Acid-soluble Material Research Articles

Expression, Purification, and Characterization of an Active RNase H Domain of the Hepatitis B Viral Polymerase

The replication of the hepatitis B viral DNA genome proceeds through a pregenomic RNA intermediate. This pregenomic RNA subsequently serves as the template for the formation of the viral DNA by the reverse transcriptase activity of the viral P gene product. The P gene product is believed to be a multifunctional enzyme with DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, and RNase H activities. Detailed biochemical studies of this protein have not been performed because of the inability to obtain sufficient amounts of the enzyme from the virus and by the inability to produce the enzyme in heterologous expression systems. The RNase H activity is essential for viral replication and is believed to be responsible for the degradation of the RNA pregenomic intermediate as well as for generating the short RNA primer that is required for DNA second strand synthesis. We have assembled an expression vector which directs the synthesis of a protein that corresponds to the putative RNase H domain of the P gene product and having a carboxyl-terminal polyhistidine tag to facilitate purification. The protein has been expressed in Escherichia coli and purified to yield 1-2 mg of protein/liter of culture. This protein has RNase H activity as defined by its ability to degrade the RNA component of RNA-DNA hybrids but not the DNA component. The RNase H has a basic optimum pH, is active only in the presence of reducing agents, and is dependent on the presence of divalent cations, with magnesium being preferred over manganese.

2',3'-Dideoxycytidine metabolism in a new drug-resistant cell line.

2',3'-Dideoxycytidine (ddC) is a nucleoside analogue that inhibits human immunodeficiency virus type 1 (HIV-1) replication in vitro and is currently used in the therapy of acquired immune deficiency syndrome (AIDS). This compound exerts a delayed cytotoxicity due to inhibition of mitochondrial DNA (mDNA) synthesis. Long-term exposure of U937 human monoblastoid cells to ddC resulted in a time- and concentration-dependent decrease in mDNA content and Rhodamine 123 fluorescence. However, after 2 months on 0.1 microM ddC, a drug-resistant cell line (U937-R) with 66% of the normal amount of mDNA was isolated. ddC transport in U937 and U937-R cell lines was similar. In contrast, U937-R accumulated ddC phosphorylated derivatives at a much lower rate and to a reduced concentration into acid-soluble material. The rate of 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) formation in U937-R cells was almost one-third of that measured in normal cells, although the rate of ddCTP catabolism was similar in both cell lines. Dideoxyliponucleotide (ddCDP-choline and ddCDP-ethanolamine) formation was also much slower (between one-half and one-third as fast) in U937-R than in control cells, although catabolism occurred at similar rates. ddC was phosphorylated by a cytoplasmic deoxycytidine kinase in both cell lines. This enzyme showed Km values for ddC of 80 +/- 7 and 140 +/- 9 microM in U937 and U937-R cells respectively. Furthermore, Vmax was 12 +/- 1.1 and 7.8 +/- 0.5 pmol/min per mg of protein in U937 and U937-R. Thus resistance to ddC toxicity may be due to cells' decreased ability to accumulate intracellular ddC anabolites, which may depend on cytoplasmic deoxycytidine kinase.

Effects of dexamethasone on protein degradation and protease gene expression in rat L8 myotube cultures

We examined the effects of a synthetic glucocorticoid (dexamethasone; Dex) on proteolysis and on protease messenger RNA (mRNA) concentrations in rat L8 skeletal myotube cultures. Protein degradation was measured as release of radioactive trichloroacetic acid-soluble material from intracellular proteins pre-labelled with [ 3H]tyrosine. Dex (1 μM) stimulated protein degradation ( P < 0.01). This effect was entirely blocked by the glucocorticoid antagonist, RU38486 (mifepristone; P < 0.01). Hence, actions of Dex on muscle protein degradation are mediated via intracellular glucocorticoid receptors. Molecular mechanisms by which glucocorticoids stimulate protein degradation in skeletal muscle are not known. Here, we investigated the regulation of protease (cathepsin B, cathepsin D, proteasome C2 subunit and m-calpain) mRNA concentrations by Dex in cultured L8 muscle cells. Cathepsin B mRNA concentration was enhanced 3.3-fold by Dex. This effect was blocked by RU38486. RU38486 alone did not affect cathepsin B mRNA concentration or mRNAs of other proteases. Concentrations of cathepsin D and m-calpain mRNAs were also increased by Dex. These effects were also abolished by RU38486. Proteasome C2 mRNA was unaffected by Dex and Dex reduced α-tubulin mRNA. Thus, glucocorticoids specifically regulate the concentrations of mRNAs encoding some proteases in muscle cells. The regulation of protease mRNA concentration is mediated via interaction between Dex with glucocorticoid receptors and is independent of the actions of Dex on mRNA encoding house-keeping proteins. These changes may underlie glucocorticoid-dependent control of proteolysis in muscle.

A conditional suicide system in Escherichia coli based on the intracellular degradation of DNA

The potential risks associated with the intentional or unintentional release of genetically engineered microorganisms led to the construction of biological containment systems by which bacteria are killed in a controlled suicide process. In previously published suicide systems, cell killing was caused by proteins destroying the cell membrane or cell wall. Here a conditional cell killing system based on the intracellular degradation of cellular DNA is presented. The nuclease gene used was that of the extracellular nuclease of Serratia marcescens. The nuclease gene was deleted for the leader-coding sequence, and the truncated gene was put under the control of the lambda pL promoter. Following thermoinduction of the nuclease gene cassette in Escherichia coli, cell survival dropped to 2 x 10(-5), and more than 80% of the radioactively labeled DNA was converted to acid-soluble material within 2.5 h in the absence of cell lysis. The majority (84%) of clones which survived thermoinduced killing turned out to be as sensitive to a second thermoinduction as the original strain. The other clones showed somewhat slower killing kinetics or slightly higher final levels of survivors. The suicide system described combines the regulated killing of cells with the destruction of intracellular DNA otherwise potentially available for horizontal gene transfer processes.

Trace metal geochemical association in sediments from the Cairns region of the Great Barrier Reef, Australia

Some physico-chemical properties and concentrations of the trace metals Cu and Zn have been measured in sediments from the Coral Sea between the coast at Cairns (146°E 17°S) and the Great Barrier Reef. Surface sediments along an 18 km transect and a mid-transect core were examined. The acid soluble material increased from 20 to 36% and Ca increased from 12 to 47 mg g −1 with increasing distance from shore. Fe passed through a maximum of 32 mg g −1 at about 10 km offshore. The results show the pattern of sedimentation, with Fe representing terrestrial input, and acid soluble material and Ca representing CaCO 3. The CaCO 3 was mainly calcite with only minimal amounts of aragonite (coral). Microscopic examination showed shell material but no coral debris. The Cu concentration in the surface sediments (2.0–17 μg g −1) decreased with increasing distance from Cairns harbour (correlation coefficient Cu: dist −0.793). The Zn concentration (40–53 μg g −1) followed Fe (correlation coefficient Zn:Fe 0.932) with a maximum about 10 km offshore. There was no correlation of Cu and Fe (Cu:Fe 0.041) or Cu and Zn (Cu:Zn 0.178) showing that Cu and Zn came from different sources. The principle source of Cu is in the Cairns harbour region and may be a result of previous use of Cu-based marine antifouling paints. The Zn is dominantly associated with terrestrial inputs. Within the sediment profile 11 km offshore, the pattern of particle size was consistent with the top 6 cm of sediment having been resuspended during a cyclone. 210Pb concentrations also showed evidence of sediment mixing. Deeper in the sediment the average sedimentation rate was estimated at about 3.5 mm yr −1. Most of the CaCO 3 down the profile was calcite with no evidence of substantial input of coral material. The different sources of Cu and Zn could not be recognized within the core at this distance offshore.

Mechanism for the changes in levels of glutathione upon exposure of cultured mammalian cells to tertiary-butylhydroperoxide and diamide.

Qualitative and quantitative changes associated with cellular glutatione (GSH) in response to oxidants were investigated in cultured Chinese hamster V79 cells. Incubation of cells with benzoylperoxide (BZP), tert-butylhydroperoxide (t-BuOOH), hydrogen peroxide or diamide for 1 h reduced the level of total GSH (GSH + GSSG). Among the oxidants, t-BuOOH and diamide caused an increase in levels of glutathione disulfide (GSSG) and a resultant increase in the ratio of the level of GSSG to the level of total GSH, suggestive of the induction within the cells of a pro-oxidant state by the oxidants. o-Phenanthroline, a chelator of divalent ion, almost completely suppressed the decrease in levels of total GSH caused by t-BuOOH while it did not suppressed either increases in levels of GSSG or increases in the ratio of the levels of GSSG to that of total GSH caused by the hydroperoxide. These results suggest that reactive oxygen radicals are involved in the decrease in levels of GSH by treatment with t-BuOOH but not in the increase in the level of GSSG. After treatment with either t-BuOOH or diamide for 1 h, the level of GSH rapidly increased to more than twice the control level during 15-45 min of post-treatment incubation. o-Phenanthroline almost completely suppressed the increase in levels of GSH caused by t-BuOOH, while it did not affect the changes caused by diamide, suggesting a difference between the mechanisms by which t-BuOOH and diamide cause increases in levels of GSH. It seems likely that reactive oxygen radicals participate not only in the decrease in levels of GSH caused by t-BuOOH but also in the rapid increase that occurs after such treatment. Hence, the first decrease in levels of GSH by the hydroperoxide may be causally related to the latter increase. The amount of [35S]-cysteine taken up by cells after treatment with t-BuOOH was about one half of that taken up by control cells. By contrast, the rate of incorporation of radioactive cysteine into acid-soluble material increased to more than twice that of the controls after treatment with t-BuOOH. The increase in the rate of incorporation of [35S]cysteine into acid-soluble material caused by t-BuOOH was not a consequence of inhibition by the hydroperoxide of utilization of cysteine for protein synthesis. Inhibition of protein synthesis by cycloheximide caused neither an increase in the incorporation of cysteine into acid-soluble material nor an increase in rate of biosynthesis of GSH.(ABSTRACT TRUNCATED AT 400 WORDS)

Elevated Activity of Cathepsin L-like Protease in the Jellied Meat of Japanese Flounder.

The cause of the jelification of Japanese flounder meat was investigated. Infection by parasites was not histologically detected, while the arrangement of myofibrils was notably disturbed. Large amounts of trichloroacetic acid soluble materials and a breakdown of myosin heavy chain suggested the occurrence of proteolytic degradation in the jellied meat.Crude extract of the jellied meat showed proteolytic activity on myosin heavy chain, which was hardly detected in the normal meat. The extract hydrolyzed Z-Phe-Arg-MCA most preferentially among the synthetic peptide substrates tested. Activities on myosin heavy chain and Z-Phe-Arg-MCA were inhibited by leupeptin, antipain, and E-64, but not by soybean trypsin inhibitor or pAPMSF (a specific inhibitor for trypsin-like proteases), suggesting that the activities were ascribed to a cysteine protease(s). Both myosin heavy chain and Z-Phe-Arg-MCA hydrolyzing activities were co-eluted from TSK G3000SWXL column at an eluting position whose molecular weight corresponded to 34, 000. Together with these results, the protease activity elevated and possibly involved in the jelification of Japanese flounder meat was taken to be due to an endogenous cathepsin L-like protease.

Persistence of Free Plasmid DNA in Soil Monitored by Various Methods, Including a Transformation Assay

The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23 degrees C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ.

Influence of eicosapentaenoic acid (20:5, n-3) on secretion of lipoproteins in CaCo-2 cells.

CaCo-2 cells, grown on filter membranes, were used to study the effects of fatty acids on cellular metabolism of triacylglycerol and phospholipids. The rate of triacylglycerol secretion was enhanced more than 2-fold, from 1 to 2 weeks after reaching confluency, in the presence of 0.6 mM fatty acids. Triacylglycerol secretion and oxidation of oleic acid increased 2- and 9-fold, respectively, with this culture system, as compared to cells grown on conventional plastic dishes. Eicosapentaenoic acid (20:5 n-3), when compared to oleic acid, did not reduce formation of triacylglycerol or enhance phospholipid synthesis in CaCo-2 cells during short term (less than 24 h) experiments, when the cells resided on membranes, regardless of what type of radioisotopes were used as precursors in the incubation media. However, the n-3 fatty acid was preferentially incorporated into phosphatidylinositol, lysophosphatidylcholine, and sphingomyelin, as compared to oleic acid. The disappearance from the apical medium and cellular uptake of labeled eicosapentaenoic and oleic acid were similar during incubations up to 24 h, and the metabolism of these fatty acids to acid-soluble materials and CO2 was equal. Light scattering analysis indicated that secreted lipoproteins of density less than 1.006 g/ml were in the same size-range as chylomicrons derived from human plasma. Assessment of secreted apolipoprotein B showed that by incubating CaCo-2 cells with oleic acid, apolipoprotein B levels increased approximately 1.4-fold when compared to cells incubated with eicosapentaenoic acid, whereas the amount of triacylglycerol and size-range of particles were similar for the two fatty acids. Our data indicate that CaCo-2 cells grown on filter membranes exhibit enterocyte-like characteristics with the ability to synthesize and secrete chylomicrons. Eicosapentaenoic acid and oleic acid are absorbed, metabolized, and influence secretion of lipoprotein particles in a similar way, except for some differences in incorporation of the fatty acids into certain phospholipid classes and a reduced secretion of apolipoprotein B. The culture conditions, including time after confluency and cellular support, are critical for the rate of secretion in the presence of eicosapentaenoic acid and oleic acid.

Autocatalytic degradation of proteins in extracts of a brewing strain of Saccharomyces cerevisiae. The role of endoproteinases and exopeptidases

Cells of a brewing strain of Saccharomyces cerevisiae NCYC 1109 were harvested after the end of fermentation, but before decline in viability had commenced, and extracted by passage through an Eaton press. Immediately after preparation the proteolytic enzymes PrA and CpY, but not PrB, could be detected in the extracts. After 24 h incubation at 25°C all three enzymes were found. PrA activity was unaffected by incubation whereas CpY activity increased up to threefold. Measurement of the release of acid-soluble material in the extracts themselves over an 18-h period at 25°C and a range of pH values using the Lowry and trinitrobenzoyl sulphonic acid methods, suggested that the endoprotease, PrB, was a major contributor to autocatalysis. Further studies using pepstatin, phenylmethylsulphonyl fluoride (PMSF), HgCl2, EDTA, bestatin and ZnCl2 confirmed the importance of PrB but also indicated that about 30% of the initial proteolytic degradation was due to another enzyme. This enzyme was inhibited by Zn2+ and Hg2+ but not by PMSF or pepstatin, and could be the recently characterised enzyme, PrE.

Preliminary Evidence that Incorporation of 5-Fluorouracil into RNA Correlates with Antitumor Response

A comparative study of two different species of a fluorouracil assay was conducted on 11 patients who had carcinoma that was deemed unresectable after the surgical operation. For these patients FU at a dose of 10 mg/kg was intravenously administered before operation, and portions of the tumors were resected within 120-150 min to assay both the (FU)RNA/RNA and FU/protein. After surgery, all patients were given FU alone either intravenously or orally. The FU, which was in an acid-soluble material (FU/protein), was not related to the antitumor effect of FU. However, the FU in RNA [(FU)RNA/RNA)] was found to be related to the antitumor effect of FU. When the concentration of (FU)RNA/RNA was above approximately 200 ng/mg, FU was effectual in unresectable carcinomas. It is probable that the (FU)RNA/RNA may be more suitable than FU/protein for predicting the antitumor effect of FU.

In vitro toxicity and metabolism of 2′,3′-dideoxycytidine, an inhibitor of human immunodeficiency virus infectivity

U937 human monoblastoid cell growth was inhibited in a concentration-dependent manner by 2′,3′-dideoxycytidine (ddCyd) (an antiretroviral drug) up to 500 μM. Cell growth inhibition was associated with a pronounced increase in cell volume, however this was not due to cell ATP or NAD + depletion that could effect osmotic balance or DNA repair. This ddCyd toxicity paralleled the accumulation of ddCyd into acid soluble material where 2′,3′-dideoxycytidine-5′-triphosphate (ddCTP) was the predominant labelled nucleotide up to an extracellular ddCyd concentration of 150 μM. At higher ddCyd concentrations, the amount of 2′,3′-dideoxycytidine-5′-diphosphate (ddCDP) became predominant over ddCTP. This increase of phosphorylated dideoxycytidine in U937 cells was also associated with an increased incorporation of the drug into cell DNA suggesting a possible toxicity mechanism. That ddCyd does indeed become cytotoxic to human cell by incorporation into DNA was shown by incubating human resting and stimulated lymphocytes with ddCyd. While the drug does not affect cell viability in resting cells it strongly affects cell proliferation upon phytohemagglutinin (PHA) addition.

Effects of the prolyl 4-hydroxylase proinhibitor HOE 077 on human and rat hepatocytes in primary culture.

Alterations in extracellular matrix occur in many chronic liver diseases leading to the formation of hepatic fibrosis. We have studied the effects of the putative hepatoselective fibrosuppressive compound HOE 077, a proinhibitor of prolyl 4-hydroxylase, on normal adult human and rat hepatocytes in primary culture. In human hepatocyte cultures, the cytotoxicity of HOE 077 was assessed after a 20-h treatment at concentrations ranging from 0.125 to 2 mg/ml of medium. No significant change was found in cell morphology, neutral red uptake, red oil staining, lactate dehydrogenase release, tetrazolium salt reduction, ethoxyresorufin O-deethylase activity and protein synthesis; however, HOE 077 slightly decreased DNA synthesis at 2 mg/ml. In rat hepatocyte cultures, the cytotoxicity of the compound was assessed by testing the same parameters after a daily exposure of cultures for 2 days or 4 days, at concentrations ranging from 0.25 to 4.5 mg/ml of medium. Whatever the concentration, the compound had no obvious morphological effect. However, hepatocytes were less spread at the concentration of 4.5 mg/ml. HOE 077 at 2 mg/ml slightly decreased neutral red uptake but was without obvious effect on protein synthesis after 2 days. By contrast, on day 4, protein synthesis was markedly reduced in hepatocyte cultures exposed to HOE 077 at 4.5 mg/ml. Hydroxyproline content determination in media from 4-day-old hepatocyte cultures incubated with HOE 077 at 0.5 to 4.5 mg/ml, showed a dose-dependent decrease in the hydroxyproline/proline ratio in acetic acid soluble material. By indirect immunoperoxidase, intracellular collagen IV was found to be inhibited in hepatocyte cultures after 4 days of exposure to 4.5 mg/ml HOE 077.(ABSTRACT TRUNCATED AT 250 WORDS)

Retroendocytosis of high density lipoproteins by the human hepatoma cell line, HepG2.

When human HepG2 hepatoma cells were pulsed with 125I-labeled high density lipoproteins (HDL) and chased in fresh medium, up to 65% of the radioactivity released was precipitable with trichloroacetic acid. Cell-internalized 125I-HDL contributed to the release of acid-precipitable material; when cells were treated with trypsin before the chase to remove 125I-HDL bound to the outer cell membrane, 50% of the released material was still acid-precipitable. Characterization of the radioactive material resecreted by trypsinized cells revealed the presence of particles that were similar in size and density to mature HDL and contained intact apolipoproteins (apo) A-I and A-II. The release of internalized label occurred at 37 degrees C but not at 4 degrees C. Monensin, which inhibits endosomal recycling of receptors, decreased the binding of 125I-HDL to cells by 75%, inhibited the release of internalized radioactivity as acid-precipitable material by 80%, and increased the release of acid-soluble material by 90%. In contrast, the lysosomal inhibitor chloroquine increased the association of 125I-HDL to cells by 25%, inhibited the release of precipitable material by 10%, and inhibited the release of acid-soluble radioactivity by 80%. Pre-incubation with cholesterol caused a 50% increase in the specific binding, internalization, and resecretion of HDL label. Cholesterol affected the release of acid-precipitable label much more (+90%) than that of acid-soluble material (+20%). Taken together, these findings suggest that HepG2 cells can bind, internalize, and resecrete HDL by a retroendocytotic process. Furthermore, the results with cholesterol and monensin indicate that a regulated, recycling, receptor-like molecule is involved in the binding and intracellular routing of HDL.

In vivo release and clearance of rat platelet phospholipase A 2

Intravenous injection of ADP into rats produced a rapid increase of plasma phospholipase A 2 activity with a concomitant decrease in platelet count. Phospholipase A 2 activity in plasma reached a peak within 1 min and thereafter declined sharply. This phospholipase A 2 activity was reactive with a monoclonal antibody specific for rat platelet-derived phospholipase A 2. These findings, together with the fact that ADP is a stimulant specific for platelets, suggest that the phospholipase A 2 observed may be released into plasma from activated platelets in vivo. When platelet-derived phospholipase A 2 was purified, labeled with 125I, and injected intravenously into rats, the radioactivity in the plasma decreased rapidly: the half-life of the enzyme in the blood stream was less than 30 s. Addition of either heparin or anti-phospholipase A 2 monoclonal antibody directed against the domain of the enzyme responsible for binding to heparin retarded the clearance of the enzyme, suggesting that phospholipase A 2 might be adsorbed onto heparan sulfate proteoglycans on the endothelial cell surface. Examination of the distribution of radioactivity in vivo revealed that most of the enzyme was rapidly taken up by the liver and degraded to acid-soluble materials.

A new lethal brittle bone syndrome with increased amount of type V collagen in a patient

A new lethal brittle bone disease is described in three patients with slender long bones, thin ribs, hypomineralized calvaria, and normal facial appearance. In spite of several limb fractures this syndrome can be differentiated from the lethal forms of osteogenesis imperfecta and is better related to the thin-bone group of lethal dysplasias. Biochemical investigation of collagen from one of the patients by the use of gel electrophoresis and high-pressure liquid chromatography analyses failed to demonstrate any evident defect in the structure of type I collagen chains. Nevertheless collagen extractability from the dermis was altered owing to an increase in the proportion of acid-soluble material. Tritium-proline labeling of cultured fibroblasts confirmed the reduction in total collagen synthesis. This was attributed to a lower type I and type III amount whereas type V collagen level was markedly increased in the cell layer. RNA analysis of the three collagen types with the appropriate cDNA probes confirmed the protein data. Electron microscopic examination of bone and skin showed morphologically abnormal fibroblasts and osteoblasts with an abundant distended rough endoplasmic reticulum and an altered plasma membrane. Unexpected thin fibrils with a banding pattern and surrounding the type I fibrils were observed. They might represent type V collagen. We suggest that, in this patient, the moderate decrease in type I collagen amount is insufficient to account for the radiological findings and that type V collagen overproduction could play a role in the bone brittleness by interfering with the process of mineralization.

Gastric luminal digestion of lactoferrin and transferrin by preterm infants

Lactoferrin, a milk iron-binding protein, may play antimicrobial, iron-absorptive and growth-promoting roles in the developing gastrointestinal tract. To perform such functions, lactoferrin must survive digestive processes in the gut lumen in an active form. We investigated the gastric digestion of lactoferrin in addition to that of the other milk proteins, transferrin and casein, in preterm infants by measuring their degradation during incubation in vitro at 37°C with gastric fluid at pH 1.8, 3.2 and 5.8. Fluid was obtained 1 h after a milk feeding, a time of maximum peptic activity, from 12 infants with a mean gestational age of 29.7 ± 0.8 weeks at birth and a postnatal age of 24.7 ± 3.2 days at sampling. Hydrolysis of all three proteins as indicated by generation of trichloroacetic acid soluble material from iodinated substrate was maximal at acid pH and declined by greater than 75% at pH 5.8, lactoferrin was less rapidly degraded than casein at low pH and transferrin breakdown was intermediate. Analysis of reaction mixtures by SDS-polyacrylamide gel electrophoresis showed degradation of lactoferrin and transferrin to low molecular weight products at pH 3.2 but minimal breakdown at pH 5.8. Several discrete fragments were generated at low pH, including species with molecular weights of 41,000–42,000 which may represent half-molecules. We conclude that dietary lactoferrin and transferrin may be degraded by preterm infant gastric fluid to discrete species, but that hydrolysis may be minimal at the prevailing postprandial pH. Consequently they may be rendered available for possible subsequent biological action within the infant.

Hexose Transport and Metabolism in Cultured Fibroblasts Derived from Normal and Alzheimer Disease Affected Individuals

ABSTRACTThe transport and metabolism of glucose was compared in cultured skin fibroblasts derived from normal and Alzheimer disease affected individuals. No significant differences were observed in sugar transport, CO2production, lactate production, trichloroacetic acid soluble and precipitable material between the test groups. It is concluded that altered glucose uptake or metabolism is not a general characteristic of all Alzheimer disease cultured fibroblasts.

Preferential effect of bleomycin on newly replicated chromatin in nuclei from L1210 cells

This study determined whether nascent chromatin in nuclei from leukemia L1210 cells constitutes a preferential target for bleomycin. No differences were seen in fragmentation of nascent and bulk DNA as judged by DNA double-stranded cleavage and the release of acid-soluble material or subnucleosomal (under 8 S) fragments. In contrast, bleomycin-induced chromatin aggregation (Woynarowski, J.M., Gawron, L.S. and Beerman, T.A. (1987) Biochim. Biophys. Acta 910, 149–156) occurred preferentially in nascent chromatin as indicated by a retarded solubilization of nascent chromatin and generation of a fast-sedimenting material (above 45 S) in the sedimentation profiles of drug-released nascent chromatin. This preferential aggregation disappeared completely when chromatin became older than 10 min. The drug aggregation activity did not distinguish nascent and mature presolubilized oligonucleosomes. The results suggest that bleomycin recognizes higher-order structures of nascent chromatin.

Accurate measurement of psoralen-crosslinked DNA: Direct biochemical measurements and indirect measurement by hybridization

This paper evaluates methods to measure crosslinkage due to psoralen plus light in total DNA and in specific sequences. DNA exposed in cells or in vitro to a bifunctional psoralen and near ultraviolet light accumulates interstrand crosslinks. Crosslinkage is the DNA mass fraction that is attached in both strands to a crosslink. We show here biochemical methods to measure psoralen photocrosslinkage accurately in total DNA. We also describe methods to measure photocrosslinkage indirectly, in specific sequences, by nucleic acid hybridization. We show that a single 4,5′,8-trimethylpsoralen (TMP) crosslink causes at least 50 kbp of alkali-denatured DNA contiguous in both strands with it to snap back into the duplex form when the denatured preparation is returned to neutral pH. This process was so efficient that the DNA was not nicked by the singlestrand nuclease S1 at 100-fold excess after snapping back. Uncrosslinked DNA was digested to acid-soluble material by the enzyme. Crosslinkage therefore equals the fraction of S1-resistant nucleotide in this kind of experiment. We alkali-denatured DNA samples crosslinked to varying degrees by varying TMP concentration at constant light exposure. We then measured crosslinkage by ethidium bromide (EtBr) fluorometry at pH 11.8; by EtBr fluorometry at neutral pH of S1 digests of the DNA; and by the fraction of radioactivity remaining acid insoluble in S1-digests of DNA labeled uniformly with [ 3H]deoxythymidine. These assays measure distinct physical properties of crosslinked DNA. Numerical agreement is expected only when all three measurements are accurate. Under optimum conditions, the three methods yielded identical results over the range of measurement. Using alkaline EtBr fluorescence in crude cell lysates, we detected crosslinks at frequencies in the range of 1.6 × 10 −7 per base pair. These levels were compatible with cell survival, attesting to the sensitivity of the measurement system. Crosslinkage affected hybridization as well. One crosslink prevented all alkali-denatured DNA contiguous in both strands with it from hybridizing to complementary DNA either on solid supports or in solution. Strand-length effects on crosslinkage and on reassociation caused solution hybridization levels to exceed those predicted by simple theory. In a quantitative, dot-blotting assay hybridization was linear up to membrane saturation by denatured, uncrosslinked DNA of any strand length. Longer, duplex DNA bound to the membrane, although it hybridized less than 0.03 as efficiently as single-stranded DNA and had no effect on the ability of the latter to bind or hybridize, even when present in great excess. The results here provide the basis for an accurate system to quantify TMP crosslinkage in specific sequences and in total DNA.