Lipolytic activities of a lipase immobilized on six selected supporting materials

Six different types of materials including PVC, chitosan, chitin, agarose, Sepharose, and Trisacryl were evaluated for their lipase-coupling efficiencies. Among those tested, chitosan yielded the highest amount of lipase (79 mg/mL packed gel) immobilized but with lowest oil hydrolytic activity (0.03 mg eq/mL gel). The amount of lipase immobilized was affected by the length of the hydrocarbon chain attached to the PVC matrix but not by the pore size of the supports used. On the other hand, the specific activity of the immobilized lipase was affected by the pore size but not by the chain length of the hydrocarbon attached to the support. After immobilization, the optimal reaction pH was shifted from 7.5 to 8.5 and the optimal reaction temperature from 35 to 45-55 degrees C. Lipase immobilized on PVC exhibited higher thermal stability than that on agarose. The half-life of the PVC immobilized lipase operating at 30 degrees C in a packed-bed reactor was estimated to be about 400 h.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Assignment of the sorbitol dehydrogenase locus to human chromosome 15 pter→q21

The locus for sorbitol dehydrogenase (SORD, E.C. 1.1.1.14) has been shown to segregate with hexosaminidase A and mannose phosphate isomerase in a series of human-Chinese hamster somatic cell hybrids. Cytogenetic analysis supports the assignment to chromosome 15 and further defines the gene locus to the region 15pterq21.This research was supported by the Medical Research Council of Canada (MT 4061), the Children's Hospital of Winnipeg Research Foundation, Inc., and the Department of Health, Province of Manitoba (H.S.W.).     

Regulation of proton leakage from broken chloroplasts by CF0.

Measurements of proton translocation in CF₁-depleted, N, N′-dicyclohexylcarbodiimide-resealed broken chloroplasts were made under different light intensities. Kinetic analysis of the data shows that the outward leakage of accumulated protons through CF₀ is still dependent on light intensity with a first-order rate constant equal to mR₀, where R₀ is the initial rate of proton uptake which normally increases with light intensity and m is a characteristic constant which is independent of proton gradient and light intensity. Measurements of proton translocation in these modified chloroplasts cross-linked with glutaraldehyde under illumination and in the dark respectively suggest that the light-dependent proton leakage through CF₀ is regulated by conformation change in the membrane. It is proposed that the ovserved regulation of proton leakage through the CF₁.CF₀ complex in native chloroplasts is for optimizing the steady state synthesis of ATP under different light intensities.     

The synthesis and the 1h- and 13c-nuclear magnetic resonance spectroscopy of the cyclic sulfites of some sugars

Treatment of methyl β-D-ribofuranoside with thionyl chloride in hexamethyl-phosphoric triamide gives two diastereoisomeric methyl 5-chloro-5-deoxy-β-D-ribo-furanoside 2,3-cyclic sulfites. Similar cyclic sulfites are formed from benzyl β-D-ribofuranoside and 1,4-anhydro-DL-ribitol. If acetonitrile is substituted for hexa-methylphosphoric triamide, the cyclic sulfites are the main products, and only traces of the chlorinated sugars are formed. ¹H- and ¹³C-n.m.r.-spectral analysis of these reactions demonstrated that one of the diastereomers preponderates. The structure of these cyclic sulfites was established by comparison of the ¹H-n.m.r. spectra with those of the propylene sulfites. Treatment of 1,2-O-isopropylidene-α-D-glucofuranose (14) with thionyl chloride in hexamethylphosphoric triamide yields 3-chloro-3-deoxy-1,2-O-isopropylidene-α-D-allofuranose 5,6-cyclic suffite. In contrast to the 2,3-cyclic suffites, which are stable, the cyclic sulfites derived from 14 slowly decompose at room temperature.     

Rapid mapping of restriction sites of a DNA: restriction of agarose gel and two-dimensional analysis of end-labeled DNA.

A new two-dimensional technique for the mapping of restriction sites is presented. Linear DNA labeled at both ends is first partially digested with the restriction endonuclease for which a map is desired. Following electrophoresis of the partial digest in an agarose gel, complete digestion of the fragments in the gel matrix with a second restriction enzyme is carried out. Electrophoresis in the second dimension resolves two sets of labeled spots: one set from the left and the other from the right end. For a given band of the autoradiogram of the first dimension gel, the mobility of the band gives the size of the DNA fragment, and therefore the distance of a particular restriction site from one of the ends of the original linear DNA. The mobility of the labeled spot derived from this band in the second dimension gel allows one to distinguish whether the distance deduced above is from one end or the other. Additional information about the location of one set of restriction sites for one enzyme relative to those for a second enzyme can also be obtained using the two-dimensional method. The advantages of the technique are the small amount of DNA required and the rapidity with which many maps can be constructed from one labeled DNA. As a test of the method, maps for the HindIII and HaeIII cleavage sites of circular phage PM2 DNA have been obtained, after first converting the DNA to the linear form by digestion with HpaII.     

Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and of incorporation of acetate into cholesterol in homozygous hypercholesterolemic fibroblasts by ferritin-low density lipoprotein conjugates.

Conjugates of ferritin with low density lipoproteins (LDL) were prepared and separated by sucrose gradient centrifugation. These conjugates, at cholesterol concentration of 100--132 microgram/ml, caused a greater than 90% suppression of hydroxymethylglutaryl coenzyme A reductase activity and of acetate incorporation into cholesterol in cultured skin fibroblasts from a normal subject as well as from a subject with homozygous familial hypercholesterolemia. The half maximal inhibition concentration was approx. 10 microgram/ml cholesterol for LDL and ferritin . (LDL)2 and 5 microgram/ml for (ferritin)2 . LDL in both cell lines. In contrast, native low density lipoproteins have only a minimal inhibitory effect in homozygous cells. The ability of the conjugates to stimulate the incorporation of oleate into cholesteryl esters was also equal in the two cell lines, although the conjugates were only 10% as active as low density lipoproteins in the normal cells. LDL reduced the ferritin . (LDL)2-mediated suppression of hydroxymethylglutaryl-CoA reductase activity in homozygous cells while ferritin . (LDL)2 reduced the LDL-mediated stimulation of cholesteryl ester formation in normal cells.     

A novel temperature-sensitive mammalian cell line exhibiting defective prophase progression

A temperature-sensitive mammalian cell line has been isolated which grows and divides normally at the permissive temperature of 33°C. When incubated at 39°C, the nonpermissive temperature, interphase cells continue to enter a prophase-like state. Chromatin-like material condenses and coalesces into dark-staining clumps rather than into discernible chromosomes. Disappearance of the nuclear boundary is observed, but re-formation of the boundary around the clumps fails to occur. Incorporation of labeled precursors reveals a decrease in protein synthesis which is accompanied by a slower decrease in DNA synthesis. Approximately 0.2% of the mutant cells revert in their capability of growth and cell division at 39°C. These “revertants” are found to contain a higher number of chromosomes. The isolation of this mutant is based on the initial observation that the cells become rounded at the nonpermissive temperature. The cell-rounding process characteristic of mitotic cells should serve as a useful marker in the isolation of mitotic mutants.     

Growth of Enterobacter aerogenes in a chemostat with double nutrient limitations.

The behavior of Enterobacter aerogenes during growth in chemostats limited by single and double nutrient restrictions was examined. On the assumption that different essential nutrients act to limit growth in different ways, we selected pairs of nutrients likely to affect different aspects of metabolism. Results show that macromolecular cell composition can be controlled by using more than one nutrient restriction. The polysaccharide content of the cells is readily manipulated by the ratio of carbon to nitrogen in the inlet nutrients. Also, at low dilution rates, ratios of protein to ribonucleic acid are dependent on the ratio of phosphate to nitrogen in the input nutrients. An examination of both acetic acid and metabolite production (as measured by ultraviolet absorbance of culture filtrates) showed that accumulation of these products was dependent on both dilution rate and type of nutrient limitation(s). These results were examined in terms of the problems of translation of batch to continuous culture processes and the use of selected nutrient limitations to control noncellular product formation.