CO2 Incorporation and 4-Hydroxy-2-Methylbenzoic Acid Formation during Anaerobic Metabolism of m-Cresol by a Methanogenic Consortium

The metabolism of m-cresol by methanogenic cultures enriched from domestic sewage sludge was investigated. In the initial studies, bromoethanesulfonic acid was used to inhibit methane production. This led to the accumulation of 4.0 ± 0.8 mol of acetate per mol of m-cresol metabolized. These results suggested that CO₂ incorporation occurred because each molecule of m-cresol contained seven carbon atoms, whereas four molecules of acetate product contained a total of eight carbon atoms. To verify this, [¹⁴C]bicarbonate was added to bromoethanesulfonic acid-inhibited cultures, and those cultures yielded [¹⁴C]acetate. Of the label recovered as acetate, 89% was found in the carboxyl position. Similar cultures fed [methyl-¹⁴C]m-cresol yielded methyl-labeled acetate. A ¹⁴C-labeled transient intermediate was detected in cultures given either m-cresol and [¹⁴C]bicarbonate or bicarbonate and [methyl-¹⁴C]m-cresol. The intermediate was identified as 4-hydroxy-2-methylbenzoic acid. In addition, another metabolite was detected and identified as 2-methylbenzoic acid. This compound appeared to be produced only sporadically, and it accumulated in the medium, suggesting that the dehydroxylation of 4-hydroxy-2-methylbenzoic acid led to an apparent dead-end product.     

Effect of water potential on seed germination

The response of seed germination to substrate water potential was determined for several plant species of the arctic tundra. Seeds were collected from Cape Thompson and Eagle Summit, Alaska and germinated on dialysis membranes over water solutions of polyethylene glycol with osmotic potentials of 0 to −6 bars. Germination did not occur with potentials below −3 bars, except for three fellfield species. Germination was delayed at lower osmotic potentials. Because the response of most species was similar, substrate water potential is probably not a factor affecting the establishment of most tundra plant species from seeds.     

Serum lipoproteins and albumin in the lecithin: Cholesterol acyltransferase reaction

The unique features of pig ovarian follicular fluids, i.e., presence of high density lipoprotein (HDL) only and lecithin: cholesterol acyltransferase (EC 2.3.1.43; LCAT) activity, provides a good model to study the effect of serum lipoproteins and serum albumin on the LCAT reaction. $\text{In}\text{vitro}$ cholesterol esterification is enhanced when very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions are added, but is inhibited when one or the other of these lipoproteins is absent. High concentrations of HDL₂ result in decreased activation which can be compensated for by the addition of the VLDL-LDL mixture. These findings suggest that the rate of cholesterol esterification in ovarian follicular fluid may be enhanced by providing the exogenous VLDL and LDL as the recipients of HDL-cholesteryl ester. The inhibition of LCAT activity caused by free fatty acid and lysophosphatidylcholine can be partially reversed by the addition of serum albumin, suggesting that serum albumin may regulate the LCAT reaction.     

Poly(ADPRibose) levels as a function of chick limb mesenchymal cell development as studied in vitro and in vivo.

NAD is converted into a chromatin-bound polymer, poly(ADPribose), with the excision of nicotinamide. In intact cells, the incorporation of labeled adenine, through NAD, into poly(ADPribose) has been correlated with the commitment and/or initial phenotypic expression of chick limb mesenchymal cells. Using an assay for chemical quantities of poly(ADPribose), we report here measurements of poly(ADPribose) during limb development in situ and during limb mesenchymal cell commitment and expressional events in cell culture. Substantial changes in the levels of poly(ADPribose) are observed during early phases of limb cell development either in situ (embryonic stages 22 to 26) or in culture (Days 1 to 4); during this time, we observed a threefold decrease in poly(ADPribose) per unit DNA (21 to 7 nmoles/mg DNA), as compared to relatively minor changes of 10 to 20% during later expressional events especially related to muscle development. These observations establish a correlation between cellular poly(ADPribose) levels and the early phases of chick limb mesenchymal cell differentiation and development.     

Rapid and efficient separation and identification of the two molecular forms of human adenosine deaminase by thin-layer gel filtration chromatography

Two molecular forms of adenosine deaminase have been found in human tissues. The column gel filtration method has been used for the separation of the two enzyme forms. Routine separation and analysis of the enzyme forms based on the molecular size difference can be achieved by thin-layer gel filtration on Sephadex G-200 superfine gel. The thin-layer method has been found to be more rapid and efficient than the column method. Enzymes in crude preparations can be studied effectively with the thin-layer method.     

Transcriptional diversity in myogenesis.

Duodenal morphogenesis in the chick embryo has been studied to see if changes in organization of extracellular fibers are associated with villus formation. Three major processes occur during morphogenesis; longitudinal ridges form, then these ridges become zigzag in shape, and finally a double row of villi form from each zigzag ridge. Tissues of different developmental stages were progressively trypsinized to remove cellular material and were prepared for scanning microscopy to show the organization of extracellular fibers. Results show that fiber systems of increasing complexity form as the dudoenum develops, and suggest that some cellular events such as initial ridge formation precede these changes. Tissues with longitudinal ridges contain only randomly organized fibers. In tissues with zigzag ridges, oriented fibers appear along the ridges and some extend laterally from flexures of each zigzag ridge, but interridge fibers are still randomly organized. When villi form, fibers in the body of the villus are random but fibers at the base of villi and between villi are highly oriented. Transmission microscopy and collagenase treatment of partially trypsinized tissue indicate that most, if not all extracellular fibers viewed by scanning microscopy are collagenous. Therefore, since collagen fiber organization is so closely related to morphogenetic changes, we presume it plays an important role.     

Intracellular localization of GDP-l-fucose: Glycoprotein and CMP-sialic acid: Apolipoprotein glycosyltransferases in rat and pork livers

A GDP-l-fucose:glycoprotein fucosyltransferase which transfers l-fucose to terminal β-N-acetyl-d-glucosaminyl residues of sialidase-, β-galactosidase-treated α₁-acid glycoprotein and a CMP-sialic acid:glycoprotein sialyltransferase acting on sialidase-treated apolipoprotein-Ala₁ from human very low density lipoprotein have been shown to be concentrated in rat liver Golgi apparatus preparations at enrichments of 40- and 45-fold, respectively, and in pork liver Golgi-rich fractions at enrichments of 35- and 20-fold, respectively. A second fucosyltransferase acting on sialidase-treated α₁-acid glycopretein was absent from rat liver and was enriched only 13-fold in a pork liver Golgi-rich fraction. The smooth-surfaced microsome fraction was the only other rat liver subcellular fraction with appreciable levels of the GDP-l-fucose: β-N-acetyl-d-glucosaminide fucosyltransferase and the lipoprotein sialyltransferase (enrichments of 2.6- and 5.2-fold, respectivley). This enrichment could not be attributed to the plasma membrane content of the smooth microsome fraction since plasma membrane fractions from rat liver were shown to have relatively low concentrations of these two transferases (enrichments of 0.3 or less). Rat liver plasma membrane was also shown to have similarly low relative specific activities for three other glycosyltransferases (sialyl-, galactosyl-, and N-acetylglucosaminyl-). The accurate determination of the glycosyltransferase activities of the plasma membrane fraction required the use of relatively low concentrations of plasma membrane and relatively high concentrations of nucleotide-sugars in order to avoid interference by the high nucleotide-sugar pyrophosphatase and hydrolase activities of this fraction.     

The possible differentiation of osteogenic elements in vitro from chick limb mesodermal cells. I. Morphological evidence

In the developing chick limb bud, myogenic, fibrogenic, chondrogenic, and osteogenic tissues are derived from embryonic mesenchyme. Previous reports show that when stage 24 limb mesenchymal cells are cultured in vitro, chondrocytes, fibrocytes, and myocytes can be identified on the basis of morphological and biochemical parameters. The studies reported here clearly demonstrate that, in similar cultures, crystalline calcium phosphate material is deposited in the chondrocytic and fibrocytic matrices. Such crystalline material is not observed before Day 10 of culture life. Subsequent to Day 10 of culture, first amorphous, then crystalline calcium phosphate is observed. On the basis of light and electron microscopic analysis, it appears that the in vitro calcification phenomenon closely resembles the morphological and temporal sequence of osteogenesis observed in vivo.