Carbohydrate-conjugate heterobimetallic complexes: synthesis, DNA binding studies, artificial nuclease activity and in vitro cytotoxicity

New carbohydrate-conjugated heterobimetallic complexes [C(32)H(62)N(10)O(8)NiSn(2)Cl(4)]Cl(2)(1) and [C(32)H(62)N(10)O(8)CuSn(2)Cl(4)]Cl(2) (2) were synthesized and characterized by spectroscopic (IR, (1)H, (13)C, and (119)Sn NMR, EPR, UV-vis, ESI-MS) and analytical methods. The interaction studies of 2 with CT DNA were studied by using various biophysical techniques, which showed high binding affinity of 2 toward CT DNA. The extent of interaction was further confirmed by the interaction of 2 with the nucleotides viz.; 5'-AMP, 5'-CMP, 5'-GMP, and 5'-TMP, by absorption titration. (1)H, (31)P, (119)Sn NMR spectroscopy further validated the interaction mode of 2 with 5'-GMP. The electrophoresis pattern observed for 2 with supercoiled pBR322 DNA, exhibited significantly good nuclease activity following oxidative pathway. The preferential selectivity of 2 toward the major groove was observed on interaction of 2 with pBR322 DNA, in the presence of standard groove binders viz.; DAPI and methyl green. Additionally, in vitro antitumor activity of 2 was evaluated on a panel of human cancer cell lines, exhibiting remarkable cytotoxicity activity against Colo205 (colon) and MCF7 (breast) cell lines with GI(50) values <10 μg/mL.     

The DNase activity of RNase T and its application to DNA cloning.

RNase T is one of eight distinct 3'-->5' exoribonucleases present in Escherichia coli. The enzyme plays an important role in stable RNA metabolism, including tRNA end turnover and 3' maturation of most stable RNAs because it is the only RNase that can efficiently remove residues near a double-stranded (ds) stem. In the course of study of its specificity and mechanism, we found that RNase T also has single-strand-specific DNase activity. Purified RNase T degrades both single-stranded (ss)RNA and ssDNA in a non-processive manner. However, in contrast to its action on RNA, RNase T binds ssDNA much more tightly and shows less sequence specificity. As with RNA, DNA secondary structure strongly affects its degradation by RNase T. Thus, RNase T action on a dsDNA with a single-stranded 3'-extension efficiently generates blunt-ended DNA. This property of RNase T suggested that it might be a useful enzyme for blunt-ended DNA cloning. We show here that RNase T provides much higher cloning efficiency than the currently used mung bean nuclease.     

The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm

A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 °C and 50 °C was almost half of that at the optimum temperature (37 °C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.Abbreviations GW groundwater GWA groundwater aquifer     

The effect of ultra small doses of thyroliberin on the structural changes in endoplasmic reticulum membranes in vitro

Peptides are known to have the ability of modulating the activity of important regulatory cellular systems. One of them--thyroliberin, i.e. thyreotropin-releasing hormone (TRH), causes changes in the membrane structure and morphology of rat erythrocytes, as well as activates retractive activity of lymphatic vessels in ultra low concentrations (10(-10) to 10(-16) mol/l). In this study we used an electron spin resonance (ESR) method to explore the effect of TRH in a wide range of concentrations (10(-4) to 10(-18) mol/l) on thermo-induced structural transitions and microviscosity of lipid bilayer of the endoplasmic reticulum membrane of mice (C57 bI) liver cells. Two stable free radicals were used as paramagnetic probes: 2,2,6,6-tetramethil-4-capryolyl-1-oxyl and 16-doxyl-stearic acid, that are localized in superficial and deep layers of the membrane respectively. TRH caused a statistically significant change (p < 0.001) in microviscosity of the membrane surface layer. The largest effect (up to 30% decrease) was observed at TRH concentrations of 10(-10) and 10(-16) mol/l. It was also demonstrated that an addition of 10(-4), 10(-10) and 10(-16) mol/l of TRH decreases effective activation energy and temperature (by several degrees) of the thermo-induced structural transitions. The observed changes in the parameters of the membrane surface layer induced by TRH may be essential for its physiological activity, because of the obtained negative correlation (r = 0.99; p < 0.001) between the membrane microviscosity and frequency of lymphatic vessels' contraction. Complex changes in the structure of deep hydrophobic layer of the membrane caused by TRH were observed in this study as well. Higher concentrations of TRH (10(-4) and 10(-10) mol/l) produced results that were similar to the effect of TRH on the superficial lipid layer of the membrane, whereas the effect of ultra low TRH concentration (10(-16) mol/l) was reversed for microviscosity, number and activation energy of structural transitions in contrast with the case of surface layer. The results of this study suggest presence of a nonspecific factor in the effect of TRH on structural characteristics of the lipid component of biological membranes. It is possible, that the change of structural properties of biological membranes may be a part of the mechanism of TRH action at ultra low concentrations.     

The effect of nitrosoguanidine upon DNA synthesis in vitro

Summary Both the polymerase and the exonuclease activities of DNA polymerase III^* are inactivated by treatment with nitrosoguanidine. The treatment of the DNA template with the mutagen does not affect the template in supporting DNA synthesis. No effect of nitrosoguanidine upon fidelity of replication in vitro was detected.     

Single-strand-specific nuclease from rat liver endoplasmic reticulum: characterization and mode of action

1. The characteristics and mode of action of a single-strand-specific nuclease isolated from rat liver endoplasmic reticulum are investigated with respect to its DNA and RNA substrates. 2. The RNase activity of the enzyme is slightly influenced by the presence of divalent cations but the DNase activity is enhanced by divalent cations particularly Mn2+. 3. Activity is partially inhibited by the presence of EGTA; this effect is reversed most efficiently by the addition of Mn2+. 4. The enzyme exhibits small pH dependence between pH 6-9 and maximum activity is observed at pH 7-7.5 for both DNase and RNase activities. 5. Sulfhydryl group reagents do not affect its action but histidyl group reagents exert a small but definite effect. 6. The enzyme degrades DNA and RNA endonucleolytically producing fragments which possess 3'-OH and 5'-phosphate termini. 7. Monomers are not produced even after prolonged degradation. 8. The end product of poly(U)degradation ranges between two and four building blocks but the DNA product is longer probably due to considerable percentage of secondary structure.     

gamma -glutamyltransferase and its isoform mediate an endoplasmic reticulum stress response

Although use of multiple alternative first exons generates unique noncoding 5'-ends for gamma-glutamyltransferase (GGT) cDNAs in several species, we show here that alternative splicing events also alter coding exons in mouse GGT to produce at least four protein isoforms. GGTDelta1 introduces CAG four bases upstream of the primary ATG codon and encodes an active GGT heterodimeric ectoenzyme identical to constitutive GGT cDNA but translational efficiency is reduced 2-fold. GGTDelta2-5 deletes the last eight nucleotides of exon 2 through most of exon 5 in-frame, selectively eliminating residues 96-231 from the amphipathic N-terminal subunit, including four N-glycan consensus sites, while leaving the C-terminal hydrophilic subunit intact. GGTDelta7 introduces 22 bases from intron 7 causing a frameshift and a premature stop codon so a truncated polypeptide is encoded terminating with 14 novel residues but retaining the first 339 residues of the native GGT protein. GGTDelta8-9 deletes the terminal four nucleotides of exon 8 plus all of exon 9 and inserts 24 bases from intron 9 in-frame so the C-terminal subunit of the encoded polypeptide loses residues 401-444 but gains eight internal hydrophobic residues. In contrast to the product of GGTDelta1, those derived from GGTDelta2-5, Delta7, Delta8-9 all lack transferase activity and persist as single-chain glycoproteins retained largely in the endoplasmic reticulum as determined by immunofluorescence microscopy and constitutive endoglycosidase H sensitivity in metabolically labeled cells. The developmental-stage plus tissue-specific regulation of the alternative splicing events at GGTDelta7 and GGTDelta8-9 implies unique roles for these GGT protein isoforms. The ability of the GGTDelta1 and GGTDelta7 to mediate the induction of C/EBP homologous protein-10, CHOP-10, and immunoglobulin heavy chain binding protein, BiP, implicates a specific role for these two GGT protein isoforms in the endoplasmic reticulum stress response.     

Extracellular esterase activity as an indicator of endoplasmic reticulum calcium depletion

Abstract

Context: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases.

Objective: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein.

Materials and methods: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity.

Results: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity.

Discussion: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases.

Conclusion: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.     

Granzyme A activates an endoplasmic reticulum-associated caspase-independent nuclease to induce single-stranded DNA nicks

The cytotoxic T lymphocyte protease granzyme A (GzmA) initiates a novel caspase-independent cell death pathway characterized by single-stranded DNA nicking. The previously identified GzmA substrate SET is in a multimeric 270-420-kDa endoplasmic reticulum-associated complex that also contains the tumor suppressor protein pp32. GzmA cleaved the nucleosome assembly protein SET after Lys(176) and disrupted its nucleosome assembly activity. The purified SET complex required only GzmA to reconstitute single-stranded DNA nicking in isolated nuclei. DNA nicking occurred independently of caspase activation. The SET complex contains a 25-kDa Mg(2+)-dependent nuclease that degrades calf thymus DNA and plasmid DNA. Thus, GzmA activates a DNase (GzmA-activated DNase) within the SET complex to produce a novel form of DNA damage during cytotoxic T lymphocyte-mediated death.     

Interactions of liver endoplasmic reticulum membranes and polysomesin vitro

Summary The interactions of various preparations of endoplasmic reticulum membranes and polysomes have been studied by means of a sandwich sucrose gradient that clearly isolates free ribosomes, smooth endoplasmic reticulum (S.E.R.) and rough endoplasmic reticulum (R.E.R.) from the microsomal fraction of rat liver homogenates. Reconstructed rough membranes separate well from the native R.E.R. but occupy the same position along the gradients as the S.E.R. and the rough membranes, stripped of their ribosomes by means of LiCl. Native R.E.R. and S.E.R. do not bind any added labeled polysomes at 0°C; previous treatment with LiCl does not modify the behavior of S.E>R. The presence of cell sap during the binding reaction does not increase polysome fixation by stripped-rough membranes but protects in some way the polysomes and preserves all their original functional capacity of amino acid incorporation into protein.     

The in vitro reassembly of rough endoplasmic reticulum: Ribosome binding capacity

Rough endoplasmic reticulum membranes were dissolved in 1% deoxycholate and the deoxycholate was then dialysed out for five days. Well-defined bilayer vesicles were formed only if the dialysis was performed at room temperature for the first six hours. The vesicles were separated into a pelleted fraction (Fraction I) and a fluffy layer (Fraction II) by centrifugation. As measured by amino acid incorporation ability, Fraction II bound polysomes, while Fraction I did not. When smooth endoplasmic reticulum was assembled, it was found that Fraction II so derived had a polysome binding capacity which was more sensitive to increased KCl concentrations (25 mM–100 mM KCl) and that it bound significantly more monosomes than the corresponding fraction derived from rough membranes. The SDS-polyacrylamide polypeptide patterns of the various fractions were compared.     

Structure and assembly of the endoplasmic reticulum. The synthesis of three major endoplasmic reticulum proteins during lipopolysaccharide-induced differentiation of murine lymphocytes

Monospecific rabbit antibodies have been prepared against ERp72, ERp99, and ERp60, major protein components of a detergent-solubilized extract of endoplasmic reticulum purified from mineral oil-induced plasmacytoma 315 tissue. When subcellular fractions of mineral oil-induced plasmacytoma 315 tissue were assayed by an immunoprecipitation procedure, all three endoplasmic reticulum proteins (ERps) were found to be enriched in the rough endoplasmic reticulum. In murine lymphoid cells, the three ERps represent two major structural classes of protein. Both ERp72 and ERp60 contain no endoglycosidase H-sensitive, N-linked oligosaccharides. On the other hand, ERp99 is glycoprotein containing, in all likelihood, one endoglycosidase H-sensitive oligosaccharide. Immunologically cross-reacting proteins of similar molecular weight have also been detected in other eukaryotic cell lines. The anti-ERp antibodies were used to quantitate the synthesis and accumulation of the three ERps in splenic lymphocytes cultured in the presence and absence of bacterial lipopolysaccharide (Escherichia coli serotype B5:055) (LPS). In the presence of LPS, lymphocytes differentiate from resting cells into actively secreting cells. The synthesis of ERp72 and ERp99 increased 3- and 10-fold, respectively, in response to LPS. The synthesis of ERp60 does not change significantly. The turnover rates for these three proteins are similar in both control and LPS-treated lymphocytes. As a result, membranes isolated from LPS-treated cells are enriched in ERp72 and ERp99.     

Ferritin synthesis on polyribosomes attached to the endoplasmic reticulum.

The evidence that ferritin is synthesized both on free polyribosomes and on polyribosomes attached to the endoplasmic reticulum is reviewed. Evidence that some ferritin is secreted from cells after synthesis on bound polyribosomes was found to be inconclusive.     

Heat stress enhances phytohemagglutinin synthesis but inhibits its transport out of the endoplasmic reticulum

In this study we examined the effect of heat stress (up to 6 hours at 43°C) on the biosynthesis and transport of phytohemagglutinin (PHA) in cotyledons of developing seeds of the common bean, Phaseolus vulgaris. Heat stress resulted in a decrease of total protein synthesis and an enhancement of the synthesis of heat shock proteins and PHA. Pulse chase experiments showed that a considerable proportion of the newly synthesized PHA was present in the endoplasmic reticulum (ER)/Golgi fraction and did not readily chase-out. Analysis with endoglycosidase H showed that the oligosaccharide sidechains of PHA were almost entirely in the high mannose configuration, indicating that most of the newly synthesized PHA was in the ER. However, some of the PHA became fucosylated at 43°C, indicating fucosyltransferase activity. That the biosynthesis and secretion of fucosyl-containing cell wall polymers proceeded normally at 43°C provided evidence that certain Golgi functions (i.e. transport to the cell wall) remained unaffected by heat stress. The ER obtained from these heat stress cotyledons had a greater density (1.16 g· cm⁻³ at 43°C instead of 1.14 g·cm⁻³ at 22°C) in sucrose gradients. Ultrastructural observations showed that the width of the lumen of the ER cisternae had increased from 20 nanometers at 22°C to 60 to 80 nanometers at 43°C; the lumen was filled with electrondense material presumed to be protein. The experiments are interpreted as evidence that heat stress imposes a block in the transport of PHA out of the ER. Whether heat stress affects the ER itself or alters the conformation of PHA, thereby preventing its transport, is not clear.     

Phenobarbital-induced alterations in phosphatidylcholine and triglyceride synthesis in hepatic endoplasmic reticulum

Biosynthetic pathways of phosphatidylcholine and triglyceride were studied in proliferating hepatic endoplasmic reticulum of rats pretreated with phenobarbital. Phosphatidylcholine accounted for the major increment in membrane phospholipid. In vitro measurements of hepatic microsomal enzymes which catalyze phosphatidylcholine biosynthesis revealed a significant increase in specific activity of the enzyme governing phosphatidylcholine synthesis by sequential methylation of phosphatidylethanolamine. The specific activity of phosphorylcholine-glyceride transferase, which catalyzes phosphatidylcholine synthesis from d-1,2-diglyceride and CDP-choline, was not altered. Specific activity of diglyceride acyltransferase, which catalyzes triglyceride biosynthesis, was increased to a degree comparable to the increase in specific activity found in the phenobarbital-induced drug-metabolizing enzyme which oxidatively demethylates aminopyrine. In vivo incorporation of methyl-(3)H from l-methionine-methyl-(3)H into microsomal phosphatidylcholine was significantly increased, resulting in an increased methyl-(3)H to choline-1,2-(14)C incorporation ratio of more than three times that found in control animals. A comparable increase in this incorporation ratio was noted in serum phospholipids. The in vitro enzyme studies, in agreement with in vivo incorporation data, indicate that the increase in phosphatidylcholine content of phenobarbital-induced proliferating endoplasmic reticulum is related to increased activity of the pathway of phosphatidylcholine biosynthesis involving the sequential methylation of phosphatidylethanolamine.     

Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.