S-labeled S. exigua lectin.

Development of a method for the purification of human trypsin by single step affinity chromatography suitable for human isotope incorporation studies

A method has been developed for the single step purification of human amylase from duodenal juice in order to allow measurement of amylase synthesis [ 11. Nonparallel enzyme synthesis has been reported in rats fed protein-rich or carbohydraterich diets [2]. A protein rich diet augmented proteolytic proenzyme synthesis rates and decreased amylase and lipase synthesis rates, however, a carbohydrate-rich diet only decreased the synthesis rate of anodic chymotrypsinogen. Divergent changes in rat pancreatic enzyme synthesis have been reported during infusion of optimal doses of caerulein [3] and after intraduodenal administration of oleic acid [4]. Non-parallel enzyme secretion has been demonstrated in the human [5]. Individual enzyme synthesis has not been measured in man, so it is not known whether non-parallel enzyme synthesis occurs under certain conditions. It has been debated at length whether synthesis of a particular enzyme is primarily affected in chronic pancreatitis and if so which enzyme this is. It was therefore decided to measure trypsin synthesis rates as well as amylase synthesis rates in patients with chronic pancreatitis and controls. In

Conversion of 5-S-methyl-5-thio-D-ribose to methionine in Klebsiella pneumoniae. Stable isotope incorporation studies of the terminal enzymatic reactions in the pathway.

Extracts of Klebsiella pneumoniae convert 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine and formate. To probe the terminal steps of this biotransformation, [1-13C]methylthioribose has been synthesized and its metabolism examined. When supplemented with Mg2+, ATP, L-glutamine, and dioxygen, cell-free extracts of K. pneumoniae converted 50% of the [1-13C]methylthioribose to [13C]formate. The formation of [13C]formate was established by 13C and 1H NMR spectroscopy studies of the purified formate, and by 13C and 1H NMR spectroscopy and mass spectrometry studies of its p-phenylphenacyl derivative. By contrast, no incorporation of label from [1-13C]methylthioribose into the biosynthesized methionine was detected by either mass spectrometry or 13C and 1H NMR spectroscopy. The most reasonable interpretation of these results is that C-1 of methylthioribose is converted directly to formate concomitant with the conversion of carbon atoms 2-5 to methionine. The penultimate step in the conversion of methylthioribose to methionine and formate is an oxidative carbon-carbon bond cleavage reaction in which an equivalent of dioxygen is consumed. To investigate the fate of the dioxygen utilized in this reaction, the metabolism of [1-13C]methylthioribose in the presence of 18O2 was also examined. Mass spectrometry revealed the biosynthesis of substantial amounts of both [18O1]methionine and [13C, 18O1]formate under these conditions. These results suggest that the oxidative transformation in the conversion of methylthioribose to methionine and formate may be catalyzed by a novel intramolecular dioxygenase. A mechanism for this dioxygenase is proposed.

Biosynthesis of lathyrine; a novel synthase activity

Comparison of [U- 14C]serine and [3- 14C]serine as precursors of the alanine side-chain of the pyrimidinyl amino acid lathyrine, indicate that serine donates all three of its carbon atoms as a unit. Isotope incorporation studies identified the precursor of the pyrimidine moiety as 2-amino-4-car☐ypyrimidine. Studies with enzymic extracts of Lathyrus seedlings showed the presence of a pyridoxal phosphate-dependent lathyrine synthase which simultaneously decar☐ylates the pyrimidine precursor alanylates its C-4 position. The enzyme is stimulated by biotin. Unlike a number of other plant synthases catalysing formation of heterocyclic β-substituted alanines, lathyrine synthase requires serine and not O-acetylserine as a substrate. The action of the synthase was successfully modelled in a non-enzymic pyridoxal-catalysed reaction in the presence of either Al 3+ or Ga 3+ ions. The latter were much more effective in this respect.

Effects of dietary nutrients on lipogenic enzyme and mRNA activities in rat liver during induction

By feeding a carbohydrate diet (without protein) to fasted rats, malic enzyme mRNA activity in the liver was increased to the level in rats fed a carbohydrate and protein diet, whereas the enzyme activity itself was increased to 60% of that level. It appears that malic enzyme mRNA activity was increased by dietary carbohydrate, while dietary protein contributed to an increase in the translation of mRNA. In the animals fed carbohydrate without protein, glucose-6-phosphate dehydrogenase mRNA activity increased to 50% of the level in rats fed the carbohydrate and protein diet, whereas the enzyme activity increased to only 25%. By feeding a protein diet (without carbohydrate), glucose-6-phosphate dehydrogenase activity increased to 65% of the level in rats fed both carbohydrate and protein. This enzyme induction appears to be more dependent on protein than carbohydrate. With the carbohydrate diet, acetyl-CoA carboxylase was induced up to the level in the carbohydrate and protein diet group, whereas fatty acid synthetase was induced to only 33%. Acetyl-CoA carboxylase induction appears to be carbohydrate dependent. On the other hand, isotopic leucine incorporation studies showed that the magnitudes of the enzyme inductions caused by the dietary nutrients should be ascribed to the enzyme synthesis rates rather than the degradation. By fat feeding, the mRNA activities of malic enzyme and glucose-6-phosphate dehydrogenase were markedly decreased along with the enzyme induction. Fat appears to reduce these enzyme inductions before the translation of mRNA.

The role of glutamate in modulating the synthesis and degradation of NADP-glutamate dehydrogenase from Saccharomyces cerevisiae

NADP-glutamate dehydrogenase (NADP-GDH) from Saccharomyces cerevisiae has a lower activity in yeast grown on glutamate as nitrogen source than when grown on ammonium. With the use of the immunotitration method, it was found that the difference in activity was parallel to the difference in immunoprecipitable material. By isotope incorporation studies, it was established that the decrease in NADP-glutamate dehydrogenase levels in glutamate-grown cells was brought about by an increase in the degradation rate and a decrease in the synthesis constant of the enzyme. The degradation rate of NADP-glutamate dehydrogenase is further increased in carbon-starved cells. The possible role of internal metabolites in modulating NADP-glutamate dehydrogenase degradation is discussed.

The role of glutamate in modulating the synthesis and degradation of NADP-glutamate dehydrogenase from Saccharomyces cerevisiae

NADP-glutamate dehydrogenase (NADP-GDH) from Saccharomyces cerevisiae has a lower activity in yeast grown on glutamate as nitrogen source than when grown on ammonium. With the use of the immunotitration method, it was found that the difference in activity was parallel to the difference in immunoprecipitable material. By isotope incorporation studies, it was established that the decrease in NADP-glutamate dehydrogenase levels in glutamate-grown cells was brought about by an increase in the degradation rate and a decrease in the synthesis constant of the enzyme. The degradation rate of NADP-glutamate dehydrogenase is further increased in carbon-starved cells. The possible role of internal metabolites in modulating NADP-glutamate dehydrogenase degradation is discussed.

An electron microscopic demonstration of induction of chondrogenesis in neonatal rat muscle outgrowth cells in monolayer cultures.

Second passage fibroblast-like cells grown from explants of neonatal rat muscle continue to demonstrate fibroblast-like properties for many days when cultured on plastic surfaces. Such cells can be induced to change to a chondrocyte-like mode of expression by the addition of effector materials prepared from bovine cortical bone decalcified with 0.6 N HCl. Other studies show that similar demineralized bone particles and extracts from them have, in vivo, osteoinductive properties. Optimum conditions for this differentiation in monolayer culture were found in the use of 2% fetal calf serum with Dulbecco's modified Eagles medium. At 10% fetal calf serum the chondrogenic changes could not be detected. Light microscopy showed a sequence of morphological changes, after 36 h in culture, which resembled those seen at the beginning of osteogenesis in vivo. Induced cultures showed abundant extracellular proteoglycan production. Isotope incorporation studies showed stimulation of glycosaminoglycan synthesis in response to effector materials in soluble form. Type II collagen could be detected after three days. Electron microscopic analysis of induced and control cultures showed unequivocal evidence for marked production of an extensive extracellular matrix in the region of effector particles. The cells themselves change shape and develop an abundant system of lysosome-like vesicles and a very active, highly engorged endoplasmic reticulum and Golgi apparatus. After nine days in culture, evidence for the formation of a ruthenium red stained structure on the surface of the cells in contact with inductive particles, was observed. The simple monolayer culture system described provides a direct means by which the presence of active chondrogenic fractions may be assessed, and in which the mechanism of action of the effectors can be studied.

Nitrogen source regulates glutamate dehydrogenase NADP synthesis in Neurospora crassa.

Neurospora crassa glutamate dehydrogenase-NADP (EC 1.3.1.3) has a higher activity when mycelium is grown on ammonium or nitrate as nitrogen source than when grown on glutamate or glutamine. Quantitative immunoelectrophoresis established that, under all conditions, enzyme activity corresponded to enzyme concentration. Isotope incorporation studies demonstrated that the nitrogen source exerts its regulation at the level of de novo enzyme synthesis.