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PubMedDate not supplied

Taxol metabolism. Isolation and identification of three major metabolites of taxol in rat bile.

The elimination of nonradioactive taxol in bile and urine was investigated in the rat after administration via the caudal vein (10 mg/kg). As in humans, no metabolites of taxol were detected by HPLC in rat urine, and only 10% of the injected taxol was recovered in urine over a 24-hr period. In contrast, 11.5% and 29% of the injected taxol was recovered in rat bile as unchanged taxol and metabolites, respectively. Among the nine taxol metabolites detected by HPLC, the side chain at C13, which is required for pharmacological activity, had been removed in only one minor metabolite, baccatin III. The chemical structures of the two major hydroxylated metabolites were determined by mass spectrometry (fast atom bombardment and desorption chemical ionization) and 1H-NMR spectroscopy. One was a taxol derivative hydroxylated on the phenyl group at C3' of the side chain at C13, while the other corresponded to a taxol derivative hydroxylated in the m-position on the benzoate of the side chain at C2. Although these two major taxol metabolites were as active as taxol in preventing cold microtubule disassembly, they were, respectively, 9 and 39 times less cytotoxic as taxol on in vitro L1210 leukemia growth. These results show for the first time that there is a significant hepatic metabolism of taxol.

Alkaloids↗
PubMed1998

Mutations of p53 gene can be detected in the plasma of patients with large bowel carcinoma.

AIMS: To attempt to detect p53 gene mutations in the plasma of patients with large bowel carcinoma. METHODS: Plasma was collected from 20 control patients with no history of cancer and from 17 patients with large bowel carcinoma. Corresponding tumour and benign lymph node (control) samples for each of the carcinoma patients were obtained from paraffin blocks. A Dukes' stage was determined for each tumour. DNA was extracted from the plasma samples and the paraffin embedded tissue using previously described methods. A nested primer polymerase chain reaction protocol was used for the amplification of exons 5 to 8 of the p53 gene. "Cold" single strand conformational polymorphism (SSCP) was performed on mini gels and silver stained. Abnormal bands were excised, the DNA eluted, and reamplified for automated dye termination sequencing. Any sample showing an apparent mutation was rechecked from the original extracted DNA sample at least three times. RESULTS: p53 gene mutations were not found in the control specimens. They were found in both the primary tumour and the plasma in three cases, in the primary tumour alone in one case, and in the plasma alone in two cases. One of the latter two cases also had metastatic transitional cell carcinoma of the bladder and the other had widespread metastatic deposits. One of the cases with mutant DNA in both the plasma and the primary was a Dukes' stage B tumour. The others were Dukes' C and Dukes' D. CONCLUSIONS: p53 gene mutations can be detected in the plasma of some patients with large bowel carcinoma and these are concordant with those in the primary carcinomas.

Adult↗
PubMed1995

Cold denaturation of an icosahedral virus. The role of entropy in virus assembly.

Assembly of icosahedral viruses is not completely understood at the molecular level. The main puzzle is to answer how chemically identical protein subunits take up unique positionally dependent conformations during the process of assembly. The stability of the ribonucleoprotein particles of cowpea mosaic virus (CPMV) to pressures and subzero temperatures has been studied. At room temperature, reversible pressure denaturation of CPMV is obtained only in the presence of 5.0 M urea. On the other hand, when the temperature is decreased to -15 degrees C, the ribonucleoprotein components denature, at 2.5 kbar, in the presence of 1.0 M urea. At temperatures close to -20 degrees C, denaturation is obtained even in the absence of urea. Whereas the denaturation promoted by pressure and urea at room temperature is reversible, virus particles denatured when the temperature is decreased under pressure cannot reassemble. Bis-ANS binding data suggest that this irreversibility may be related to protein release from RNA, which probably does not occur under denaturating conditions at room temperature. The contributions of enthalpy (delta H*) and entropy (delta S*) for the free energy of association of CPMV are calculated from the cold denaturation curves under pressure. The entropy change is positive and large, making the assembly of ribonucleoprotein components an entropy-driven process, suggesting that the burial of nonpolar side chains during the process of assembly is the structural foundation for CPMV assembly.

Cold Temperature↗
PubMed1979

Isolation of a collagen-binding fragment from fibronectin and cold-insoluble globulin.

Limited proteolytic cleavage of fibronectin and plasma cold-insoluble globulin with cathepsin D produced two major fragments. The smaller, Mr = 72,000 fragment bound to collagen and contained most of the cysteine in the molecule. This region contains intrachain disulfide bonds which maintain a conformation that is necessary for interaction with collagen. Cleavage of the intact protein and the 72,000-dalton fragment with plasmin localized the collagen-binding region in cold-insoluble globulin to a sequence of about 42,000 daltons. This region is located approximately two-thirds of the linear distance from the NH2 terminus of each chain in the dimeric molecule.

Amino Acids↗
PubMed2000

Effects of pinacidil on coronary Ca(2+)-myosin phosphorylation in cold potassium cardioplegia model.

The effects of the potassium (K(+)) channel opener pinacidil (Pin) on the coronary smooth muscle Ca(2+)-myosin light chain (MLC) phosphorylation pathway under hypothermic K(+) cardioplegia were determined by use of an in vitro microvessel model. Rat coronary arterioles (100-260 microm in diameter) were subjected to 60 min of simulated hypothermic (20 degrees C) K(+) cardioplegic solutions (K(+) = 25 mM). We first characterized the time course of changes in intracellular Ca(2+) concentration, MLC phosphorylation, and diameter and observed that the K(+) cardioplegia-related vasoconstriction was associated with an activation of the Ca(2+)-MLC phosphorylation pathway. Supplementation with Pin effectively suppressed the Ca(2+) accumulation and MLC phosphorylation in a dose-dependent manner and subsequently maintained a small decrease in vasomotor tone. The ATP-sensitive K(+) (K(ATP))-channel blocker glibenclamide, but not the nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine methyl ester, significantly inhibited the effect of Pin. K(+) cardioplegia augments the coronary Ca(2+)-MLC pathway and results in vasoconstriction. Pin effectively prevents the activation of this pathway and maintains adequate vasorelaxation during K(+) cardioplegia through a K(ATP)-channel mechanism not coupled with the endothelium-derived NO signaling cascade.

Animals↗
PubMed1987

Effect of acclimation temperature on the elongation step of protein synthesis in different organs of rainbow trout.

Cytosolic extracts of liver, kidney, spleen, gill, red and white muscle from rainbow trout acclimated to 4 and 17 degrees C, respectively, have been investigated in vitro with respect to their enzymic activity in stimulating the growth of nascent peptide chains (labelled polyphenylalanine) at assay temperatures from 5 to 25 degrees C using polyuracil as messenger RNA. The elongation step of protein synthesis is characterized by a Q10 value of about 2.4 (range 10-25 degrees C) in all organs from both, 4 and 17 degrees C acclimated fish. Except for the red muscle, the organs of cold acclimated trout, however, exhibit significantly higher specific elongation rates (mol phenylalanine polymerized/(g wet weight X h)) at any experimental temperature than those of warm acclimated fish. This increase of the elongation rates varies between the organs and ranges from +29% (liver) to +60% in the gill. The specific acylation rate (mol phenylalanyl-tRNA formed/(g wet weight X h] surpasses the specific elongation rate by a factor of at least 8.5. Moreover, the specific acylation rate per mg protein is independent of acclimation temperature. It is concluded that the increased specific elongation rates in 4 degrees C acclimated trout are not due to altered pool sizes of the precursor phenylalanyl-tRNA, but reflect an effective enhancement of enzymic elongation factor activities. In accordance with data taken from literature, this finding suggests a compensatory enhancement of in vivo protein synthesis to occur in trout during cold acclimation.

Acclimatization↗
PubMed2004

Posttranslational modification of brain tubulins from the Antarctic fish Notothenia coriiceps: reduced C-terminal glutamylation correlates with efficient microtubule assembly at low temperature.

We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.

Aging↗
PubMed2001

A newly synthesized, ribosome-bound polypeptide chain adopts conformations dissimilar from early in vitro refolding intermediates.

Little is known about the conformations of newly synthesized polypeptide chains as they emerge from the large ribosomal subunit, or how these conformations compare with those populated immediately after dilution of polypeptide chains out of denaturant in vitro. Both in vivo and in vitro, partially folded intermediates of the tailspike protein from Salmonella typhimurium phage P22 can be trapped in the cold. A subset of monoclonal antibodies raised against tailspike recognize partially folded intermediates, whereas other antibodies recognize only later intermediates and/or the native state. We have used a pair of monoclonal antibodies to probe the conformational features of full-length, newly synthesized tailspike chains recovered on ribosomes from phage-infected cells. The antibody that recognizes early intermediates in vitro also recognizes the ribosome-bound intermediates. Surprisingly, the antibody that did not recognize early in vitro intermediates did recognize ribosome-bound tailspike chains translated in vivo. Thus, the newly synthesized, ribosome-bound tailspike chains display structured epitopes not detected upon dilution of tailspike chains from denaturant. As opposed to the random ensemble first populated when polypeptide chains are diluted out of denaturant, folding in vivo from the ribosome may begin with polypeptide conformations already directed toward the productive folding and assembly pathway.

Antibodies, Monoclonal↗
PubMed1999

Alteration in alpha 2 integrin immunocytochemical expression on cultured human gingival fibroblasts following nicotine exposure.

BACKGROUND: Clinical epidemiologic studies carried out in smokers versus non smokers support the concept that tobacco-related factor may affect the periodontal tissues and wound healing. METHODS: In this study, the role of nicotine on the integrin alpha 2 chain immunocytochemical expression, in human gingival fibroblasts (HGF) was investigated in vitro. The kinetic induction of this type of integrin on HGF in response to increasing percentage of nicotine was been characterized. A human gingival fibroblast strain, derived from a healthy individual with non-inflamed gingiva, was used in this study. The cells were then grown on acetylated microscope slides and fixed with cold ethanol, samples were then incubated for 16 to 19 hrs at 4 degrees C to anti-human alpha 2 integrin chain monoclonal antibody. RESULTS: In control cultures and in HGF treated with 0.025 microM nicotine the initial higher expression of alpha 2 chain decreased (grade 1 in both culture) while in HGF treated with 0.075 microM increased (grade 3). After 48 hours HGF exposed to 0.075 microM nicotine, increased further their expression of alpha 2 chain. CONCLUSIONS: The results obtained demonstrate a dose-time dependent nicotine influence on immunocytochemical expression of alpha 2 integrin chain. These data suggest that nicotine may promote a collagene breakdown via an increase of alpha 2 integrin chain expression.

Cells, Cultured↗
PubMed1995

Solution X-ray scattering analysis of cold- heat-, and urea-denatured states in a protein, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI), a homo-dimeric protein with a subunit of 113 residues with two disulfide bonds, is known to exist at low pH in at least three distinct thermodynamic states namely, the native (N), cold-denatured (D') and heat-denatured (D). Small-angle X-ray scattering was used to analyze and to compare overall chain conformations of SSI in typical, N, D', D and urea-denatured states (Durea). Molecular masses were determined from scattering intensities extrapolated to a scattering angle of zero, which showed that SSI exists as a homo-dimer in the N state, but as dissociated monomers in the D', D and Durea states. From Guinier plots of the scattering intensities, radii of gyration (Rg) were determined to be 20.1(+/- 1.8) A for N, and 20.7(+/- 1.3), 25.8(+/- 1.5) and 32 to 35 A for D', D and Durea, respectively. Kratky plots for both N and D' exhibited a bell-shape indicating that the polypeptide chain has a globular part not only in N but also in D', while Kratky plots for D and Durea showed that the polypeptide chain has no globular part either in Durea or D. Combined with the results from circular dichroism and 1H NMR spectra, a picture emerges for the polypeptide chain conformation of SSI such that in N it is a globular dimer close to that in the crystal, in Durea it is totally disordered and expanded nearly to a fully random chain with restrictions only from the disulfide bridges, in D the entire chain is disordered and expanded but with considerable local intra-chain interactions, and in D' the chain consists of a part with a unique tertiary structure and a part disordered and expanded to a degree comparable to D.

Bacterial Proteins↗
PubMed1984

Anesthetic-membrane interaction: a 2H nuclear magnetic resonance study of the binding of specifically deuterated tetracaine and procaine to phosphatidylcholine.

The binding of the local anesthetics tetracaine and procaine with multilamellar dispersions of egg phosphatidylcholine has been studied by 2H nuclear magnetic resonance (NMR). The 2H-NMR line shapes of specifically deuterated local anesthetics are found to be very dependent on the attainment of a true equilibrium. The equilibrium could be most properly reached by the use of repeated freeze-thaw-vortex cycles. The data for tetracaine are consistent with the three-site exchange model proposed earlier. Tetracaine is in slow exchange between a strongly bound site and a weakly bound site and in fast exchange between the weakly bound site and free in solution. The slow exchange rate is estimated, from temperature and dilution studies, to be approximately 1.5 X 10(3) S-1 at pH 5.5 and slightly faster at pH 9.5. Comparisons of the quadrupole splitting with those seen for our earlier work in egg phosphatidylethanolamine suggest that the location of the strongly bound site in phosphatidylcholine is dependent on the anesthetic charge. This is in contrast to egg phosphatidylethanolamine, where molecular shapes appear to be the determining factor for the location of the anesthetic. Procaine bound very weakly to the model membranes, to yield only a broad resonance and no quadrupole splitting. It appears that procaine, unlike tetracaine, is not bound by the ordered acyl chains.

Chemical Phenomena↗
PubMed2006

Cloning and characterization of a gene encoding a cold-shock protein in Lactobacillus casei.

One csp-like gene, called cspA, from the lactic acid bacterium Lactobacillus casei was identified by an inverse polymerase chain reaction approach based on degenerate primers. cspA encodes a protein of 66 amino acid residues, which displays at least 74% identity with Csp proteins of Lactobacillus genera. Northern blot analysis revealed that cspA is transcribed monocistronically and that its expression is induced after a temperature downshift from 37 degrees C to 20 degrees C. The transcriptional start site has been determined and is situated 98 bp upstream of the initiation codon. A cspA mutant strain was constructed and it showed reduced growth rate compared with the wild type at both optimal and low temperatures, demonstrating that CspA plays an important role in the physiology of L. casei.

Amino Acid Sequence↗
PubMed1991

Effects of phosphorylation by myosin light chain kinase on the structure of Limulus thick filaments.

The results discussed in the preceding paper (Levine, R. J. C., J. L. Woodhead, and H. A. King. 1991. J. Cell Biol. 113:563-572.) indicate that A-band shortening in Limulus muscle is a thick filament response to activation that occurs largely by fragmentation of filament ends. To assess the effect of biochemical changes directly associated with activation on the length and structure of thick filaments from Limulus telson muscle, a dually regulated tissue (Lehman, W., J. Kendrick-Jones, and A. G. Szent Gyorgyi. 1973. Cold Spring Harbor Symp. Quant. Biol. 37:319-330.) we have examined the thick filament response to phosphorylation of myosin regulatory light chains. In agreement with the previous work of J. Sellers (1981. J. Biol. Chem. 256:9274-9278), Limulus myosin, incubated with partially purified chicken gizzard myosin light chain kinase (MLCK) and [gamma 32P]-ATP, binds 2 mol phosphate/mole protein. On autoradiographs of SDS-PAGE, the label is restricted to the two regulatory light chains, LC1 and LC2. Incubation of long (greater than or equal to 4.0 microns) thick filaments, separated from Limulus telson muscle under relaxing conditions, with either intact MLCK in the presence of Ca2+ and calmodulin, or Ca2(+)-independent MLCK obtained by brief chymotryptic digestion (Walsh, M. P., R. Dabrowska, S. Hinkins, and D. J. Hartshorne. 1982. Biochemistry. 21:1919-1925), causes significant changes in their structure. These include: disordering of the helical surface arrangement of myosin heads as they move away from the filament backbone; the presence of distal bends and breaks, with loss of some surface myosin molecules, in each polar filament half; and the production of shorter filaments and end-fragments. The latter structures are similar to those produced by Ca2(+)-activation of skinned fibers (Levine, R. J. C., J. L. Woodhead, and H. A. King. J. Cell Biol. 113:563-572). Rinsing experimental filament preparations with relaxing solution before staining restores some degree of order of the helical surface array, but not filament length. We propose that outward movement of myosin heads and thick filament shortening in Limulus muscle are responses to activation that are dependent on phosphorylation of regulatory myosin light chains. Filament shortening may be due, in large part, to breakage at the filament ends.

Actin Cytoskeleton↗
PubMed2002

Purification and characterization of the raffinose oligosaccharide chain elongation enzyme, galactan : galactan galactosyltransferase (GGT), from Ajuga reptans leaves.

Galactan: galactan galactosyltransferase (GGT), an enzyme involved in the biosynthesis of the long-chain raffinose family of oligosaccharides (RFOs) in Ajuga reptans, catalyses the transfer of an alpha-galactosyl residue from one molecule of RFO to another one resulting in the next higher RFO oligomer. This novel galactinol (alpha-galactosyl-myo-inositol)-independent alpha-galactosyltransferase is responsible for the accumulation of long-chain RFOs in vivo. Warm treatment (20 degrees C) of excised leaves resulted in a 34-fold increase of RFO concentration and a 200-fold increase of GGT activity after 28 days. Cold treatment (10 degrees C/3 degrees C day/night) resulted in a 26- and 130-fold increase, respectively. These data support the role of GGT as a key enzyme in the synthesis and accumulation of long-chain RFOs. GGT was purified from leaves in a 4-step procedure which involved fractionated precipitation with ammonium sulphate as well as lectin affinity, anion exchange, and size-exclusion chromatography and resulted in a 200-fold purification. Purified GGT had an isoelectric point of 4.7, a pH optimum around 5, and its transferase reaction displayed saturable concentration dependence for both raffinose (Km = 42 mM) and stachyose (Km = 58 mM). GGT is a glycoprotein with a 10% glycan portion. The native molecular mass was 212 kDa as determined by size-exclusion chromatography. Purified GGT showed one single active band after native PAGE or IEF separation, respectively, which separated into three bands on SDS-PAGE at 48 kDa, 66 kDa, and 60 kDa. The amino acid sequence of four tryptic peptides obtained from the major 48-kDa band showed a high homology to plant alpha-galactosidase (EC 3.2.1.22) sequences. GGT differed, however, in its substrate specificity from alpha-galactosidases; it neither hydrolysed nor transferred alpha-galactosyl-groups from melibiose, galactinol, UDP-galactose, manninotriose, and manninotetrose. Galactinol, sucrose, and galactose inhibited the GGT reaction considerably at 10-50 mM.

Journal Article↗
PubMed1973

Changes in rabbit-liver lysosomes and fructose 1,6-bisphosphatase induced by cold and fasting.

Exposure of rabbits to cold or fasting results in marked increases in the number and size of liver lysosomes, associated with increases in the total and "free" proteolytic activity. Changes in lysosomal morphology also indicate increased activity and fragility. These effects of cold and fasting are correlated with decreases in the levels of liver fructose bisphosphatase (EC 3.1.3.11) activity and changes in the molecular properties of the purified enzyme, including the loss of a small peptide containing the only tryptophan residue in the peptide chain.

Animals↗
PubMed1997

Critical role of anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures.

Listeria monocytogenes is a food-borne pathogen capable of growth at refrigeration temperatures. Membrane lipid fatty acids are major determinants of a sufficiently fluid membrane state to allow growth at low temperatures. L. monocytogenes was characterized by a fatty acid profile dominated to an unusual extent (> 95%) by branched-chain fatty acids, with the major fatty acids being anteiso-C15:0, anteiso-C17:0, and iso-C15:0 in cultures grown in complex or defined media at 37 degrees C. Determination of the fatty acid composition of L. monocytogenes 10403S and SLCC 53 grown over the temperature range 45 to 5 degrees C revealed two modes of adaptation of fatty acid composition to lower growth temperatures: (i) shortening of fatty acid chain length and (ii) alteration of branching from iso to anteiso. Two transposon Tn917-induced cold-sensitive mutants incapable of growth at low temperatures had dramatically altered fatty acid compositions with low levels of i-C15:0, a-C15:0, and a-C17:0 and high levels of i-C14:0, C14:0, i-C16:0, and C16:0. The levels of a-C15:0 and a-C17:0 and the ability to grow at low temperatures were restored by supplementing media with 2-methylbutyric acid, presumably because it acted as a precursor of methylbutyryl coenzyme A, the primer for synthesis of anteiso odd-numbered fatty acids. When mid-exponential-phase 10403S cells grown at 37 degrees C were temperature down-shocked to 5 degrees C they were able, for the most part, to reinitiate growth before the membrane fatty acid composition had reset to a composition more typical for low-temperature growth. No obvious evidence was found for a role for fatty acid unsaturation in adaptation of L. monocytogenes to cold temperature. The switch to a fatty acid profile dominated by a-C15:0 at low temperatures and the association of cold sensitivity with deficiency of a-C15:0 focus attention on the critical role of this fatty acid in growth of L. monocytogenes in the cold, presumably through its physical properties and their effects, in maintaining a fluid, liquid-crystalline state of the membrane lipids.

Betaine↗