Differential effect of subspecies of lipoprotein containing apolipoprotein A-I on cholesterol efflux from cholesterol-loaded macrophages: functional correlation with lecithin: cholesterol acyltransferase.

Two species of lipoprotein containing apoA-I, one containing only apoA-I (LpA-I), and the other containing apoA-I and apoA-II (LpA-I/A-II), were tested for their effects on macrophage foam cells. Rat macrophages were converted to foam cells by incubation with radiolabeled acetylated LDL. Incubation with LpA-I or LpA-I/A-II decreased the cellular cholesteryl esters (CE) mass. However, the free cholesterol (FC) mass was only reduced by LpA-I. All the radioactivity excreted into the medium was associated with LpA-I or LpA-I/A-II; 39% of the excreted radioactivity was esterified in LpA-I and 10% in LpA-I/A-II. Upon complete inactivation of lecithin: cholesterol acyltransferase (LCAT) activity with dithiobisnitrobenzoic acid, the cholesterol reducing capacity of LpA-I was weakened significantly. However, the CE mass reducing capacity of LpA-I/A-II was not affected. When LpA-I and LpA-I/A-II were combined, the cholesterol reducing capacity of the mixture was similar to that of LpA-I alone. However, LpA-I re-isolated from the medium showed a lower esterification rate than did the re-isolated LpA-I/A-II, thereby indicating that the cholesterol esterified in LpA-I was transferred to LpA-I/A-II. These results suggest that (i) the function of LpA-I is closely linked to the LCAT activity while that of LpA-I/A-II is not, and (ii) LpA-I in concert with LpA-I/A-II induces a series of extracellular events; LCAT-mediated esterification of excreted FC by LpA-I and a subsequent CE transfer to LpA-I/A-II. These mechanisms might be important for net cholesterol efflux from macrophage foam cells in physiological states.


Homology-derived three-dimensional structure prediction of Candida cylindracea lipase.

We propose a structural model of Candida cylindracea lipase (CCL) based on the reported X-ray structure of the highly homologous Geotrichum candidum lipase (GCL). The network of interactions around the active site, the salt and disulfide bridge pattern is conserved in the proposed structure. Functional, structural and evolutionary aspects of the peculiar usage of CTG codons by C. cylindracea ATCC 14830 are discussed.


Enhancement of platelet functions by low density lipoproteins.

Platelet suspensions, that secreted about 50% of their dense granule contents upon stimulation with alpha-thrombin, showed a dose-dependent increase in secretion after 30 min preincubation with 0.5-3.0 g low density lipoprotein (LDL) protein/1. A 1-5 min preincubation had no effect. The enhancement by LDL only occurred at about 20% secretion or more, indicating that a minimal degree of activation was required for LDL to become effective. Lysine-modified LDL was equally effective as native LDL. The effect of LDL on secretion was accompanied by enhanced thromboxane B2 formation caused by stimulation of the liberation of arachidonate from phosphatidylcholine and/or phosphatidylinositol. However, when thromboxane formation was inhibited or the prostaglandin H2-thromboxane A2-receptor was blocked, LDL remained a potent stimulator of the secretion response. Thus, LDL enhances platelet secretion by a thromboxane A2-dependent and a thromboxane A2-independent mechanism via an effect that is independent of specific binding sites on the platelet.


Activation of phospholipases in platelets by polyclonal antibodies against a surface membrane protein.

In a previous paper we demonstrated using immunochemical techniques that propolypeptide of von Willebrand factor was present on the surface of resting platelets. In the present paper we show that polyclonal antibodies against propolypeptide of von Willebrand factor induce activation of phospholipase(s) in platelets and lead to platelet aggregation. The antibody-stimulation of platelets induced the synthesis of thromboxane A2 (TXA2). Furthermore, the aggregation was inhibited by aspirin and an antagonist of TXA2. Aspirin inhibited not only the aggregation but also the activation of arachidonic acid liberation from phospholipids, but the effect of aspirin on arachidonic acid liberation was cancelled by the combined effect of the antibodies and a TXA2 mimetic agonist, which itself did not activate arachidonic acid liberation. The antibody-induced activation of arachidonic acid liberation and the aggregation were blocked by cytochalasin B. All these results obtained with antibodies were quite similar to the results obtained with collagen.


Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias. I. Increased phosphatidylcholine and reduced sphingomyelin in patients with elevated levels of triacylglycerol-rich lipoproteins.

The composition of red blood cell membrane and plasma phospholipids has been analyzed in patients with hyperlipidemias. In red cells of patients with elevated levels of triacylglycerol-rich lipoproteins, phosphatidylcholine (PC) was raised and sphingomyelin (SM) reduced, resulting in a 20% increase of the membrane PC/SM ratio. In plasma phospholipids of these patients PC and SM levels were also higher and lower, respectively and the plasma PC/SM ratio was elevated by more than 50%. Close positive correlations between plasma and membrane phospholipids were obtained for PC, SM and the PC/SM ratio in normolipidemic and hyperlipidemic donors. Plasmalogen phosphatidylethanolamine (PE), a supposed endogenous protector against lipid oxidation, was reduced by about 20% in red cell membrane lipids in hyperlipidemic patients. Also plasmalogen-PE in plasma tended to be reduced in hyperlipidemic donors. Plasma HDL levels were positively related to the content of plasmalogen PE in the red cell membrane. In conclusion, there are closely related increases in PC/SM ratios in plasma and the red cell membrane in patients with elevated levels of triacylglycerol-rich lipoproteins. It is speculated that decreases in red cell membrane plasmalogen-PE in hyperlipidemic patients could be related to impaired antioxidant protection, possibly as a consequence of reductions in plasma HDL levels.


Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias. II. Increases in distinct molecular species of phosphatidylethanolamine and phosphatidylcholine containing arachidonic acid.

The molecular species composition of red blood cell diacyl-phosphatidylcholine (PC), diacyl-phosphatidylethanolamine (PE) and alkenylacyl-PE (plasmalogen PE) has been analyzed in normolipidemic and hyperlipidemic donors. In all three phospholipid subclasses the percentages of the species 16:0/20:4 were increased in hyperlipidemic patients. In diacyl-PE, 18:1/20:4 was also elevated. No changes were observed in the other quantitatively important molecular species containing arachidonic acid at sn-2, namely 18:0/20:4. The rise in 16:0/20:4 in diacyl-PC and diacyl-PE of hyperlipidemic donors was accompanied by a fall in molecular species with linoleic acid (18:2) at sn-2 (in particular 18:1/18:2). In alkenylacyl-PE the elevation of 16:0/20:4 was compensated by a decrease in species with docosatetraenoic acid (22:4) at sn-2 in particular by a fall in 16:0/22:4. Among all donors, the percentages of 16:0/20:4 in diacyl-PC and PE were positively associated with plasma total cholesterol levels. The changes in molecular species composition of PC and PE in hyperlipidemia are expected to alter the function of erythrocyte membrane transport proteins and--if present also in other cell types--to affect eicosanoid metabolism.


Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycerol.

Phospholipid conjugates of antiretroviral nucleoside analogs have been proposed to have several advantageous features when compared to the parent drugs (Hostetler, K.Y. et al. (1990) J. Biol. Chem. 265, 6112-6117). Here we report on the synthesis of one such type of lipid conjugates, i.e., nucleosides diphosphate diacylglycerols. The syntheses of 3'-azido-3'-deoxythymidine diphosphate diacylglycerol, 3'-deoxythymidine diphosphate diacylglycerol and 2',3'-dideoxycytidine diphosphate diacylglycerol (with different acyl chains) were performed starting from phosphatidic acid and the antiviral nucleoside. A high-performance liquid chromatography procedure for a single step purification of the compounds is presented. The compounds were characterized biochemically, using rat liver enzymes and chemically by phosphorus, fatty acid, ultraviolet, IR and 1H-NMR analyses. Preliminary data on the behaviour in aqueous solution of some of the compounds are presented.


A new glycolipid from Mycobacterium avium--Mycobacterium intracellulare complex.

From a nonpolar lipid fraction of Mycobacterium avium--Mycobacterium intracellulare complex cell mass, a new glycolipid was obtained, which was shown to be 5-mycoloyl-beta-arabinofuranosyl-(1-->2)-5-mycoloyl-alpha-ar abinofuranosyl- (1-->1')-glycerol. When examined by TLC, all the 12 strains of this species tested, including clinical isolates, were found to contain this glycolipid. But the glycolipid was not detected in Mycobacterium bovis BCG or Mycobacterium tubrculosis H37Rv.


Identification and characterization of apolipoprotein(AII-E2-AII) complex in human plasma lipoprotein.

A new apolipoprotein complex designated as the apo(AII-E2-AII) complex was identified in the lipoprotein fractions of human plasma with apoE phenotypes containing apoE2 (E4/E2, E3/E2, and E2/E2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by an immunoblotting assay using anti-apoE or anti-apoAII antibodies, established that the apo(AII-E2-AII) complex, with a molecular weight of 58,000, was identical to the complex consisting of apoE and apoAII, and that it also dissociated following reduction with beta-mercaptoethanol. This new complex was also demonstrated to be distinct from the apo(E-AII) complex and apoE monomer by isoelectric focusing, in the samples that were not treated with beta-mercaptoethanol. In apoE phenotype E3/E2, the apo(AII-E2-AII) complex was primarily included in the high-density lipoprotein (HDL, 1.063 < d < 1.21 g/ml) fraction, but was also observed in a small quantity in the very-low-density lipoprotein (VLDL, d < 1.006 g/ml) fraction. For further characterization, the apo(AII-E2-AII) complex was isolated by preparative SDS-PAGE, and no contamination of apo(E-AII) complex and apoE monomer was detected by immunoblotting assay using an anti-apoE antibody. It was confirmed by an enzyme-linked immunosorbent assay (ELISA) system that a molecular ratio between apoAII monomer and apoE in the isolated apo(AII-E2-AII) complex was approx. 2, when the apo(E-AII) complex was used as a standard with the ratio of 1:1. It indicates that the apo(AII-E2-AII) complex is formed from two molecules of apoAII monomer and one molecule of apoE.(ABSTRACT TRUNCATED AT 250 WORDS)


Protein transfer between A-I-containing lipoprotein subpopulations: evidence of non-transferable A-I in particles with A-II.

Transfer of apolipoproteins (apo) between the two subpopulations of apo A-I-containing lipoproteins in human plasma: those with A-II [Lp(AI w AII)] and those without [Lp(AI w/o AII)], were studied by observing the transfer of 125I-apo from a radiolabeled subpopulation to an unlabeled subpopulation in vitro. When Lp(AI w AII) was directly radioiodinated, 50.3 +/- 7.4 and 19.5 +/- 7.7% (n = 6) of the total radioactivity was associated with A-I and A-II, respectively. In radioiodinated Lp(AI w/o AII), 71.5 +/- 6.8% (n = 6) of the total radioactivity was A-I-associated. Time-course studies showed that, while some radiolabeled proteins transferred from one population of HDL particles to another within minutes, at least several hours were necessary for transfer to approach equilibrium. Incubation of the subpopulations at equal A-I mass resulted in the transfer of 51.8 +/- 5.0% (n = 4) of total radioactivity from [125I]Lp(AI w/o AII) to Lp(AI w AII) at 37 degrees C in 24 h. The specific activity (S.A.) of A-I in the two subpopulations after incubation was nearly identical. Under similar incubation conditions, only 13.4 +/- 4.6% (n = 4) of total radioactivity was transferred from [125I]Lp(AI w AII) to Lp(AI w/o AII). The S.A. of A-I after incubation was 2-fold higher in particles with A-II than in particles without A-II. These phenomena were also observed with iodinated high-density lipoproteins (HDL) isolated by ultracentrifugation and subsequently subfractionated by immunoaffinity chromatography. However, when Lp(AI w AII) radiolabeled by in vitro exchange with free [125I]A-I was incubated with unlabeled Lp(AI w/o AII), the S.A. of A-I in particles with and without A-II differed by only 18% after incubation. These data are consistent with the following: (1) in both populations of HDL particles, some radiolabeled proteins transferred rapidly (minutes or less), while others transferred slowly (hours); (2) when Lp(AI w AII) and Lp(AI w/o AII) were directly iodinated, all labeled A-I in particles without A-II were transferable, but some labeled AI in particles with A-II were not; (3) when Lp(AI w AII) were labeled by in vitro exchange with [125I]A-I, considerably more labeled A-I were transferable. These observations suggest the presence of non-transferable A-I in Lp(AI w AII).


Mevinolin, a competitive inhibitor of hydroxymethylglutaryl coenzyme A reductase, suppresses enterocyte esterification of exogenous but not endogenous cholesterol.

Mevinolin (lovastatin), a competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase, directly inhibited acyl-CoA cholesteryl acyltransferase in rabbit intestinal microsomes at a dose of 20 micrograms/ml or more. Lineweaver-Burk analysis showed a competitive type of inhibition with respect to oleoyl-CoA. In cultured intestinal Caco-2 cells, mevinolin reduced [14C]oleate incorporation into cholesteryl-esters by 86% of controls at doses as low as 0.1 micrograms/ml. However, in cells whose activity of acyl-CoA cholesteryl acyltransferase was stimulated 7-fold by 10 mM mevalonolactone, a significant inhibitory effect on cholesteryl-ester formation could not be detected, even at 40 micrograms/ml of mevinolin. In contrast, cells supplied with liposomal cholesterol or cholesterol derived from low-density lipoproteins showed a marked reduction of cholesteryl-ester formation in the presence of 10 or 0.1 micrograms/ml of mevinolin, respectively. It is concluded that the observed suppressive effects of mevinolin on cholesterol esterification in cultured Caco-2 cells are indirect and possibly caused by changes in the acyl-CoA cholesteryl acyltransferase substrate pool or intracellular cholesterol transport.


Regulation of intestinal apo A-IV mRNA abundance in rat pups during fasting and refeeding.

The amount of intestinal apolipoprotein (apo) A-IV mRNA was examined in rat pups during fasting and refeeding. When 14-day old pups were fasted for 15 h, apo A-IV mRNA levels in the whole intestine decreased to 20% of the prefasting level. Refeeding casein and lactose, and the artificial milk composed of Intralipid, casein and lactose, caused an elevation of the apo A-IV mRNA after 3 h, without accompanying an elevation of serum triacylglycerols and apo A-IV (fat-independent elevation of apo A-IV mRNA). Refeeding Intralipid alone simultaneously elevated the apo A-IV mRNA, and serum triacylglycerols and apo A-IV after 3 h (fat-dependent elevation of apo A-IV mRNA). Administration of physiological saline during fasting partly suppressed the reduction of the apo A-IV mRNA (40% of the prefasting level), and the dietary fat-independent elevation of the message disappeared. Refeeding dam's milk to the pups, fasted without water administration, increased the apo A-IV mRNA after 3 and 15 h, although the elevation of serum triacylglycerols and apo A-IV occurred only after 15 h. Refeeding the milk increased the apo A-IV mRNA after 3 h and 15 h. Refeeding dam's milk to the pups fasted with saline administration accelerated the fat-dependent elevation of the apo A-IV mRNA. Simultaneously refeeding Intralipid and Pluronic L-81, an inhibitor of lymphatic fat transport, delayed the elevation of the apo A-IV mRNA and serum triacylglycerols and apo A-IV. Transcription rates of the apo A-IV mRNA, determined by nuclear run/on assay, were similar before and after fasting and refeeding Intralipid. During fasting, administration of puromycin, as compared with actinomycin D, enhanced the disappearance rate of the apo A-IV message. Intestinal mRNA for apo B, but not for apo A-I and beta-actin, similarly changed to the apo A-IV message. Thus, it can be concluded that: (1) dietary fat-dependent and -independent factors are involved in the elevation of the intestinal apo A-IV message; (2) the elevation of the message is not mediated by lipid uptake in the enterocytes but rather stimulated by the events leading to secretion of chylomicrons; and, (3) dietary fat-dependent elevation of the message appears to be due to the stabilization of the message.


Characterization of recombinant human and rat pancreatic phospholipases A2 secreted from Saccharomyces cerevisiae: difference in proteolytic processing.

An expression plasmid for human pancreatic phospholipase A2 in Saccharomyces cerevisiae was constructed by insertion of cDNA encoding its preprophospholipase A2 into a yeast expression vector pAM82. The resulting product secreted in the yeast culture medium was mainly prophospholipase A2, which was the same as the natural proenzyme in all aspects examined, including the higher order structure. However, when the rat preprophospholipase A2 cDNA was manipulated in the same manner, the active phospholipase A2 of the intact mature form was secreted with the proenzyme being hardly detected in the medium. This unexpected favorable result would occur due to cleavage of rat phospholipase A2 pro-peptide by a trypsin-like proteinase in S. cerevisiae. Based on this finding, we constructed a plasmid carrying the sequence coding for the prepro-peptide of rat pancreatic phospholipase A2 behind the PHO5 promoter in the pAM82 vector, which leads to the secretion of heterologous proteins as their mature form. The use of this plasmid led to secretion of biologically active human pancreatic secretory trypsin inhibitor and a glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600, which are eukaryote and prokaryote proteins, respectively, in the culture medium of S. cerevisiae.


Molecular cloning and sequence analysis of guinea-pig NADPH-cytochrome P-450 oxidoreductase [corrected].

A cDNA clone coding for cytochrome P-450 oxidoreductase was isolated from a guinea-pig liver cDNA library. The cDNA, MSr2, contained a complete coding region of 678 amino acids. The amino acid sequence of the guinea-pig cytochrome P-450 oxidoreductase showed approx. 90% identities with those of rat, human, rabbit, pig enzymes indicating conservation of primary structure of the enzyme during evolutionary divergence of species. The high conservation of acidic residues of the enzyme sustained the importance of them to maintain its function [corrected].


Nucleotide sequence of hamster spermidine/spermine-N1-acetyltransferase cDNA.

The nucleotide sequence of a cDNA encoding hamster spermidine/spermine-N1-acetyltransferase, a key enzyme in polyamine degradation and excretion, has been determined. The cDNA consists of a 1016 base pair insert including 120 nucleotides of the 5' untranslated region and the complete 3' untranslated region. The deduced amino acid sequence is very similar to the human spermidine/spermine-N1-acetyltransferase with only 8 differences in 171 amino acids and the corresponding nucleotide sequence shows 91% identity. The 5' untranslated regions are even more closely related with 97% identity suggesting that this region may play a role in the regulation of acetyltransferase activity. Translation of the acetyltransferase mRNA in a reticulocyte lysate was not altered by the addition of N1,N12-bis(ethyl)spermine.


Nucleotide sequence of the 5' end including the initiation codon of cDNA for the E1 alpha subunit of the human branched chain alpha-ketoacid dehydrogenase complex.

The 5' end including the start AUG codon has been defined for the human E1 alpha subunit of the branched chain alpha-ketoacid dehydrogenase complex by rapid amplification of cDNA ends. Considering conservative substitutions the amino acid sequence in the mitochondrial targeting sequence for the human clone is 73% identical to this sequence in rat and 84% identical to the bovine sequence. This peptide also shows similarity to the targeting sequence for the human beta subunit but not with targeting sequence for the other subunits of the complex.


Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis.

A new approach for the analysis of hotspots of mutations is described. It is based on the classification of hotspot site sequences. Using this approach, the consensuses RGYW and TAA of hotspot sites were revealed in the V gene. Correlation between somatic mutations and these consensuses is investigated by the statistical weight method in 323 somatic substitutions in 14 V genes. Assuming the absence of any correlation, the probability of observing such data in the sample would be very low (0.0003). These results support the idea that emergence of somatic mutation is significantly influenced by neighbouring base sequences. This idea was also supported by the analysis of 296 somatic mutations in flanking sequences of V genes. It is supposed that this influence is an important feature of somatic hypermutagenesis.


Cloning and characterization of the secY gene from the cyanobacterium Synechococcus PCC7942.

The secY gene product is an essential component of the Escherichia coli cytoplasmic membrane, which mediates the protein translocation across the membrane. We found a gene homologous to secY in the genome of the cyanobacterium Synechococcus PCC7942. The deduced amino acid sequence, 439 amino acids long, shows 43% homology with that of the E. coli secY. The hydrophobic profile suggests that the Synechococcus SecY protein is an integral membrane protein containing ten membrane-spanning segments, which are closely related to the E. coli counterpart. The SecY protein may participate in the protein translocation across the cytoplasmic or thylakoid membrane in Synechococcus PCC7942.


Cloning and nucleotide sequence of the cDNA encoding human erythrocyte-specific AMP deaminase.

The nucleotide sequence of cDNA encoding human erythrocyte AMP deaminase has been determined by screening of human spleen cDNA library and by utilizing polymerase chain reaction (PCR) techniques. The 3.7 kb cDNA contains an open reading frame of 2301 bp which encodes 767 amino acids chain resulting in 89 kDa protein. The polyadenylation consensus signal (5'-AATAAA) located at 1212 bp 3' downstream from the stop codon. The homologies to human and rat muscle-specific AMP deaminases showed 64.1% and 65.2% identities, respectively, at the nucleotide level in the area of open reading frame, and 60.2% and 59.8% similarities at the deduced amino acid level.


rel, NFKB, and the Brachyury T gene.

Comparing the sequences of the murine Brachyury T gene protein with the NKFB, rel and dorsal protein family reveals weak sequence similarities, including a DNA-binding/dimerization motif. These similarities may reflect homologies between these proteins or may only be of phylogenetic relevance.


Gender related differences in the expression and characteristics of glutathione S-transferases of human colon.

In the present study, the expression of glutathione S-transferase (GST) isozymes was compared in human male and female colon tissues. GST isozymes were purified and quantified in five male and five female colon tissue samples. Noticeable differences were observed in the isoelectric focusing profiles (IEF) of the GSTs, from male and female colon tissues. Both male and female colon tissues had three common GST peaks with pI values of 9.2, 6.7 and 4.8. An additional GST peak with a pI value of 6.2 found in all females was not found in males. Based on kinetic, immunological and structural properties, these isozymes were classified into alpha (pI 9.2), mu (pI 6.7 and 6.2) and pi (pI 4.8) classes. Activity of the alpha-class GST in male colon was approx. 2-fold higher than the corresponding isozyme in female colon. The pi-class GST 4.8 was the most predominant GST in both the sexes and its activity with CDNB as substrate was more abundant (about 1.6-fold) in female colon as compared to that in male colon. Significant differences were seen in substrate specificities between male and female colon GST 4.8. Sex related differences were also observed in the inhibition kinetics of GST pi from male and female colon in the presence of hematin. In addition, GST pi isolated from female colon was more thermostable as compared to the corresponding male isozyme. The thermostability of purified GST pi isozyme from males or females was not affected by incubation of the enzyme with either estrogen, testosterone or progesterone.


Stress factors affecting expression of O6-methylguanine-DNA methyltransferase mRNA in rat hepatoma cells.

O6-Methylguanine-DNA methyltransferase (MGMT) is decisively involved in protecting mammalian cells against genotoxic effects of alkylating carcinogens. We analysed regulation of MGMT expression after exposing rat hepatoma H4IIE cells to various 'stress' factors. Treatments that damage DNA such as alkylation, hydrogen peroxide, ultraviolet or X-ray exposure, as well as restriction enzymes introduced into cells by electroporation or arrest of replication by hydroxyurea significantly induced MGMT mRNA (2.5 to 5-fold). Slight induction (up to 2.5-fold) was observed after heat shock or cadmium/zinc treatment. No or only a very weak induction (less than 1.5-fold) was observed after treatment with 6-thioguanine, 5-azacytidine, transfection of methylated DNA, depletion of MGMT by feeding with O6-methylguanine or O6-benzylguanine, serum starvation and feeding of starved cells, cAMP, TPA and dexamethasone treatment. Inhibitors of protein kinases, H8 and H9, induced MGMT mRNA. On the other hand, an inhibitor of phosphatases (sodium vanadate) prevented induction of MGMT by N-methyl-N'-nitro-N-nitrosoguanidine. The data indicate that DNA breaks are an ultimate signal for MGMT mRNA induction and that protein phosphorylation is involved in regulating MGMT expression.


Involvement of AP1 and PEA3 binding sites in the regulation of murine tissue inhibitor of metalloproteinases-1 (TIMP-1) transcription.

Transcription of tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted protein that regulates the activities of the metalloproteinases, collagenase and stromelysin, is activated by serum growth factors. Transient transfection experiments have revealed several regions of cis-acting regulatory sequences involved in the response of the murine TIMP-1 gene to serum. One area is in the vicinity of the promoter, consisting of a non-consensus binding site (5'-TGAGTAA-3' at -59/-53) for transcription factor AP1 and an adjacent 24 bp region of dyad symmetry that contains a PEA3-binding site. A second is an upstream region (-1020 to -780) that acts as an enhancer when linked to a heterologous promoter, and contains a consensus AP1 binding site (at -803/ -797). Gel retardation assays revealed differences between nuclear factors in mouse C3H10T1/2 cells that bound to the TIMP(-59/ -53)AP1 site and a consensus collagenase TRE (TPA-response element). The TIMP(-59/ -53)AP1 site is a promiscuous motif that binds c-Fos/c-Jun AP1 translated in vitro and is an effective competitor for binding of nuclear AP1 factors to the consensus TRE, but in addition it binds factors that do not associate with the consensus TRE. The TIMP(-59/ -53)AP1 motif and the dyad symmetry region stimulated expression from a thymidine kinase promoter in an additive fashion, and competition experiments showed that excess copies of these factor binding sites reduced expression from a reporter plasmid driven by the TIMP-1 promoter. Our data show that binding sites for AP1 and PEA3 transcription factors are involved in the regulation of TIMP-1 transcription, which suggests that the coordinated induction of TIMP-1, collagenase and stromelysin may be achieved through the actions of a shared set of nuclear transcription factors. However, the properties of the TIMP-1(-59/ -53)AP1 motif likely reflect an additional type of transcriptional regulation restricted to TIMP-1.


Interaction of human and Escherichia coli tRNA(Phe) with human 80S ribosomes in the presence of oligo- and polyuridylate templates.

Human placenta and Escherichia coli Phe-tRNA(Phe) and N-AcPhe-tRNA(Phe) binding to human placenta 80S ribosomes was studied at 13 mM Mg2+ and 20 degrees C in the presence of poly(U), (pU)6 or without a template. Binding properties of both tRNA species were studied. Poly(U)-programmed 80S ribosomes were able to bind charged tRNA at A and P sites simultaneously under saturating conditions resulting in effective dipeptide formation in the case of Phe-tRNA(Phe). Affinities of both forms of tRNA(Phe) to the P site were similar (about 1 x 10(7) M-1) and exceeded those to the A site. Affinity of the deacylated tRNA(Phe) to the P site was much higher (association constant > 10(10) M-1). Binding at the E site (introduced into the 80S ribosome by its 60S subunit) was specific for deacylated tRNA(Phe). The association constant of this tRNA to the E site when A and P sites were preoccupied with N-AcPhe-tRNA(Phe) was estimated as (1.7 +/- 0.1) x 10(6) M-1. In the presence of (pU)6, charged tRNA(Phe) bound loosely at the A and P sites, and the transpeptidation level exceeded the binding level due to the exchange with free tRNA from solution. Affinities of aminoacyl-tRNA to the A and P sites in the presence of (pU)6 seem to be the same and much lower than those in the case of poly(U). Without a messenger, binding of the charged tRNA(Phe) to 80S ribosomes was undetectable, although an effective transpeptidation was observed suggesting a very labile binding of the tRNA simultaneously at the A and P sites.


The unique structure of the Paramecium caudatum hemoglobin gene: the presence of one intron in the middle of the coding region.

The gene encoding hemoglobin (Hb) in the ciliated protist, Paramecium caudatum, has been cloned and sequenced. A single Hb locus was detected on an isolated genomic fragment with a size of approx. 15 kbp. This gene contained 443 bp. The 5' transcriptional regulatory region contained TATA box- and CAAT box-like sequences which resemble the structure of the promoter region in higher eukaryotic genes. The amino acid sequence deduced from the gene agreed precisely with that of the major Hb component. It was an unusual structure, much smaller than Hbs from many other organisms. Furthermore, all of the untranslated regions in this gene were also small. Particularly, one intervening sequence (intron) being only 23 bp was shorter than any other introns reported for eukaryotic genes encoding protein. The position of the intron in the P. caudatum Hb gene corresponded closely to that of the second intron which had been reported only for plant Hb genes. Possible routes for the evolution of Hb genes were discussed.


Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.


Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.


The two splice variants of collagen XII share a common 5' end.

The short splice variant of collagen XII is composed of 1960 amino acid residues. This polypeptide contains the same signal peptide and the same carboxy-terminus as the long splice variant, but lacks an internal fragment of 1164 amino acid residues. Thus, the two variants of collagen XII are not created by the use of two different transcription initiation sites as generally assumed, but result from the inclusion or skipping of several exons located within the collagen XII gene.


Nucleotide sequence of rat erythropoietin.

The cDNA for the rat erythropoietin (EPO) has been cloned and sequenced. The deduced amino acid sequence consists of 166 amino acid residues, which has a 79% and 95% homology with human and mouse EPOs, respectively. Many short stretches, highly conserved in primate and rodent EPOs, are found in the 3'-noncoding region when insertions and deletions are taken into consideration.


Linkage position determination in a novel set of permethylated neutral trisaccharides by collisional-induced dissociation and tandem mass spectrometry.

A set of neutral permethylated trisaccharides identical in the non-reducing (A----B) disaccharide and linkage isomeric in the reducing terminal (B----C) disaccharide has been synthesized. Collision-activated tandem mass spectrometry was used for analysis of the B----C linkage position. The trisaccharides, gal(beta 1----4)glc(beta 1----X)glc, where X = 3, 4 and 6, were synthesized and examined using fast atom bombardment collision-induced dissociation tandem mass spectrometry. Results were rationalized using molecular modeling. We have previously reported results for determination of the A----B linkage position with isomeric sets of synthetic trisaccharides containing internal amino sugars. The neutral trisaccharides were synthesized to isolate electronic effects of the amino group. An approach of relating daughter ion to parent ion ratios and collision energy offset was used to generate slopes that predict linkage position in glc beta 1----X glc reducing end glycoside form of terminal trisaccharides.


Measurement of muscle protein fractional synthetic rate by capillary gas chromatography/combustion isotope ratio mass spectrometry.

The measurement of skeletal muscle protein fractional synthetic rate using an infusion of (1-13C)leucine and measuring the isotopic abundance of the tracer in skeletal muscle protein by preparative gas chromatography (GC)/ninhydrin isotope ratio mass spectrometry (IRMS) is laborious and subject to errors owing to contamination by 12C. The purpose of this study was to compare muscle (13C)leucine enrichment measured with the conventional preparative GC/ninhydrin IRMS approach to a new, continuous-flow technique using capillary GC/combustion IRMS. Quadriceps muscles were removed from four Sprague-Dawley rats after each was infused at a different rate with (1-13C)leucine for 6-8 h. Muscle leucine enrichment (at. % excess) measured by both methods differed by less than 4%, except at low (13C)leucine enrichments (less than 0.03 at. % excess). In addition, capillary GC/combustion IRMS was used to assess muscle (13C)leucine enrichment and fractional muscle protein synthesis rate in ten normal young men and women infused with (1,2-13C2)leucine for 12-14 h. This approach reduced the variability of the isotope abundance measure and gave estimates of muscle protein synthesis rate (0.050 +/- 0.011% h-1 (mean +/- SEM); range = 0.023-0.147% h-1) that agree with published values determined using the standard analytical approach. The measurement of (13C)leucine enrichment from skeletal muscle protein by capillary GC/combustion IRMS provides a simple, acceptable and practical alternative to preparative GC/ninhydrin IRMS.


Liquid chromatographic/atmospheric pressure chemical ionization mass spectrometric analysis of synthetic elastase inhibitor peptide.

A liquid chromatographic/atmospheric pressure chemical ionization mass spectrometric method for the determination of tert-butyloxycarbonyl-Tyr-Leu-Val-CH2Cl (Boc-YLV-CH2Cl) was investigated. Boc-YLV-CH2Cl was eluted from a Cosmosil 5C8 column in a solvent system of 0.1% acetic acid-methanol (30: 70, v/v). The positive ion spectrum of Boc-YLV-CH2Cl showed a quasi-molecular ion at m/z 526 with fragment ions which provided structural information. Boc-YLV-CH2Cl was purified from blood and tissue samples with Sep-Pak C18 cartridges. The analysis in biological samples was performed by focusing the characteristic ion at m/z 526. The calibration graphs were linear over the concentration range studied (injected amounts of 9.5-190 pmol). The method was applied to the determination of Boc-YLV-CH2Cl in biological samples.


Mass spectrometric quantification of cysteine-containing leukotrienes in rat bile using 13C-labeled internal standards.

Leukotriene C4 and N-acetyl leukotriene E4 were determined in rat bile using a procedure based on high-performance liquid chromatographic fractionation, hydrogenation to 5-hydroxyeicosanoic acid, and gas chromatographic/mass spectrometric selected ion monitoring analysis of the pentafluorobenzyl ester, trimethylsilyl ether derivatives. 13C4-Labeled analogs of the leukotrienes were synthesized and used as internal standards. The concentrations of both leukotrienes in rat bile were markedly elevated after administration of endotoxin to anesthetized animals; N-acetyl leukotriene E4 was the more abundant product. The presence of leukotriene C4 in rat bile after endotoxin challenge was confirmed by fast atom bombardment/tandem mass spectrometry with precursor ion scanning. Quantitative determination of leukotriene C4 using the tandem mass spectrometric procedure was consistent with the gas chromatographic/mass spectrometric data but the latter procedure gave a substantially lower detection limit.


Design and optimization of a micropump-based electrospray device for preparation of nitrocellulose substrates for plasma desorption time-of-flight mass spectrometry.

Details of a nitrocellulose (NC) substrate preparation technique in 252Cf plasma desorption time-of-flight mass spectrometry were investigated using an electrospray device, in which a micropump was employed for solution delivery. The molecular ion yields for three standard proteins (porcine insulin, chicken-egg lysozyme and chymotrypsinogen A) were studied as a function of the electrosprayed NC layer thickness, spray rate, NC solution concentration and some other parameters. Optimal parameters of the NC substrate preparation procedure were determined, which include deposition of a layer of 250-750 micrograms cm-2 at up to 15 microliters min-1 spray rate and up to 15 micrograms microliters-1 solution concentration.


Determination of L-654,066, a new 5 alpha-reductase inhibitor in plasma by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry.

L-654,066 is a new 5 alpha-reductase inhibitor. A sensitive and specific assay based on combined liquid chromatography/mass spectrometry has been developed for the determination of this drug candidate in plasma. The analyte was isolated from plasma by solid-phase extraction on a C-18 cartridge. A related substance, L-683,838, was used as the internal standard. Extracts were chromatographed on a 5 cm C18 reverse-phase high-performance liquid chromatography column interfaced via the heated nebulizer probe to a corona discharge chemical ionization source. The mass spectrometer was operated in the positive ion tandem mode. The method has sufficient sensitivity, precision, accuracy and selectivity for the analysis of clinical samples containing L-654,066 at concentrations in the range 0.5-20 ng ml-1. The chromatographic run time is less than 2 min.


Simultaneous determination of tenidap and its stable isotope analog in serum by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry.

A specific high-performance liquid chromatographic/atmospheric pressure chemical ionization tandem mass spectrometric assay for the quantitative determination of tenidap and its D3 analog in human serum is described. The assay exhibited a linear dynamic range of 0.1-25 micrograms ml-1. Its precision for the two analytes over the range was 17% or better. The assay validity and its utility to investigate changes in tenidap bioequivalence and pharmacokinetics are presented.


Water as a useful calibrant for thermospray mass spectrometry.

The present report demonstrates that thermospray liquid chromotography/mass spectrometry (LC/MS) calibration can be easily achieved by the observation of water cluster ions in filament-on or discharge ionization modes. Cluster ions of sizes ranging from 1 to 44 water molecules are readily observed and thus are useful over the mass range from 17 to 792 Da.


Optimization of short-column gas chromatography/electron ionization mass spectrometry conditions for the determination of underivatized anabolic steroids.

A gas chromatographic/mass spectrometric method based on the use of short capillary gas chromatograph columns (3-5 m) and electron ionization mass spectrometry has been optimized and evaluated for the determination of underivatized anabolic steroids. The short-column gas chromatographic/mass spectrometric method was shown to result in short analysis times and to require minimal sample preparation, but suffered from some loss in sensitivity and chromatographic resolution compared with conventional gas chromatographic/mass spectrometric techniques for derivatized steroids. Therefore, short-column gas chromatographic conditions were optimized to maximize the sample transfer efficiency (sensitivity) from the gas chromatograph into the ion source of the mass spectrometer, while maintaining chromatographic integrity and minimizing thermal decomposition. Mass spectrometric conditions were optimized to maximize ionization efficiency with respect to the intensity of the molecular ion and degree of fragmentation such that positive identification of each steroid could be made based on the resulting mass spectra. Under optimized conditions, we have shown that underivatized anabolic steroids spiked into urine samples can be determined at low-nanogram levels using short-column chromatography/full-scan electron ionization mass spectrometry.


Utility of liquid chromatography/fast atom bombardment mass spectrometry and liquid chromatography/thermospray mass spectrometry for structure identification of metabolites of a fluorinated herbicide.

This study demonstrates a useful application of on-line microbore high-performance liquid chromatography (HPLC) fast atom bombardment (FAB) and thermospray (TSP) mass spectrometry techniques for identification of metabolites from the in vitro metabolism of an experimental Monsanto herbicide: 2-chloro-N-(ethoxymethyl-N-[2-methyl-6- (trifluoromethyl)phenyl]acetamide, 'chloroacetanilide'. The microbore HPLC FAB technique on a high-resolution sector mass spectrometer accelerated identification of polar metabolites from the in vitro metabolism study of the herbicide. It provided good chromatographic resolution and excellent FAB sensitivity with strong protonated molecular ions. Scanning high-resolution LC/FAB mass spectrometry also provided molecular formulae for structural elucidation of unknown metabolites. Sample purification and concentration were minimized. Identification of less polar metabolites was carried out using LC/TSP mass spectrometry with a quadrupole mass spectrometer. LC/TSP mass spectrometry provided useful structural information for both polar and less polar metabolites because their spectra showed more fragmentation than FAB spectra. Glutathione conjugation was the major reaction observed during in vitro incubation of the herbicide. Oxidation of the chloroacetanilide by rat liver enzymes was also a significant metabolic reaction. Seven metabolites were identified, of which four were glutathione conjugates.


Electron impact and fast atom bombardment mass spectrometric analysis of the food-borne carcinogens 2-amino-3-methylimidazo [4,5-f] quinoline, 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline and their metabolites.

Electron impact (EI) and fast atom bombardment (FAB) mass spectrometry were used to characterize the heterocyclic aromatic amines, 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and their metabolites. The carcinogenic N2-hydroxy metabolites and several non-conjugated detoxification products were analysed directly by EI mass spectrometry, while several polar sulphate and beta-glucuronic acid conjugates were analysed by FAB mass spectrometry. Analysis of beta-glucuronic acid conjugates was also achieved by EI mass spectrometry following silylation.


[Radioimmunoscintigraphy: fundamentals (1)].

Radioimmunoscintigraphy (scintigraphy of benign and malignant diseases with radioactively labelled monoclonal antibodies) is being performed via clinically controlled studies in Germany since 1985. In this review the immunological fundamentals and diagnostic possibilities of this new diagnostic approach are given described.


[Radioimmunoscintigraphy of colorectal tumors: state of the art (2)].

Radioimmunoscintigraphy (RIS) of colorectal carcinomas is under investigation in controlled clinical studies in Germany since 1985. With the availability of 99mTc labelled monoclonal antibodies against the carcinoembryonic antigen (CEA), there is an increasing acceptance of this new technique; nevertheless, the merits of RIS in comparison to established methods of staging and restaging of patients with colorectal carcinomas as computed tomography, endoscopy and sonography are still under discussion. In this review the current "State of the Art" of radioimmunoscintigraphy of colorectal carcinomas is given in respect of whether there is additional information which can be gained only by Ris but not by the established diagnostic procedure.


[A debatable issue: indications for the selection and use of absorber materials].

In response to the present occasion, the areas of application of different radiation-absorbent test device materials used in practice are presented in an overview. The test methods recommended for acceptance and constancy testing, the absorbers suitable for this as well as the dependence of a few test parameters relevant to image quality and their tolerances regarding the absorber material are described. The test results are verified and it is shown that in the use of technical test devices, unusually high dose tolerances or high densities of the test device exposures, for example, are by all means in agreement with good technically induced medical imaging quality.


[Magnetic resonance angiography: physical principles and clinical applications].

Within the last four years magnetic resonance angiography (MRA) developed very rapidly towards a well accepted screening technique for vascular examinations as a fast add on to conventional MR. This review describes the basic physical principles as well as the different methods like time-of-flight and phase-sensitive MRA for visualization of blood vessels. Different applications of 3D, 2D sequential and 3D multi-volume MRA are shown from various regions of the head and body. A short outlook to quantitative flow measurements is given in the last chapter including some interesting applications of these techniques which show the still expanding potential of magnetic resonance.


["Pseudo-dissection" in an abdominal aortic aneurysm].

Sonography of 52 patients with aneurysms of the abdominal aorta revealed in 7 cases hypoechoic crescent-like zone between parietal thrombus and the aortic wall which simulated aortic dissection. Duplex-sonography was performed in all cases and no flow could be detected in this area. In two patients intraoperatively several milliliters of a serous fluid could be aspirated from this zone which confirmed the suggestion of seroma within the parietal thrombus. This observation may be found rather frequently and should not be a reason for immediate surgery.


[The importance of CT for the diagnosis of adrenal gland tumors: myelolipoma].

Myelolipoma is a rare benign tumour of the adrenal cortex. 20% of the patients have intratumoral, mostly tiny foci of calcifications. In most cases CT, as the gold standard for imaging the adrenal gland, is able to diagnose the myelolipoma. MR yields no additional information. Even large tumours with or without calcifications can remain without surgical intervention if there is a regular follow-up. The indications for surgical management are reduced by the characteristic appearance of this tumour on CT scan.


[Complications in the kidney pelvis and the ureter following kidney transplantation].

Development of complications of the pelvis and the ureter, especially obstruction and necrosis with an urinary fistula was seen in 35 of 751 patients after kidney transplantation (4.6%). In this study they occurred significantly often in patients with multiple renal arteries of the donor kidney, for example pole arteries. An arterial angiography of the aorta abdominalis in living donors before transplantation is not be neglected. The diagnosis of urological complications includes sonography, in cases of ureteral obstruction percutaneous antegrade urography, and in a given case computed tomography.


[Bladder tumors--Magnevist-assisted magnetic resonance tomography].

Magnetic resonance imaging (MRI, Magnetom 0.5 Tesla) using a high-resolution surface coil was performed on 10 healthy volunteers, 16 patients with bladder tumours, 2 patients with cystitis and 15 patients with bladder tumours and cystitis. Lesion delineation versus urine, bladder wall and surrounding organs was evaluated in plain and contrast-enhanced (IV Magnevist, 0.1 mmol/kg) T1-weighted, plain proton-density and T2-weighted spin-echo images. Surface-coil MRI proved very sensitive since all lesions were found in plain scans. Cystitis and 27 of 31 tumours exhibited contrast enhancement, however, differentiation of tumours and cystic lesions was not improved. Plain T2 and contrast-enhanced T1-weighted images demonstrated bladder wall disruption and perivesical tumour spread with equal sensitivity. Post-contrast images permitted unequivocal visualisation of perivesical tumour spread in surrounding peritoneal fat. The authors conclude that T2-weighted imaging may be restricted to non-enhancing bladder tumours thus significantly reducing imaging time.


[The clinical relevance of sonography in intensive care units].

336 sonographic examinations were performed with a portable ultrasound scanner on 217 patients in intensive-care units. In 302/336 (90%) cases, the region of clinical interest could be demonstrated completely or mostly by ultrasound. The clinical question could be answered by ultrasound in 270/310 (87%) cases partially. In 17/210 (5%) cases the clinical question could not be answered by ultrasound, in 26/336 (8%) cases there was no clinical question at all. 211/336 (63%) sonographic examinations were retrospectively classified as clinically relevant. A large part of clinically relevant examinations was found at the sonographic evaluation of the liver (100%), of the pericardium (96%), of the kidneys (89%), of the blood vessels (80%), of the gallbladder (78%), of the pleural space (76%) and on sonographic evaluation of patients with blunt abdominal trauma (91%). A relatively small part of clinically relevant examinations was found at sonographic evaluation of circumscribed or free intraabdominal fluid (59% resp. 57%), of cholestasis (53%) and of suspected neoplasm (11%). Sonographic examinations of the pancreas (40%) or the spleen (33%) and examinations without any clinical question (50%) also showed little clinical relevance.


[Bladder outflow obstruction due to a small, median prostatic cyst--diagnosis and interventional radiology treatment].

We describe a rare case of a bladder outlet obstruction due to a midline prostatic cyst. In the following clinical appearance, diagnostic imaging and therapy by CT-guided punction are reported. Differential diagnosis and therapy are discussed.


[Detection of a pheochromocytoma in the urinary bladder wall].

We present the case of a 23-year old female patient with clinical signs and biochemical results of a pheochromocytoma. Abdominal ultrasound gave no hints on localisation. Angiography demonstrated a richly vasculated tumour in the region of the superior urinary bladder. Computed tomography (CT) was able to localise the tumour but could not differentiate the tumour origin in bladder or uterus. The selective venous catheter blood sampling confirmed the localisation of the norepinephrine producing pheochromocytoma in the inferior pelvic region. Intraoperative diagnosis turned out to be an extra-adrenal pheochromocytoma of unknown tumour status in the dorsal bladder wall.


A national survey of physicians' and nurses' attitudes toward endoscope cleaning and the potential for cross-infection.

To elicit information on endoscope cleaning practices and the perceived risk of cross-infection, questionnaires were mailed to 500 physicians and 1,000 gastroenterology nurses and associates in November and December 1991. The survey results indicated that while the majority (83% of nurse/associates and 82% of physicians) expressed satisfaction with current cleaning methods, levels of confidence in endoscope reprocessing varied in several key areas. Concern about the potential for device-borne human immunodeficiency virus transmission was expressed by 82% of nurses and 87% of physicians, while 75% of nurses and 53% of physicians feared being liable should cross-infection occur. While most respondents (86% of physicians and 30% of nurses) were unaware of cross-infection incidents, 6% of nurses reported outbreaks in their institutions. According to survey responses, the risk of cross-infection may be attributable to variable cleaning practices--failure to clean scopes before placing them in disinfectant, abbreviated overall cleaning times, and inconsistent disinfectant immersion times--that may be compounded by unreliable automatic disinfectors. At least once a year endoscopes were blocked by debris according to 74% of nurses and 72% of physicians. Reactions to glutaraldehyde had been experienced by 87% of nurses and 65% of physicians. Gastroenterology professionals may be unaware of the actual incidence of cross-infection, but recognize its potential. Lapses in cleaning practices and the difficulty in monitoring postendoscopy nosocomial infections add to their uncertainty.


Endoscopic retrograde hemorrhoidal sclerotherapy.

This article describes an endoscopic retrograde hemorrhoidal sclerotherapy technique that is well tolerated and associated with high patient satisfaction, low complication rates, and satisfactory long-term results. This therapy is best used when colonoscopy is performed, and the need for treatment of hemorrhoidal disease is identified prior to the procedure. Since conventional sclerotherapy equipment is used, the demands on the endoscopy staff and finances are lessened. This article also discusses the implications for nursing care and patient teaching associated with this procedure.


Primary biliary cirrhosis: current diagnosis and treatment.

Primary biliary cirrhosis is a progressive noninflammatory destruction of the interlobular bile ducts within the liver, leading to cholestasis and eventual cirrhosis. Ninety percent of affected patients are women. Most patients are initially without symptoms or have mild symptoms such as fatigue or pruritus. A minority of patients have the classical triad of jaundice, pruritus, and xanthelasmas. Almost all patients will have positive anti-mitochondrial antibody test results and an elevation of the serum alkaline phosphatase level. Primary biliary cirrhosis is thought to be an autoimmune disorder with additional liver injury being mediated by the subsequent cholestasis and accumulation of toxic bile acids. New treatment modalities include colchicine, ursodeoxycholic acid, and methotrexate. All patients, including those with only minor symptoms, have increased mortality compared with age-matched controls, thereby emphasizing the need for early diagnosis.


Droperidol (Inapsine).

Droperidol may be used to bring about conscious sedation in therapeutic and diagnostic endoscopy. Droperidol is a neuroleptic tranquilizer that produces marked tranquilization and sedation. Droperidol may be used to allay apprehension and provide a state of mental detachment while maintaining reflex alterness necessary in endoscopic interventions. Adverse reactions to droperidol are generally self-limiting or readily managed with hydration to correct hypotension.


Age-related modifications in neural cardiovascular control.

Integrated cardiovascular responses to a range of different stimuli, as well as the overall, spontaneously occurring variability in blood pressure and heart rate, undergo complex changes with aging. A general trend is that homeostatic control mechanisms lose part of their ability to modulate heart rate and to buffer the concomitant blood pressure variations; the two phenomena are possibly linked by a cause-effect relationship. A detailed analysis of the age-related changes in the major reflex systems reveals a clear-cut impairment in arterial baroreceptor control of the heart rate, but much less pronounced changes in its control of blood pressure, on the other hand, both the hemodynamic and humoral components of the cardiopulmonary reflex appear to be markedly attenuated. The experimental evidence of the mechanisms underlying these changes is still largely incomplete, and it appears that the gaps will have to be filled by a systematic, detailed analysis, i.e., that no generalizations or extrapolations will be possible. Indeed, the data available so far indicate that the age-related alterations are highly non-uniform, some functions undergoing a definite impairment but others being much better preserved and some being even enhanced; thus aging is by no means associated with a generalized decline in cardiovascular functions and should instead be viewed as a complex, highly selective process. These peculiar biological features of the aging phenomena merit further investigation in both the cardiovascular and the other organ systems, in order to verify the possibility that currently unrecognized homeostatic potentials in the elderly subject may be exploited to advance his/her clinical management in health and disease.


Stress, glucocorticoids, and aging.

Attention has long been focused on the relationship between stress and aging, both under the guise of stress as an accelerator of normal aging and of aging as a time of impaired ability to cope with stress. This review examines the considerable amount of evidence in support of these views. We address these ideas with respect to glucocorticoids, the adrenal steroid hormones secreted during stress. In particular, we concentrate on three model systems: 1) programmed senescence in marsupial mice and semelparous fish as mediated by glucocorticoid excess; 2) glucocorticoid hypersecretion in rats and its role in damaging the aging brain; and 3) potential human and primate adrenocortical dysfunction during aging. We discuss physical and cognitive consequences of adrenocortical dysfunction in these systems, and how they may relate to human aging.


Age-related changes in tissue contents of immunoreactive calcitonin gene-related peptide.

Age-related changes in calcitonin gene-related peptide (CGRP) content were found in a number of rat tissues, and most can be explained by the physiological and pathological changes associated with advancing age. In some specific areas of the brain, and some cardiovascular tissues such as atria and arteries, the CGRP content declined with age (p < 0.01), while the contents were increased in the thyroid gland, plasma and in veins (p < 0.01). In the substantia nigra and striatum, the immunoreactive-CGRP (i-CGRP) levels found in older rats were about 50% of those found in adolescent rats (p < 0.01). The i-CGRP levels found in the penis, bladder, kidney, testis and adrenal gland gradually increased up to maturity, and then rapidly declined in the aging rats. However, in the lungs and sensory ganglia (dorsal root and trigeminal), the highest content of i-CGRP was found in immature 3-week-old rats, and then declined with age. Changes of i-CGRP observed with age may support multiple physiological roles for CGRP at various stages of development. The changes described here, particularly in the lungs, substantia nigra, striatum and the cardiovascular system, should help in further understanding the physiological and pathophysiological role of CGRP in health and in age-related disorders.


Changes in posture and balance with age.

Changes in posture with age are of concern because of their association with impaired mobility and the possibility of falls. In this study alignment of the joint centers and balance of body segments were measured in 41 women, aged 65 and older, and correlated with weight, age, change in height, and activity. Balance was assessed by calculating the whole-body center of gravity, and the partial centers of gravity above the knee and hip. Compared to a younger reference population, the older subjects had greater kyphosis, a more posterior hip position, and leaned forward more from the hips (more anterior center of gravity above the hips). Among the 41 elderly subjects, larger kyphoses correlated with greater decrease in height. Elderly subjects who were inactive tended to learn more, but forward lean was not correlated to other variables. These findings suggest two separate sets of changes: an osteoporosis-related stooping, and also a forward lean that may relate to muscular weakness or to fear of instability. Posture among the elderly was quite varied and did not follow any one pattern of change.


Norepinephrine sensitivity of the endocrine pancreas in aging F344 rats.

The effect of norepinephrine (NE) infusion on glucose-stimulated insulin secretion was evaluated in whole perfused pancreases isolated from 6-, 12-, and 26-month-old male Fischer 344 (F344) rats. Glucose-stimulated insulin secretion was significantly inhibited by NE in all age groups. There was no significant effect of aging on the sensitivity or magnitude of inhibition at any of the concentrations of NE. In contrast, vascular pressure attained during NE infusion was significantly less in the 26-month-old rats compared to 6- and 12-month-old animals. These results suggest that the adrenoceptor neuroeffector mechanism of the smooth muscle declines at a faster age-related rate than does the beta-cell's adrenoceptor mechanism.


Effect of cholinergic tone on growth hormone-releasing hormone-induced secretion of growth hormone in normal aging.

Aging is associated with an impairment in the GH response to GHRH and to several other stimuli of GH secretion. We evaluated the effect of pyridostigmine (PD) or placebo pretreatment (Protocol A: placebo or 120 mg PD orally at 8 a.m.; Protocol B: placebo or 60 mg PD twice orally at 9 p.m. and 7 a.m.) on GH responsiveness to GHRH (1 micrograms/kg BW bolus i.v. at 9 a.m.) in 15 normal elderly males (65-92 years) and in 14 normal young adults (20-37 years). GH response to GHRH was significantly reduced in elderly subjects compared to young adults (p < 0.05). PD (Protocol A) increased GH release in both elderly and young subjects. In elderly men, PD enhanced GH response to GHRH. The phenomenon was more evident when PD was administered according to Protocol B (p < 0.01). The area under the curve of GH was significantly greater after PD plus GHRH than it was after placebo plus GHRH (p < 0.01). In young adults, PD induced an increase in GH responsiveness to GHRH when administered according to Protocol A (p < 0.05) but not Protocol B. Both GH peak and AUC of GH after PD plus GHRH (Protocols A and B) in elderly subjects were not significantly different from the same parameters found in young subjects after placebo plus GHRH. Our data confirm that pituitary somatotrophin responsiveness to GHRH in man changes with aging. PD restores GH responsiveness to GHRH in elderly subjects. The effect of PD on GH secretion suggests that cholinergic mechanisms may be involved in GH control in normal aging.(ABSTRACT TRUNCATED AT 250 WORDS)


Clinical trials in cognitive impairment in the elderly.

The pharmacological treatment of dementia is particularly difficult because of the uncertainty of the diagnosis and of the underlying pathogenetic processes, and because of the variable severity of the syndrome. This paper suggests several lines for future therapeutic trials in dementia. These include the need to work with homogenous populations and to take into account the natural history of the disease, the pharmacologic characteristics of the drugs, and the particular ethical considerations pertaining to demented patients. We review the diagnostic criteria for dementia, Alzheimer's disease (AD) and vascular dementia. We discuss the different types of therapeutic trial design, the problem of the duration of the protocol, the tools to evaluate the cognitive effects and the activities of daily living, and the use of a Clinical Global Impression of Change (CGIC). The results of some therapeutic trials in AD and vascular dementia are given as examples.


[Unevenness of distribution of historical events throughout an 11-year solar cycle].

Tchizhevsky hypothesis (1922) of historical process heliotaraxia (helios--sun, taracsio--perturb) was empirically tested. Samples of near 13 and 4.6 thousand events mentioned in Chronology sections of two largest Soviet historical handbooks were analyzed. Events were classified into 4 groups on the basis of "strength" and "social contradictions meaning" of their names, called tolerance and polarity: tolerant--intolerant (e.f. riot--roform) and polar--neutral (e.f. civil war-external war). It was found that frequency and polarity of historical events increased in maximum of sunspot cycle and in the next year as compared with minimum and the year before minimum. The probability of revolution (the most polar and intolerant name of historical event) is the highest in maximum and the lowest in the year before minimum. Intolerance of polar events increased and neutral events decreased in maximum. All these relations were highly significant (P < 0.001). It was concluded that heliotaraxic phenomena exist and are basically associated with year of sunspot maximum.


[Biological detector of weak cosmic fields].

Possibility of use of biological object as a detector of faint cosmophysical fields is discussed. The signals of free induction and electric echo caused by a short-term effect of light with an intensity of approximately 0.2 mWt/sm2 on biological systems were observed. The experiments with Blatella germanica, Muska domestica were performed in vivo and with red and green seaweeds and yeast cells in vitro. The signals were detected by means of the dielectric permeability measurement in a dynamic regime. Main features of the observed responses are described and assumption on mechanisms of their origin are given. An example of registration of the Lunar eclipse on a detector with in active element of Blatella germanica is given.


[Temporal analysis of calls of the psychiatric emergency medical aid team].

6075 calls for the psychiatric emergency ambulance unit were reviewed. An analysis of the hourly and 24-hour number of calls showed the existence of 24-hour, lunar and seasonal rhythms in the frequency of calls. The correlation between clinical symptoms and the number of calls points to aggravations of diseases. The accumulated data indicate that there is a connection between the dynamics of the psychopathological processes and ecological factors which are determined by periodic fluctuation of gravitation fields.


[Sensitivity of biological objects to the effects of the geomagnetic field].

It is shown on the basis of calculations of energy sublevels of the hyperfine structure that the effect of the geomagnetic field upon the impurity atoms in the volume of living cells should be considered in relation to the value of geomagnetic field induction pulses delta B. When delta B > or = 10 pT and the dielectric constant epsilon > or = 10, magnetodipole transitions between sublevels of the hyperfine structure within one term are possible in impurity atoms in their 2P-state. During magnetic storms with delta B > or = 100 nT magnetodipole or magnetoquadrupole forced transitions from 2P1/2 and 2P3/2 states to 2S1/2 metastable state are possible in the resonant zones formed by intersection of hyperfine energy sublevels of the corresponding excited levels.


[The effect of weak variable extremely low frequency magnetic fields on the infra-day rhythm of the rat sympathoadrenal system].

It was found in open test that changing magnetic fields with 8 Hz frequency and 5 mcTl induction in daily 3 hours exposition on rats with a middle level of the mobile activity move the phase of diuresis daily rhythms and epinephrine and norepinephrine yield as well. In rats with desinchronosis provoked by hypokinesia the changing magnetic fields restore the synchronism of the studied processes.


[Cosmophysical correlations in live and non-live nature as a manifestation of weak interactions].

Determination of weak effects is proposed, their relationship with cosmophysical correlations is advanced. The experimental results are presented on the intensification coefficient of weak effects (10(4)) for polarized electromagnetic radiation. The magnetoresonance mechanism of weak effects is suggested. The experimental evidence in favor of this mechanism and its application to the problems of cosmophysical correlations are given.


[The synchronizing effect of infralow frequency magnetic fields on biological systems].

Time-organization of lipid peroxidation and thiol groups exchange was examined. It was shown that one-divisible 3-hours effect of the extremely low magnetic fields depending on the frequency ensured formation of definite rhythm of the investigated processes. The obtained results are considered as an evidence of frequency-dependent adoption of the magnetic fields by the living systems according to the mechanism of forced synchronization of elementary cyclic processes underlain by integral oscillations.


[Connection between the sector structural of the interplanetary magnetic field and hematologic indicators in normal and neoplastic processes].

Interplanetary magnetic field (IMF) sector polarity was compared to hematologic indexes of normal mice and mice after single injection of carcinogen 1,2-Dimethylhydrazine. It has been established that the hemoglobin level and the number of leucocytes in the standard blood volume depend on IMF sector polarity: both indexes are higher at +IMF, than at -IMF. Mice injected with carcinogen have mainly higher hemoglobin level at +IMF and mainly lower one at -IMF than the control mice.


[Cosmophysical fluctuations and development of the human embryo].

An analysis of the dependence of embryonic pathology development on heliocosmic factors is conducted and regular changes of the appearance of different kinds of pathology depending on heliocosmic situations in different months of the year are shown. Periods of conception of increasing amount of children with different disturbances of development coincide with values of the Wolf's number and sun-radiation stream density of certain ranges in comparison with the mean values, the same indexes are in conformity with the periods of conception of children having haemolytic disease and diabetic foetopathy.


[Effect of an infralow frequency magnetic field on nerve cell rhythm and their resistance to hypoxia].

The direct effect of infralow-frequency (0.05, 0.1, 0.25 Hz, 100 nT) magnetic fields (MF) was demonstrated on the brain cellular--tissue model--surviving slices of mouse cerebellum. MF influence is a trigger for the nervous cells. MF-5 Hz revealed two-phases response: inhibition and excitation of the impulse activity of neurons. Besides that we recorded convulsive effect of MF. The experiments with simultaneous exposure of hypoxia and MF revealed a prohypoxia effect of MF, when the oxygen concentration was very low and also after reoxygenation. The surviving slices may be used as a model for studying the fine mechanisms of influence of different intensity MF on the nervous cells.


[Deviation from a Poisson distribution in a series of identical tests of E. coli cultures as a result of the effect of correlating factors of exo- and endogenous natures].

Vital cells number (VCN) in the sampling of E. coli populations was experimentally measured and distribution histograms were obtained. In most cases distributions show considerable deviation from Poisson model. VCN distribution histograms are polymodal, dispersion/arithmetical mean ratio may essentially differ from 1. The more essential differences from Poisson distribution were observed for populations with the higher cell concentration. Computer simulation of the VCN histograms indicated that additional parameters (such as those describing cellular interaction of different nature and/or other factors that influence random behaviour of cells) should be introduced into Poisson model to explain observed variations in VCN distribution histograms.


[Correlation of the probability of occurrence of breast neoplasms with solar activity].

A contingent with high incidence of precancerous breast diseases (1080 persons) was studied to assess the relationship between solar activity and 1) alterations in the relationship between women with early menarche and women with middle and late menarche (high and low risk of breast cancer respectively) and 2) incidence of benign breast diseases. It was established that population content alterations were wave-like and had statistically significant periods 3,5; 7 and 12 years, and correlate with Wolf number changes. Incidence of benign breast diseases in the group aged 20-50 years also changed periodically (T = 11 years) depending on the birth-year and correlated conversely with Wolf numbers at the birth-years. Incidence of benign breast diseases changes periodically in the population of women with early menarche with the period of 3.6 years, and in those with middle and late menarche--with the periods of 10.5, 3.2 and 4 years. There is a statistically significant correlation between the incidence of benign breast diseases and Wolf numbers in population of women with middle and late menarche.


[Processing biorhythmic data modified by a cosinor analysis method].

Modified Cosinor-Analyses method used in processing of biorhythmological data is considered. Advantages of modified method and its practical results are shown. Assumption of oscillations with the periods of 4-6 hours in humans daily physiological activity is confirmed.


[The possible effect of parameters of the gravitational field on indicators of functional asymmetry of the brain].

Correlation of the brain functional asymmetry (BFA) in 2400 humans born in 1930-1984 and numbers of the joining of planets, moon and the Sun was investigated in corresponding years of conception and birth. A significant correlation with BFA was found in men, while there was no such correlation in women.


[Modulation of energy metabolism of lymphocytes in children by natural physical factors].

The aim of this work is exploration of natural physical factors influencing change of age-dependent energy exchange parameters (e.g. activity of SDG) in healthy and premature children. 35 healthy and 35 premature children were investigated over 11 years from early antenatal and postnatal period. Cytochemical investigations were carried out 1-2 time a year until 6 years of age. Solar activity, geomagnetic excitability, air temperature and atmospheric pressure and its diurnal variation on index represented environmental factors. It is suggested that quiet magnetic field might be a predictor of higher level of SDG activity and vice versa. Higher activity of magnetic field leads to displacement of maximum-age of SDG activity to an earlier age. Lymphocytes react to magnetic storm in a phasic manner. Other physical environmental factors might have reciprocal effect so that some of them could negate the effect of others. If intrauterine development took place in a year of higher solar activity, early-development results. However, later development is characterized by lower level of SDG activity. On premature children, as well as in healthy subjects, the more influencing factors on energetic age-dependent parameters are geomagnetic field and variation of atmospheric SDG activity in healthy children is within normal range (and has a training effect), while in premature children depression of hydrogenase in the end stage.


[Natural and technogenic electromagnetic background and its effect on the environment].

Causes of the initiation of natural and technogenic electromagnetic background, its effect on the man and environment are considered. Ideas are discussed of eliminating negative consequences of the effects of electromagnetic waves of different range on the man.


[Reaction of rats with various constitutional features to the effect of weak variable magnetic fields of extremely low frequency].

The influence of 8 Hz frequency and 5 mcTl induction changing magnetic fields on the functional state of blood neutrophils, sympathoadrenal system and brain integrative activity in rats with low, middle and high level of mobility was investigated in open test. It was found out that changing magnetic fields provoke the stress-reaction development in rats with low mobile activity. The adaptation which develops in rats with high and middle mobile activity comes later by 5-7 days in high mobility rats.


A non-nucleotide-based linking method for the preparation of psoralen-derivatized methylphosphonate oligonucleotides.

A method is reported for conjugating an analog of 4'-(aminomethyl)-4,5',8- trimethylpsoralen to methylphophonate oligonucleotides. This method enables the psoralen moiety to be coupled to the phosphonate backbone between any two desired bases in a sequence. When hybridized to a target mRNA, the psoralen moiety can be directed toward a uridine base and, in turn, can undergo a photo-addition reaction with the target under UV irradiation at 365 nm. Several different non-nucleotide-based amino-linker reagents have been prepared for incorporation into methylphosphonate oligonucleotides by standard phosphonamidite chemistry. In addition, an N-hydroxysuccinimide activated ester analog of 4'-[(3-carboxypropionamido)methyl]-4,5',8- trimethylpsoralen has been synthesized for conjugation to the amino-linker moieties. Using this approach, we have prepared a number of psoralen-methylphosphonate-oligonucleotide conjugates which are complementary to the chimeric bcr/abl mRNA associated with chronic myelogenous leukemia. Solution hybridization studies with a 440-base subfragment of the bcr/abl RNA have shown that the psoralen moiety does not adversely affect duplex stability. Polyacrylamide gel electrophoresis analyses have demonstrated that the psoralen-oligonucleotide conjugates undergo photo-addition to the RNA in a sequence-specific manner. Optimal photo-addition occurs when the psoralen moiety is inserted adjacent to one or more adenine residues in the oligonucleotide sequence, particularly between adenine and thymine (5'-3'). This internal labeling approach greatly increases the number of potential target sites available for photo-cross-linking experiments.


A novel method for the incorporation of glycoprotein-derived oligosaccharides into neoglycopeptides.

We describe a new method for the transfer of carbohydrate moieties to polypeptides in which complex carbohydrate, in the form of glycosyl amino acid, is removed from an available glycoprotein, derivatized, and reacted with a polypeptide via an iodoacetylated alpha-amino group. A family of oligomannose chains, N-linked to the side chain of Asn, was obtained from ovalbumin by pronase digestion and purified as previously described. A reactive sulfhydryl group was specifically placed on these molecules by reaction of 2-iminothiolane with the Asn alpha-amino group. Separately, the alpha-amino group of the peptide GGYR was specifically iodoacetylated by reaction with iodoacetic anhydride at pH 6. Reaction of the thiol-containing carbohydrate with iodoacetylated peptide at pH 8 gave in high yield the corresponding oligomannosyl-peptides, whose structures were confirmed by mass spectrometry. A peptide inhibitor of HIV protease was also oligomannosylated by this procedure. The principle advantage of this method is the efficiency of the reaction even when performed with stoichiometric amounts of the two molecules at low concentration. It should be feasible to extend this chemistry to larger polypeptides.


Genetic lesions in preleukemia.

Preleukemia is thought to be a clonal disorder of hemopoietic stem cells. The conversion of a normal cell into a preleukemic and ultimately leukemic state is a multistep process requiring the accumulation of a number of genetic lesions. The myelodysplastic syndromes have become a paradigm for human preleukemia, where nonrandom chromosomal abnormalities, including complete or partial deletions of chromosomes five and seven, trisomy eight and Y chromosome loss suggest specific changes. Of particular significance are 5q deletions, as many genes important in hemopoiesis are located in this region, including the proto-oncogene FMS, which encodes the receptor for the macrophage colony-stimulating factor, CSF-1. Genetic damage such as point mutations in the RAS and FMS genes has been detected in preleukemia patients. The RAS gene family (N, K and H) encodes membrane-bound G proteins, which, like other proto-oncogenes, are components of the intracellular signal transduction pathways controlling mitogenesis and differentiation. The characterization of such lesions may ultimately identify those patients at greatest risk of leukemic transformation.


A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.


The ras/cholesterol connection: implications for ras oncogenicity.

The frequent detection of mutated ras genes in a variety of cancers (reviewed in Bos, 1988, 1989; Der, 1988) suggests that ras makes a significant contribution to human malignancies (reviewed in Barbacid, 1987; Lacal and Tronick, 1988; Der, 1989). While the role of ras in malignancy is unclear, it is well-established that the association of ras protein with the inner surface of the plasma membrane is critical for triggering ras oncogenicity. The trafficking of ras proteins to the plasma membrane requires a series of three closely linked posttranslational modifications (farnesylation, proteolysis, and carboxymethylation) that are signaled by the consensus C-terminal CAAX motif present on all ras proteins (reviewed in Rine and Kim, 1990; Gibbs, 1991; Der and Cox, 1991). THe recent discovery that an essential intermediate in cholesterol biosynthesis, the isoprenoid farnesol, is attached covalently to ras proteins has stimulated considerable interest and has identified important new directions for studies of ras function. First, understanding the role of farnesol-linked interactions with the plasma membrane may identify the biochemical basis for the oncogenic actions of ras proteins. Second, the enzymes that catalyze the processing steps that trigger membrane association of ras proteins are promising targets for pharmacologic intervention in ras-associated disease. In this review, we summarize our current knowledge of the role of posttranslational processing in ras-membrane interaction and transforming activity. We also provide an update of recent studies addressing the role of isoprenoid modification in the function of ras and of other isoprenoid-modified proteins (reviewed in James and Olson, 1990; Glomset et al., 1990; Maltese, 1990). While this role is likely to be specific for each protein, ras proteins can provide an excellent prototype for understanding the role of isoprenoid modification in protein function.


Preparation and characterization of recombinant proricin containing an alternative protease-sensitive linker sequence.

The aim of this study was to determine the feasibility of utilizing a factor Xa-specific cleavage site within a recombinant protein containing the ricin A chain (RTA) sequence. Release of RTA is believed to be an essential step during the intracellular phase of ricin intoxication. Failure to incorporate such cleavage sites in fusions containing RTA results in a loss of toxin action (O'Hare, M., et al. (1990) FEBS Lett. 273,200. Kim, J., and Weaver, R.F. (1988) Gene 68,315). In this report we describe the introduction of a factor Xa-specific site in the linker of proricin, which we use here as a model substrate. Upon purification of the recombinant mutant proricin after expression in Xenopus oocytes, we demonstrate that the protease does have access to the engineered recognition sequence (albeit at low efficiency) and that the presence of the latter does not interfere with disulfide bond formation or the lectin activity of the ricin B chain moiety. Upon cleavage and reduction, the RTA polypeptide displays ribosome-inactivating ability, indicating that the presence of the modified linker at its C-terminus does not interfere with its catalytic activity. The general applicability of using such a cleavage site in recombinant fusions with RTA is discussed.


A model for predicting the risk of cancer consequent to retroviral gene therapy.

Theoretical estimates of the risk of cancer resulting from accidental insertion of retroviral gene therapy vectors into oncogenically vulnerable genomic sites may prove an important supplement to experimentally derived data in estimating risk/benefit ratios for future gene therapy trials. We have approached risk assessment by considering either a single vector insertion event or a single natural mutation to be potentially oncogenic, should either occur in a cell that would otherwise end with one less than the total number of mutations required for frank neoplasia. Estimates of the relative probabilities of these two phenomena yield a relative risk assessment, which in conjunction with epidemiologic data on natural cancer frequencies can be converted into an assessment of absolute risk. This approach yields an estimated range of relative risk over 10 years of about 1.00000026 to 25 for cells bearing single copies of inserted vectors; the upper limit is higher for multiple copies. These estimates, if accurate, imply that small experimental human or animal gene therapy cohorts will rarely, if ever, manifest vector-related cancers and that more precise future risk assessments will require additional data on natural and vector-induced mutation rates.


Preparation of 5- and 6-(aminomethyl)fluorescein.

5(6)-Carboxyfluorescein is protected as the diacetate then reduced to 5(6)-(hydroxymethyl)fluorescein diacetate. The separated isomers are subjected to a Mitsunobu reaction with dibenzyl imidodicarbonate, yielding diprotected 5- and 6-(aminomethyl)fluorescein diacetate. Methanolysis of the acetates followed by deprotection with HBr/acetic acid gives 5- and 6-(aminomethyl)fluorescein hydrobromide.


A convenient and flexible approach for introducing linkers on bifunctional chelating agents.

The synthesis of a protected bifunctional analog of ethylenediaminetetraacetic acid (EDTA) is described. The molecule contains an aminobenzyl moiety that allows the easy attachment of the chelating agent to a wide variety of groups. Examples of reaction with the C-termini of two peptides are given. In the following paper, the two peptides are used to study the enzymatic cleavage of metal chelates from a monoclonal antibody.


Transplantation of genetically modified autologous hepatocytes into nonhuman primates: feasibility and short-term toxicity.

Ex vivo gene therapy directed to the liver is being developed for the treatment of inherited metabolic diseases. Transplantation of hepatocytes that have been transduced with a low-density lipoprotein (LDL) receptor gene is a potential form of therapy for familial hypercholesterolemia (FH). We have demonstrated efficacy of ex vivo gene therapy for familial hypercholesterolemia in a rabbit animal model of this disease. In preparation for human trials, we describe in this report experiments in baboons for documentation of the feasibility and safety of autologous hepatocyte transplantation. Three baboons underwent a partial hepatectomy and their hepatocytes were isolated, cultured, and transduced with a retrovirus containing the human LDL receptor gene. The hepatocytes were harvested and infused into an indwelling catheter that had been placed into the inferior mesenteric vein at the time of liver resection. The baboons tolerated the procedures well and are being maintained and clinically evaluated for an indefinite time period. Follow-up evaluations have ranged from 3 to 8 months. Clinical evaluations have been unremarkable and blood chemistry and hematology determinations have stayed within normal limits.


Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides.

Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5'- and/or 3'-terminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC), a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The Tmax and the thermodynamic parameters (delta H, delta S, delta G) of complex formation were determined. According to these data, coupling of a dye with the 5'-terminal phosphate group is the most advantageous: delta G(37 degrees C) is increased by 3.59 +/- 0.04 kcal/mol compared to 2.06 +/- 0.04 kcal/mol for 3'-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3',5'-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 +/- 0.04 kcal/mol. The association constant of this modified complex at 37 degrees C is 9.5 x 10(6) M-1, whereas the analogous value for the unmodified complex is only 3 x 10(3) M-1.


Influence of a peptide linker on biodistribution and metabolism of antibody-conjugated benzyl-EDTA. Comparison of enzymatic digestion in vitro and in vivo.

Insight into the metabolism of radiolabeled antibodies is important for the design of better radioimaging and therapy agents. To test the effect of linkers that can be cleaved in vivo, we introduced Ala-Leu-Ala-Leu between the antibody Lym-1 and an 111In-labeled benzyl-EDTA. For comparison, we studied a conjugate without the linker. Digestion of the two conjugates in vitro showed that the one with Ala-Leu-Ala-Leu was cleaved rapidly by the liver protease cathepsin B1 (T1/2 approximately 6 h). After 100 h of digestion, reversed-phase HPLC product analysis of the Ala-Leu-Ala-Leu conjugate showed that 48% of the total radioactivity had the same retention time as (p-aminobenzyl)-EDTA(In), and 37% of the total radioactivity had the same retention time as [p-(Ala-Leu-amido)benzyl]-EDTA(In). After 97 h of digestion, the conjugate without the linker had 79% of the radioactivity activity still attached to the protein. We also tested the two conjugates in mice. Ala-Leu-Ala-Leu had only a moderate effect on the whole body and liver clearance in vivo. The excretion of the radioactivity was about 6% per day with the linker and about 3% per day without the linker. HPLC analysis of the urine of a single mouse showed products similar to the in vitro study; 54% of the excreted radioactivity had the same retention time as (p-aminobenzyl)-EDTA(In), while 10% had the retention time of [p-(Ala-Leu-amido)benzyl]-EDTA(In).


Public acceptance of human gene therapy and perceptions of human genetic manipulation.

Clinical trials of gene therapy are underway in different countries, and further countries can be expected to use gene therapy soon. Little remains known, however, about public perceptions of gene therapy. Nationwide mail response opinion surveys were conducted in Japan in August-October, 1991. A total of 54% of the public, 65% of the high school biology teachers, and 54% of the scientists who responded said that they would be willing to use gene therapy, and 66%, 73%, and 62%, respectively, said that they would be willing to use gene therapy on their children. There appears to be growing acceptance of gene therapy in Japan, although about one-quarter of the population are against it. The underlying reasoning behind the acceptability of human genetic manipulation and perceived benefits and risks are presented, and these were found to be generally similar to reasoning expressed in a similar survey conducted in New Zealand in May, 1990. Public perceptions are also compared to those in Europe and the United States. People perceive both benefits and risks from genetic manipulation. There appears to be more teaching of ethical, social, and environmental issues associated with genetic engineering in senior high school biology classes in New Zealand than in Japan. In Japan and New Zealand, about 90% of the public would support including discussion of social issues associated with science and technology in the curriculum.


Immobilized proteins as chromatographic supports for chiral resolution.

This review examines the role of protein-bonded chiral stationary phases (CSPs) in enantiomeric separation and investigates the performance characteristics and desired properties of protein CSPs for separation and large-scale operation. The review also discusses the ability of protein-based CSPs to examine the stereochemistry of drug metabolism processes.


The S-oxidative degradation of a novel corticosteroid tipredane (INN). Part I: Preliminary investigations into the hydrogen peroxide S-oxidation of tipredane.

The C-17 dithioketal moiety of the corticosteroid tipredane (INN, I) has been shown to undergo peroxide oxidation yielding an array of sulphoxide epimers. All epimers have been isolated and characterized by spectroscopic techniques. Oxidation of the C-17 beta methylthio-substituent appeared to occur exclusively giving a 4:1 ratio of the S:R configuration at the sulphoxide moiety. Both methylthiosulphoxide epimers (V) have been shown to be susceptible to thermolysis yielding the monoethylthio- derivative (VI) via elimination of methylsulphenic acid. As expected from stereochemical considerations at the C-17 position the S epimer has been found to be more susceptible than its corresponding R epimer towards thermolysis. The monomethylthiosulphoxide epimers (III) were produced in a 1:1 ratio, indicating that they were formed from the oxidation of the corresponding monomethylthio- derivative (VII). This probably arose from the elimination of ethylsulphenic acid from the alpha-ethylthiosulphoxide (VIII).