Effect of hydroquinone on meiotic segregation in Drosophila melanogaster females

The induction of sex chromosomes meiotic nondisjunction (ND) by hydroquinone (HQ) given orally was investigated in Drosophila melanogaster 2-7, 8-22, 24, 48, 72 and 96 h-old females. ND was assessed by a system where exceptional females (XXY) and only 1/4 of the expected regular progeny are viable. Oocytes were treated at different stages of development. 4% HQ tested only in 72 h-old females induced ND in oocytes sampled in brood I (mostly mature oocytes at metaphase I). 6% HQ increased ND in brood I of 8-22 h-old females, while other broods, (including cells treated at early prophase) were also affected in older flies, the highest significance being attained in the 48 h-old series. Newly hatched females (2-7 h-old) were refractory to the treatment, though oocytes sampled in the first three subcultures are comparable to cells showing enhancement of ND in series run with older females. Toxicity of 2, 4 and 6% HQ increased with concentration and females' age: (a) 2% was not toxic; (b) 4% was toxic only to 72 h-old females; (c) 6% was increasingly toxic to females 24, 48 and 72 h-old. The results indicate that age plays a significant role on both chromosomal segregation and toxicity and suggest that in Drosophila HQ is metabolized to its reactive species. The lack of toxic and aneugenic effect in very young females could reflect a more efficient detoxification due to the known high specific activity of glutathione-S-transferase (GST) after eclosion. The decline in GST activity around day 2 of adult life coincides with the high effect of HQ in 48 h-old females.     

Effects of 4-methyl umbelliferyl-beta-D-xylopyranoside on chondrogenesis and proteoglycan synthesis in chick limb bud mesenchymal cell cultures

When chick limb bud mesenchyme cells from stage 23 to 24 embryos are plated at high density, they rapidly divide and a large proportion initiate chondrogenic expression during the first 2 to 3 days in culture. Between Days 4 and 8, the emergent chondrocytes mature and elaborate a cartilaginous matrix. The proteoglycans synthesized by the newly emergent Day 3 to 4 chondrocytes differ from those synthesized by either the prechondrogenic mesenchyme cells or the mature Day 8 chondrocytes. Cultures were grown from initial plating (Day 0) or from Day 2 in the continuous presence of 1 mM 4-methyl umbelliferyl-beta-D-xyloside, which acts intracellularly as a competitive acceptor with the endogenous core protein of proteoglycans for chondroitin sulfate synthesis. The proteoglycans synthesized by Day 8 cultures which had been maintained on xyloside or to which xyloside was added only 1 h prior to labeling were essentially identical. They were able to form aggregates, and they contained the same number of keratan sulfate chains, but only about 40% as many chondroitin sulfate chains, as normal. Additionally, both the chondroitin sulfate and keratan sulfate chains were 25% shorter than in the normal proteoglycans. The proteoglycans synthesized by cells in a culture maintained on xyloside until Day 8, and then switched to medium with no xyloside 1 h prior to labeling, were characteristic of those synthesized by normal mature Day 8 chondrocytes. These data suggest that stage 23 to 24 mesenchyme cells undergo normal chondrogenic maturation in culture in the presence of xylosides even though (a) most of the polysaccharides are synthesized onto the exogenously supplied xyloside substrate and released into the medium, (b) the proteoglycans that are synthesized are greatly reduced in polysaccharide content, and (c) the extracellular matrix as a consequence is greatly depleted in chondroitin sulfate content and, therefore, is abnormal in general morphology.     

Issues in the culture of the extremely thermophilic methanogen, Methanothermus fervidus

Basic issues in the culture of the extremely thermophilic archaeon, Methanothermus fervidus, have been investigated, including culture medium formulation, substrate yield and product yield coefficient, growth rate and stoichiometry, and H(2) uptake kinetics. The pH optimum for growth of this organism was estimated at 6.9. Growth medium buffered with PIPES instead of bicarbonate supported both increased growth rate and maximum biomass concentration. Substitution of titanium(III) citrate for the reducing agent sodium sulfide improved culture performance as well. However, independent adjustment of iron and nickel concentrations from 11 to 111 muM, respectively, and carbon dioxide partial pressure from 5 to 20 psia did not impact the culture of M. fervidus significantly. An elemental balance approach was utilized to aid in design of a defined medium to support growth to a target maximum biomass concentration of at least 1.0 g dry wt/L. The growth of this organism was limited by H(2) availability in this reformulated culture medium. The maximum growth rate and biomass concentration achieved in anaerobic vials with the defined medium was 0.16 h(-1) and 0.74 g dry wt/L, respectively. This maximum biomass concentration was a 72% improvement over that obtained with a literature-based defined medium. The Monod parameter, K(s), with H(2) as limiting substrate, was estimated at 1.1 +/- 0.4 psia (55 +/- 20 muM in the broth), based on a H(2) consumption study. Representative values for the substrate yield, Y(X/CO(2) ), and product yield coefficient, Y(CH(4)/) (X), were determined experimentally to be 1.78 +/- 0.04 g dry wt/mol CO(2), and 0.52 +/- 0.01 mol CH(4)/g dry wt, respectively. A bench-scale fermentation system suitable for the culture of extremely thermophilic anaerobes was designed and constructed and proved effective for the culture of M. fervidus. (c) 1993 Wiley & Sons, Inc.     

4-Methylumbelliferyl-alpha-glycosides of partially O-acetylated N-acetylneuraminic acids as substrates of bacterial and viral sialidases

4-O-Acetylated, 7-O-acetylated, and 9-O-acetylated 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acids (Neu4,5Ac2-MU, Neu5,7Ac2-MU, Neu5,9Ac2-MU) were tested as substrates of sialidases of Vibrio cholerae and of Clostridium perfringens. Both sialidases were unable to hydrolyse Neu4,5Ac2-MU. This compound at 1 mM concentration did not inhibit significantly the cleavage of Neu5Ac-MU, the best substrate tested. The 4-O-acetylated sialic acid glycoside is hydrolysed slowly by the sialidase from fowl plague virus. The relative substrate specificity, reflected in V/Km of the Vibrio cholerae sialidase is Neu5Ac-MU much greater than Neu5,7Ac2-MU approximately Neu5,9Ac2-MU and of the clostridial enzyme it is Neu5Ac-MU greater than Neu5,9Ac2-MU greater than Neu5,7Ac2-MU. The affinities of both enzymes for the side-chain O-acetylated sialic acid derivatives are higher than for Neu5Ac-MU. The artificial, well-defined substrates, described here, provide the opportunity to quantify the influence of sialic acid O-acetylation on the hydrolysis of sialoglycoconjugates without the side effects introduced by other parts of more complex glycans.     

DNA replication and head assembly in bacteriophage T4.

Phage DNA was accumulated in cells of E. coli B, infected with the phage T4DtsLB3 (gene 42), without the synthesis of late proteins (in the presence of chloramphenicol). Then (stage II), chloramphenicol was removed and further replication of the phage DNA suppressed with hydroxyurea and by simultaneously raising the temperature to 40 degrees. The media M9 or M9 with 1% amino acid were used; the times of addition of chloramphenicol and the hydroxyurea concentration were also varied. It was also shown that in medium M9, at stage II, chiefly early proteins were synthesized. In the medium containing amino acids, at stage II the following was observed: 1) DNA synthesis was entirely suppressed and a degradation of DNA occurred; 2) both early and late proteins were synthesized, with a predominance of the latter; 3) an assembly of the elements of the phage tails and capsids occurred without the neck and flagellum, and a small number of phage particles were also found; 4) the capsids, isolated in a sucrose density gradient after lysis with chloroform, contained the proteins Palt, P20, P23, P24, several unidentified proteins, and did not contain Pwac, P23, and P22, 5) the yield of viable phage varied from 0.05 to 15% per cell. Thus, the entire morphogenesis of T4 phage can occur without accompanying replication of phage DNA.     

Production of a Novel Compound, 7,10,12-Trihydroxy-8(E)-Octadecenoic Acid from Ricinoleic Acid by Pseudomonas aeruginosa PR3

The production and its potential use of a novel trihydroxy unsaturated fatty acid, 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD), were investigated. TOD was formed by Pseudomonas aeruginosa PR3 (NRRL B-18602) in a culture supplied with exogenous ricinoleic acid. The yield of TOD production was always higher in a rich culture medium than in minimal screening medium. Extending the conversion time from 48 to 72 h prior to lipid extraction led to a 65% reduction in yield, indicating that TOD was further metabolized by strain PR3 and that control of reaction time is important to achieving a maximum yield. The optimum culture density, reaction time, pH, temperature, and substrate concentration for the production of TOD were: 20–24 h culture growth, 48 h, 7.0, 25°C, and 1% (vol/vol), respectively. Under optimum conditions, the yield of TOD production was greater than 45%. TOD was found to be an antifungal agent most active against the fungus that causes blast disease in rice plants, the most important fungal disease affecting rice production worldwide. Received: 4 January 2001/Accepted: 6 February 2001     

山羊卵母细胞的减数分裂进程

The meiotic progression of goat oocytes from follicles of different diameters was investigated in this study. The results were summarized as follows: (1) The in vitro meiotic maturation capacity was different among oocytes from follicles of different diameters. And thus oocytes from < or = 0.5 mm follicles were unable to resume meiosis; oocytes from 0.8-1.2 mm follicles were capable to resume meiosis, but could develop only to MI stage (60% at 24 h); oocytes from 1.5-5 mm follicles had acquired full-meiotic maturation capacity and 91% of them developed to M II stage at 24 h of culture. (2) The percentage of oocytes with intact-germinal vesicles from 1.5-5 mm follicles decreased significantly during 2-8 h of in vitro maturation and the decrease was even more rapid during 4-6 h of culture (from 60% to 19%, p < 0.0005). The percentage of oocytes at M I-stage increased from 24% to 61% during 6-12 h of in vitro maturation, and it then decreased. By 24 h of culture, only 2% oocytes remained at M I-stage. Twenty one percent of the oocytes in this group developed to M II-stage at 16 h of culture, and by 24 h of culture, 91% were at M II-stage. (3) Statistic analysis of the meiotic progression (the duration of each cell cycle stage) of oocytes from 1.5-5 mm follicles showed that GV stage lasted from 0 to 3 h of culture, prometaphase-I stage was from 3.0 to 7.0 h, metaphase-I stage was from 7.0 to 14.6 h, anaphase-I/telophase-I was from 14.6 to 18.4 h and metaphase-II stage lasted from 18.4 to 24 h. (4) Whether the oocytes capable of GVBD and entrance of M I developed to M II, the timing of meiotic progression prior to M I was similar. In summary, our results provided necessary data for studies on the mechanisms and control of meiosis in mammalian oocytes.     

Overcoming the 2-cell block by modifying standard components in a mouse embryo culture medium.

The 2-cell block may be caused by inappropriate concentrations of commonly used constituents of embryo culture media. Almost all zygotes obtained by fertilizing CF1 ova with hybrid B6D2F1/CrlBR sperm did not develop beyond the 2-cell stage when cultured in Whittingham's medium M16. This 2-cell block was overcome by lowering the concentrations of NaCl, KCl, KH2PO4, glucose, and pyruvate, either individually or in combination. The effects of changing the concentration of either NaCl or KCl depend on the concentration of NaHCO3 in the medium. Although a high percentage of embryos grew to the 4-cell stage in several media with lowered concentrations of certain components, the media are not optimal for complete preimplantation embryo development since the yield of blastocysts is low.     

Factors Influencing the Production of a Novel Compound, 7,10-Dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic Acid,by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3 (NRRL B-18602) in Batch Cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002     

Regio- and enantio-selective glycosylation of tetrahydroprotoberberines by <Emphasis Type="Italic">Gliocladium deliquescens</Emphasis> NRRL1086 resulting in unique alkaloidal glycosides

The microbial transformation of a series of tetrahydroprotoberberines (THPBs, 1–5) by Gliocladium deliquescens NRRL1086 was investigated. In this research, the novel glycosylation of tetrahydroberberrubine (1) was observed with fast rate and high regio- and enantio-selectivity. One pair of unique enantiomorphic alkaloidal glycosides T-1 and T-2 was isolated and their structures were elucidated unambiguously by HR-MS, CD, 1D and 2D NMR spectrum. It is interesting that different amounts of glucose in the potato broth medium could influence the ratio of T-1 and T-2; in the 1.5% glucose medium, the ratio was about 15:1 and the yield of the S-form product T-1 may reach the theoretical maximum yield of about 50% which could provide one practical method to prepare the enantiomerically pure product and one alternative resolution method of tetrahydroberberrubine. The preliminary enzymatic research by using sodium dodecyl sulfate (SDS) and imidazole as glycosyltransferase and glycosidase inhibitors revealed that glycosyltransferase may contribute to glycosylation process. This is the first successful approach to glycosylation of tetrahydroprotoberberines.     

Acute-phase-response induction in rat hepatocytes co-cultured with rat liver epithelial cells.

The response of rat hepatocytes co-cultured with rat liver epithelial cells to conditioned medium (CM) from lipopolysaccharide (LPS)-activated monocytes was investigated by measuring the concentration of alpha 2-macroglobulin (alpha 2M), alpha 1-acid glycoprotein (AGP), albumin and transferrin, as well as the changes in glycosylation of alpha 1-acid glycoprotein. During an initial 8-day treatment with CM, concentrations of alpha 2M and AGP increased markedly over those of control culture, whereas concentrations of albumin and transferrin decreased. The glycosylation pattern of AGP indicated an important relative increase of the concanavalin A-strongly-reactive (SR) variant upon treatment. When CM addition to hepatocyte culture medium was stopped, the concentrations of the four proteins and the glycosylation pattern of AGP reverted to those of control cultures. Further addition (on day 15) to cultures of CM increased the concentration of alpha 2M and decreased albumin and transferrin concentrations. Although AGP concentrations did not increase above those of controls, the appearance of the SR variant was again stimulated by CM. These results show that, in co-culture, rat hepatocytes remain able to respond to repeated inflammatory stimuli.     

Role of sulfation in post-translational processing of gastric mucins.

1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.     

Increasing glucose in KSOMaa basal medium on culture Day 2 improves in vitro development of cloned caprine blastocysts produced via intraspecies and interspecies somatic cell nuclear transfer

This study was conducted to evaluate the efficiency of potassium simplex optimization medium with amino acids (KSOMaa) as a basal culture medium for caprine intraspecies somatic cell nuclear transfer (SCNT) and caprine-bovine interspecies somatic cell nuclear transfer (iSCNT) embryos. The effect of increased glucose as an energy substrate for late stage development of cloned caprine embryos in vitro was also evaluated. Enucleated caprine and bovine in vitro matured oocytes at metaphase II were reconstructed with caprine ear skin fibroblast cells for the SCNT and iSCNT studies. The cloned caprine and parthenogenetic embryos were cultured in either KSOMaa with 0.2 mM glucose for 8 days (Treatment 1) or KSOMaa for 2 days followed by KSOMaa with additional glucose at a final concentration of 2.78 mM for the last 6 days (Treatment 2). There were no significant differences in the cleavage rates of SCNT (80.7%) and iSCNT (78.0%) embryos cultured in KSOMaa medium. Both Treatment 1 and Treatment 2 could support in vitro development of SCNT and iSCNT embryos to the blastocyst stage. However, the blastocyst development rate of SCNT embryos was significantly higher (P < 0.05) in Treatment 2 compared to Treatment 1. Increasing glucose for later stage embryo development (8-cell stage onwards) during in vitro culture (IVC) in Treatment 2 also improved both caprine SCNT and iSCNT embryo development to the hatched blastocyst stage. In conclusion, this study shows that cloned caprine embryos derived from SCNT and iSCNT could develop to the blastocyst stage in KSOMaa medium supplemented with additional glucose (2.78 mM, final concentration) and this medium also supported hatching of caprine cloned blastocysts.     

Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes

Meiotic spindle structure and chromosome alignment were examined after porcine oocytes were cooled at metaphase II (M II) stage. Cumulus-oocyte complexes (COCs) collected from medium size follicles were cultured in an oocyte maturation medium at 39 degrees C, 5% CO(2) in air for 44 hr. At the end of culture, oocytes were removed from cumulus cells and cooled to 24 or 4 degrees C for 5, 30, or 120 min in a solution with or without 1.5 M dimethyl sulfoxide (DMSO). After being cooled, oocytes were either fixed immediately for examination of the meiotic spindle and chromosome alignment or returned to maturation medium at 39 degrees C for 2 hr for examination of spindle recovery. Most oocytes (65-71%) cooled to 24 degrees C showed partially depolymerized spindles but 81-92% of oocytes cooled at 4 degrees C did not have a spindle after cooling for 120 min. Quicker disassembly of spindles in the oocytes was observed at 4 degrees C than at 24 degrees C. Cooling also induced chromosome abnormality, which was indicated by dispersed chromosomes in the cytoplasm. Limited spindle recovery was observed in the oocytes cooled to both 4 and 24 degrees C regardless of cooling time. The effect of cooling on the spindle organization and chromosome alignment was not influenced by the presence of DMSO. These results indicate that the meiotic spindles in porcine M II oocytes are very sensitive to a drop in the temperature. Both spindle and chromosomes were damaged during cooling, and such damage was not reversible by incubating the oocytes after they had been cooled.     

Increasine in the rate of cell hybridization by the cytotoxic action of strophanthin (ouabain)]

Incorporation of ouabain (G-strophantin) in the culture medium 24 h after that of Sandai virus, at a final concentration of 2.10(-6)M/1, increases considerably the yield of man/mouse or primate/mouse cellular hybridization.     

Effect of Low Sulfate Concentrations on Lactate Oxidation and Isotope Fractionation during Sulfate Reduction by Archaeoglobus fulgidus Strain Z

The effect of low substrate concentrations on the metabolic pathway and sulfur isotope fractionation during sulfate reduction was investigated for Archaeoglobus fulgidus strain Z. This archaeon was grown in a chemostat with sulfate concentrations between 0.3 mM and 14 mM at 80°C and with lactate as the limiting substrate. During sulfate reduction, lactate was oxidized to acetate, formate, and CO₂. This is the first time that the production of formate has been reported for A. fulgidus. The stoichiometry of the catabolic reaction was strongly dependent on the sulfate concentration. At concentrations of more than 300 μM, 1 mol of sulfate was reduced during the consumption of 1 mol of lactate, whereas only 0.6 mol of sulfate was consumed per mol of lactate oxidized at a sulfate concentration of 300 μM. Furthermore, at low sulfate concentrations acetate was the main carbon product, in contrast to the CO₂ produced at high concentrations. We suggest different pathways for lactate oxidation by A. fulgidus at high and low sulfate concentrations. At about 300 μM sulfate both the growth yield and the isotope fractionation were limited by sulfate, whereas the sulfate reduction rate was not limited by sulfate. We suggest that the cell channels more energy for sulfate uptake at sulfate concentrations below 300 to 400 μM than it does at higher concentrations. This could explain the shift in the metabolic pathway and the reduced growth yield and isotope fractionation at low sulfate levels.     

Nutrient-limited continuous culture in the phauxostat

Stable steady-state growth of Escherichia coli B limited by succinate, phosphate, or sulfate ion over the range of specific growth rates of 0.025-0.51 h(-1) was achieved using pH-controlled auxostasis in the phauxostat. The concentration of the growth-limiting substrate in the growth vessel could be varied at will in the region of the Monod half-maximal saturation constants by adjusting the concentration of that substrate in the reservior (at constant buffering capacity) or by varying the population density of the culture through changing the buffering capacity of the medium in the reservoir.     

Metabolism of Deoxycorticosterone by Human Fecal Flora

21-Dehydroxylation, a feature of metabolism of corticoids in humans, was observed in mixed cultures of fecal flora of normal individuals on a Western diet. The model substrate, 11-deoxycorticosterone (DOC), was metabolized to 3alpha-21-dihydroxy-5beta-pregnan-20-one (THDOC), 3alpha-hydroxy-5beta-pregnan20-one (pregnanolone), and to two unidentified structures, metabolites X and Y. DOC was not metabolized in all media supporting growth of fecal flora. Conversion required an initial pH between 6.0 and 8.0. 21-Dehydroxylation occurred within 4 days of incubation in media inoculated with 10-minus 1 to 10-minus 7 fecal suspensions. In higher dilutions, containing obligatory anaerobes only, DOC was converted to metabolite X and sometimes also to metabolite Y. The yield of pregnanolone was related to the promptness with which the specimen was processed, to the presence of cysteine in the medium, and to the concentrationof substrate (optimum, 16 to 64 mug of DOC per ml). The yield of THDOC was related to the delay in the processing of the specimen, the concentration of substrate (maximum at 256 mug/ml), and aeration of the culture. Pure cultures of aerobic organism of fecal origin either failed to metabolize DOC or converted it to metabolite Y. Pure cultures of fecal anaerobes converted DOC to metabolite X and sometimes also to metabolite Y. Neither THDOC nor pregnanolone was produced by pure cultures.     

Sulfation and glucuronidation of acetaminophen by cultured hepatocytes reproducing in vivo sex-differences in conjugation on matrigel and type 1 collagen

Summary The sulfate and glucuronide conjugation of acetaminophen (APAP) by hepatocytes cultured on Matrigel or type 1 collagen was compared to APAP metabolism in vivo. The metabolic fate of low (15 mg/kg), medium (125 mg/kg), and high (300 mg/kg) doses of APAP injected intraperitoneally were determined in male and female rats. Males excreted more APAP as the sulfate conjugate than females, which correlated with the twofold greater APAP sulfotransferase activity in the male vs. females (301±24 vs. 156±18 pmol · mg⁻¹ protein · min⁻¹). Also, as sulfate conjugation became saturated, there was a dose-related shift in APAP metabolism from sulfate to glucuronide conjugation in both sexes. After death, the livers of the same animals were perfused with collagenase and the hepatocytes cultured in modified Waymouth’s medium on either Matrigel or rat-tail collagen, with various doses of APAP (0, 0.125, 0.25, 0.5, and 1.0 mM). Sex differences in APAP sulfation and glucuronidation persisted in culture for up to 4 days, with sulfation predominating in the male similar to in vivo. With increasing APAP concentration (dose), there was a saturation of sulfate conjugation and a shift to glucuronidation as observed in vivo. Sex differences in APAP sulfation and glucuronidation were no longer significant by Day 4 in culture. Sulfation, and to a lesser extent, glucuronidation, were more stable on Matrigel than collagen. We concluded that APAP metabolism of freshly isolated hepatocytes could replicate in vivo sex differences in conjugation, and that Matrigel was superior to collagen as substrate.     

Metabolism of NAD and N1-methylnicotinamide in growing and growth-arrested cells

Nicotinamide is metabolized primarily into NAD and N1-methylnicotinamide in cultured cells of normal rat kidney. The metabolic pathways for the nicotinamide metabolites are independently regulated and are influenced by the growth stage of the cells. N1-Methylnicotinamide levels are 1.5--2-fold elevated in cells growth-arrested by treatment with histidinol, thymidine, or picolinic acid, or by serum starvation. This increase is due to a more rapid rate of synthesis rather than decrease in excretion. The rates of both synthesis and degradation of NAD are increased in serum-starved cells so that the NAD concentration is the same as it is in growing cells. NAD and N1- methylnicotinamide levels are not significantly increased when the intracellular nicotinamide concentration is increased 20-fold by addition of excess nicotinamide to the culture medium, demonstrating that the size of the nicotinamide pool does not limit synthesis of these compounds. In medium containing normal amounts of nicotinamide, the apparent first-order rate constant for the decay of NAD, radioactively labeled in the nicotinamide moiety, is about 4 h-1. Labeled N1-methylnicotinamide is not metabolized, but rather is excreted into the medium with a first-order rate constant of 3.9 h-1. The rate of loss of label from NAD, but not from N1-methylnicotinamide, is increased about twofold by addition of excess nicotinamide to the culture medium. This could be explained by a dilution of a labeled nicotinamide pool which is formed during NAD degradation and which is recycled into NAD but not into N1-methylnicotinamide. The results demonstrate a rapid turnover of NAD at the bond joining nicotinamide and ADP-ribose, in agreement with previous studies. In addition, the results show that nicotinamide is metabolized into N1-methylnicotinamide with what appears to be a carefully regulated synthetic mechanism. The existence of significant amounts of N1-methylnicotinamide in cultured cells raises the question of the physiological importance of this compound.