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PubMed1999

Self-inactivating lentiviral vectors with U3 and U5 modifications.

Lentiviral vectors have gained much attention in recent years mainly because they integrate into nondividing host-cell genomes. For clinical applications, a safe and efficient lentiviral vector system is required. Previously, we have established a human immunodeficiency virus type 1 (HIV-1)-derived three-plasmid lentiviral vector system for viral vector production which includes a packaging vector pHP, a transducing vector pTV, and an envelope-encoding plasmid pHEF-VSVG. Cotransfection of these three plasmids into TE671 human rhabdomyosarcoma cells routinely yields 10(5)-10(6) infectious units per milliliter in 24 h. Here we have extensively modified long terminal repeats (LTRs) of pTV to generate a safer lentiviral vector system. The 5' U3 was replaced with a truncated cytomegalovirus (CMV) immediate early (IE) enhancer/TATA promoter and the 3' U3 (except for the integration attachment site) was also deleted. These modifications resulted in a vector with 80% wild-type vector efficiency. Further deletion of 3' U5 impaired vector function; however, this problem was solved by replacing the 3' U5 with bovine growth hormone polyadenylation (bGHpA) sequence. The pTV vector containing all these modifications including the 5' promoter substitution, the 3' U3 deletion, and the substitution of 3' U5 with bGHpA exhibited a self-inactivating (SIN) phenotype after transduction, transduced both dividing and nondividing cells at similar efficiencies, and produced vector titers twice as high as that of the wild-type construct. Thus, both safety and efficacy of the HP/TV vector have been improved by these LTR modifications. Further deletion of 5' U5 impaired vector efficiency, suggesting that the 5' U5 has critical roles in vector function.

Animals↗
PubMed1976

Phospholipid-induced modifications in the brain of animals fed with cholesterol-supplemented diet.

1) Cholesterol - enriched diet induces changes in the phospholipid fatty acids of particulate fractions and modifications in the membrane-bound enzyme: 2) BC-PL (100 mg/kg i.p., as shown in rabbits) prevent cholesterol-induced modifications at brain level: 3)BC-PL (100 mg/kg i.v., as shown in mice) have a recovery effect on the cholesterol-induced modification at brain level; 4) It is suggested that some atherosclerotic lesions are analogous to those reproduced by anoxia; 5) It is suggested that the effect of BC-PL is a direct action at the level of the brain cellular membranes.

Adenosine Triphosphatases↗
PubMed1986

Modifications and degradation of high density lipoproteins.

It is evident that lipoprotein modifications, degradation and clearance from plasma and interstitial compartments involves both cellular and extracellular processing. Cellular uptake of the intact particle as a whole and/or selective removal of constituent apoproteins and lipids by various parenchymal cells goes on continuously. Regulation of these processes undoubtedly varies tissue to tissue and much remains to be clarified in human tissues in vivo. The metabolic effects of chemical, proteolytic, and lipolytic modification of lipoproteins secondary to transient cellular encounters (e.g. during transit through endothelial barriers, or reversible binding to cells) on apolipoprotein clearance remains to be defined. It is likely that multiple post-secretory modifications occur and together represent subtle regulatory events that modulate lipid shuttle functions and cellular targetting properties of HDL particles.

Adipose Tissue↗
PubMed1987

Cadmium binding and metal cluster formation in metallothionein: a differential modification study.

Mammalian metallothioneins (MT) contain 20 Cys in a total of 61 amino acid residues and bind 7 Cd and/or Zn ions. The metal is localized in two clusters made up of three and four metal-thiolate complexes in the NH2- and COOH-terminal half of the chain, respectively. The formation of these oligonuclear complexes designated as Cd4- and Cd3-cluster has now been monitored in MT reconstituted with varying amounts of Cd using differential modification of Cys with 14C-iodoacetamide. At ratios below 3 moles Cd/mole MT bound, no differential protection of Cys by the metal and, hence, no preferred binding is detectable. At Cd-to-protein ratios between 3 and 5 moles Cd/mole MT the modification profiles reveal preferred and cooperative binding in the COOH-terminal half of the chain indicating formation of the Cd4-cluster. At still higher ratios formation of the Cd3-cluster is initiated in the NH2-terminal section of the polypeptide chain. Comparison of the differential modification data of Cd6-MT and Cd7-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence. The extent of labelling of the different Cys in Cd7-MT indicates that the ligands of the Cd3-cluster are three times as accessible to iodoacetamide as those of the Cd4-cluster, suggesting a greater thermodynamic stability of the latter.

Animals↗
PubMed1993

Structural modification patterns from agonists to antagonists and their application to drug design--a new serotonin (5-HT3) antagonist series.

Structural variations from agonists to their selective antagonists seemed to follow certain patterns. To analyze the variation patterns in the structural modification processes in past examples as well as to utilize the "common" variation patterns as possible principles to design new selective antagonistic drugs, the structures of agonists and their antagonists were superimposed on a two-dimensional grid template composed of regular hexagons and the topological similarities and dissimilarities of substructural elements between agonists and antagonists were examined. Between several pairs of neurotransmitter amines and their "selective" antagonists, similar patterns were disclosed in their structural modification processes. The generalized structural modification patterns were successfully applied as guiding principles to design and identify a new prototype structure of the 5-HT3 antagonist. The prototype structure was optimized by use of QSAR procedures leading to a compound which shows a potent antiemetic activity as well as a powerful gastrointestinal-motility modulation.

Animals↗
PubMed1996

Heat-shock induced protein modifications and modulation of enzyme activities.

Upon heat stress, the cell physiology is profoundly altered. The extent of the alterations depends on the severity of the stress and may lead to cell death. The heat shock response is an array of metabolic changes characterized by the impairment of major cellular functions and by an adaptative reprogramming of the cell metabolism. The enhanced synthesis of the HSPs is a spectacular manifestation of this reprogramming. Numerous post translational modifications of proteins occur in response to heat stress and can be related to altered cellular functions. Some proteins are heat-denatured and temporarily inactivated. Heat-denaturation is reversible, chaperones may contribute to the repair. The extent of heat-denaturation depends on the cell metabolism: (a) it is attenuated in thermotolerant cells or in cells overexpressing the appropriate chaperones (b) it is enhanced in energy-deprived cells. Covalent modifications may also rapidly alter protein function. Changes in protein glycosylation, methylation, acetylation, farnesylation, ubiquitination have been found to occur during stress. But protein phosphorylation is the most studied modification. Several protein kinase cascades are activated, among which the various mitogen activated protein kinase (MAP kinase) cascades which are also triggered by a wide range of stimuli. As a possible consequence, stress modifies the phosphorylation status and the activity of components from the transcriptional and translational apparatuses. The same kinases also target key enzymes of the cellular metabolism. Protein denaturation results in constitutive hsp titration, this titration is a signal to trigger the heat-shock gene transcription and to activate some of the protein kinase cascades.

Animals↗
PubMed1983

Alteration of tRNA modification in eukaryotes: causes and consequences.

To evaluate the role of the modified nucleosides in tRNA function, especially their involvement in regulatory mechanisms of development, differentiation, or neoplastic transformation we use the following organisms: eubacteria, the slime mold D. discoideum, the topminnow Xiphophorus, and mice. Ribosylthymine, a common modified nucleoside at position 54 in tRNAs of prokaryotes and the major class of eukaryotic elongator tRNAs, is involved in the binding to the ribosomal A-site and is important for the proper functioning of tRNA during translation. Alterations in the extent of this modification occur early in the development of D. discoideum. The fully methylated species are found on polysomes, actively synthesizing protein. The partially methylated tRNAs accumulate in the nuclei, and might be involved in regulatory mechanisms at the transcriptional level. The Q base, a modified deazaguanine derivative, is present at position 34, the first position of the anticodon of tRNAAsn, tRNATyr, and tRNAHis. Alterations in the extent of this modification occur in corresponding tRNAs during the first minutes after the onset of development in D. discoideum and before final differentiation into spores, indicating that Q is important for developmental processes. Changes in the modification of G34 to Q34 in specific tRNAs of the melanophoric system of the topminnow Xiphophorus further support the view that Q is necessary in differentiation. In plasmacytomas and in Ehrlich ascites tumor cells of mice, the amount of unmodified G34 in corresponding tRNAs is correlated to the growth rate, density, or age of the tumor cells.

Animals↗
PubMed1983

Specific effects of 5-fluoropyrimidines and 5-azapyrimidines on modification of the 5 position of pyrimidines, in particular the synthesis of 5-methyluracil and 5-methylcytosine in nucleic acids.

5-Fluoropyrimidines and 5-azapyrimidines were found in our laboratory to be specific inhibitors of modification reactions taking place at the 5 position of pyrimidines in nucleic acids. Thus, 5-fluorouracil and 5-fluorouridine specifically inhibit the formation of 5-methyluracil, pseudouridine, and 5,6-dihydrouracil in tRNA. 5-Fluorocytidine, which is partially biotransformed to 5-fluorouracil derivatives in mammalian cells, inhibits the formation of 5-methyluracil, pseudouridine, 5,6-dihydrouracil, and 5-methylcytosine, and 5-azacytidine is a specific inhibitor of the formation of 5-methylcytosine in tRNA and DNA. Inhibitory effects on tRNA modifications require RNA synthesis, as shown by the observation that various inhibitors of RNA synthesis block the drug effects. An inhibitory low-molecular-weight (4-7S) RNA, consisting mainly of tRNA and pre-tRNA, was isolated from livers of mice after treatment with 5-azacytidine. This RNA, when added to an in vitro tRNA methyltransferase assay, specifically interfered with the formation of 5-methylcytosine in substrate tRNA. Similarly, a DNA inhibiting the synthesis of 5-methylcytosine in an in vitro DNA methylation assay was isolated from L1210 leukemic cells treated with a high dose of 5-azacytidine for a short time. Our data are consistent with the hypothesis that incorporation of 5-azacytosine into positions that are normally occupied by C residues destined to become methylated is required for the inhibition to occur, and a similar situation probably applies to the 5-fluoropyrimidine analogs. Analog base moieties occupying such sites are likely to bind strongly, perhaps irreversibly, to the active sites of the particular modifying enzymes. All our observations with the 5-fluoro- and 5-azapyrimidines are in accord with this hypothesis. It was also observed that administration of 5-azacytidine to mice led to strong inhibition of tRNA cytosine-5-methyltransferase, while at the same time the activities and capacities of purine-specific tRNA methyltransferases became strongly elevated after an initial lag period. We speculate that such increases may represent a response of the cell to the methylation defect induced by the drug. Undermodified tRNAs present in neoplastic cells may also trigger an increased synthesis of modifying enzymes. A scheme has been presented which explains increased tRNA turnover and increased activities of modifying enzymes in neoplastic cells as a consequence of a primary defect in tRNA modification.

5-Methylcytosine↗
PubMed1983

Alterations in post-transcriptional modification of the Y base in phenylalanine tRNA from tumor cells.

Various tumor cells contain chromatographically distinct isoacceptor tRNA species. To decide whether the tumor-specific species represent an expression of a separate tRNA gene or only an undermodified form of normal tRNAPhe, nucleotide sequences of tRNAPhe isolated from neuroblastoma and normal mouse liver were determined by postlabeling techniques. The results showed identical sequences except for the changes of post-transcriptional modifications in the anticodon loop. Normal mouse liver tRNAPhe contained Cm32, Gm34, and the hypermodified YOH next to the 3' end of the anticodon. On the contrary, tRNAPhe from neuroblastoma contained C32, G34, and, instead of YOH base m1G. A small proportion of tRNAPhe species contained an undermodified YOH base. For the examination of the conditions leading to the undermodified tRNAPhe, Vero cells derived from the kidney of African green monkey in culture were used. In these cells, deprivation of methionine or lysine resulted in changes in tRNAPhe modification similar to those in tumor cells. Ehrlich ascites tumor cells were examined to determine whether the presence of altered tRNAPhe species in various tumors is also the result of starvation of some nutritional factors. Results obtained with these cells showed that tRNAPhe species lacking the Y base disappeared in tumor-bearing mice after intraperitoneal injection with a mixture of amino acids and vitamins. Thus it is concluded that tumor-specific tRNAPhe species are the products of aberrant post-transcriptional modification, not the transcripts of different, normally repressed genes.

Animals↗
PubMed1991

Modifications of temporal approaches: anatomical aspects of a microneurosurgical approach.

All subtemporal approaches have in common the risk of temporal lobe damage. To reduce the retraction of the temporal lobe we combine two synergistic modifications of temporal approaches to reach the prepontine space. The first is the temporary resection of the zygomatic arch which allows to bring the temporalis muscle more caudally and subsequently allows an anterior subtemporal approach with only minimal temporal lobe retraction. The second modification is the resection of the apex of the petrous bone after incision of the tentorium. This provides an excellent view into the posterior fossa between the trigeminal nerve medially, the internal carotid artery caudally and the internal auditory canal laterally. The anatomical aspects of a microneurosurgical approach regarding these modifications are reported and discussed.

Brain Neoplasms↗
PubMed1994

High-yield production of human big endothelin-1 by a combination of chemical modification and proteolysis of a fusion protein in Escherichia coli.

A protein modification method has been developed for the production of human big endothelin (ET)-1. Production of a large quantity of big ET-1 by the method described here is expected to facilitate future experiments such as X-ray crystallography and nuclear magnetic resonance studies, aimed at understanding the tertiary structure of big ET-1 and its dynamics. The plasmid pETB-50 used for the synthesis carries the gene for a fusion protein consisting of 34-amino acid (aa) residues of an N-terminal portion of beta-galactosidase and the 38-aa residues of big ET-1. The fusion protein ETB-50P contains an arginine residue in the big ET-1 portion at its second C-terminal site and three lysine residues including the C-terminal site in the beta-galactosidase portion, all of which are susceptible to trypsin. Tryptic digestion of the fusion protein quantitatively produced big ET-1 (1-37), which is depleted in the C-terminal serine. However, a treatment of the fusion protein with 1,2-cyclohexanedione prior to tryptic digestion gave full-length big ET-1 with N7, -N8-(1,2-dihydroxycyclohex-1,2- ylene)-arginine. This modification was reversed to the intact arginine residue when the modified big ET-1 was incubated in 0.5 M TRIS-HCl buffer, pH 8.0. Consequently, a combination of such a reversible protein modification and tryptic digestion gave 1.74 mg of recombinant big ET-1 from 2.01 of culture broth. The procedure described here may be applied to produce other arginine-containing peptides from fusion proteins.

Amino Acid Sequence↗
PubMed1986

Phase I study of MVE-2 evaluating toxicity and biologic response modification capability.

A total of 21 patients were treated in a phase I trial using the biological response modifier MVE-2, a low molecular weight component of pyran copolymer. All patients received weekly IV MVE-2 infused over 2 h. Proteinuria, sometimes of nephrotic proportions, was the dose limiting toxicity, and was seen with increasing incidence as the cumulative dose of MVE-2 exceeded 2500 mg. Other toxicity with MVE-2 was minimal. Biologic response modification at tolerable doses was inconsistent, although several assays, particularly natural cell-mediated cytotoxicity, indicated enhanced activity at higher dosages of MVE-2. No objective tumor responses were observed. MVE-2 is not useful as a biological response modifier using our initial method of administration, since the dose limiting toxicity occurred at lower levels than were necessary to induce consistent biologic response modification. Following completion of the phase I study, we administered MVE-2 by 30-min infusion to 8 additional patients and did not detect proteinuria, in spite of large cumulative doses. It is possible that alternate schedules of MVE-2 administration could minimize proteinuria and allow the administration of dosages necessary for immunologic modification.

Adjuvants, Immunologic↗
PubMed1993

Chemically-induced histone modification as a predictor of carcinogenicity.

The interaction between carcinogens and DNA is believed to initiate neoplastic transformation, but evidence suggests that epigenetic mechanisms may also be of importance. Because the histone proteins have important roles in chromatin structure and cellular function, they provide a reasonably well understood epigenetically-based system for the detection of carcinogens. In this study, human foreskin fibroblastic cells were exposed to one of several mutagens and/or carcinogens for 3, 12, or 24 h to determine if induced histone modification may be a means of predicting chemical carcinogenicity. Butyric acid (5 mM), known to result in acetylation of histones H3 and H4, and 12-O-tetradecanoylphorbol-13-acetate (3 microM), known to result in phosphorylated histone H1, were tested initially. Electrophoresis of the histone fractions was capable of resolving multiple forms of histones H1, H3, and H4. Propane sultone (0.1 mM) induced a broadening of the H2A and H2B bands after a 24 h exposure and carbon tetrachloride (1 mM) induced the formation of new histone forms in the H1 fraction after 24 h and in the H3 fraction after 3 h. Experimental variability limited the statistically significant modifications to carbon tetrachloride and propane sultone, two known carcinogens, where new forms of modified histone were detected. Therefore, the histone modification assay, with further experimentation, may be an alternate method of detecting carcinogens, especially when conventional genotoxic tests prove unreliable.

Carbon Tetrachloride↗
PubMed1992

Changing patterns of binocular visual connections in the intertectal system during development of the frog, Xenopus laevis. III. Modifications following early eye rotation.

In the frog, Xenopus laevis, a system of intertectal connections underlies the visual projection from an eye to its ipsilateral tectal lobe and is involved in the topographic representation of binocular visual space. Rotation of one eye in early life may be followed by a radical rearrangement of the connections in this system. The modified pattern which later emerges is that which keeps the visual projection through the ipsilateral eye in topographic registration with the direct visual projection from the contralateral eye to the same tectal lobe. This plasticity requires visual experience. In this paper we describe the time-course and sequence of events by which this plasticity is effected. Following rotation of one eye in larval animals or in animals undergoing metamorphic climax, the earliest evidence of intertectal modification was found 3-4 weeks after metamorphosis. With increasing intervals after metamorphosis an increasing proportion of animals displayed modified intertectal systems. At intermediate intervals many animals showed partial modifications, which were interpreted as transitional stages in the modification process. Analysis of these transitional stages indicated that the sequence of events involved in the elaboration of a modified intertectal system following the experimental alteration of eye alignment exhibits features in common with rearrangements of the system that occur during normal development in response to growth-related alterations in eye alignment.

Animals↗
PubMed1992

A monoclonal antibody monitoring band 3 modifications in human red blood cells.

Morphologic and metabolic erythrocyte modifications are thought to be the basis of cell removal from circulating blood. A significant role has been ascribed to the immunological network which may remove aged or misshapen erythrocytes through the binding of specific autoantibodies. Along this line recent observations indicate that a senescence antigen appears in consequence of postsynthetic modifications of band 3, one of the most important erythrocyte membrane proteins, which accounts for many functional activities of the red cells. On this basis, we raised a mouse hybridoma anti-band 3 monoclonal antibody (B6 MoAb) of the IgG2a class which monitors band 3 differences among normal red blood cells separated by Percoll density gradient. These differences are outlined by the decrease of B6 MoAb binding to band 3 monomer, the appearance of an 80-90 kDa new band, lighter than band 3, and the increase of low molecular weight fragments in the 4.5 region. The B6 MoAb appears to be very useful in detecting modifications of band 3 since it bind to a 19 kDa Chy-Try fragment estimated to be sensitive to aging.

Aging↗
PubMed1995

A kinematic and kinetic analysis of locomotion during voluntary gait modification in the cat.

As part of a study to characterize the postural reactions that occur during voluntary gait modification, we examined the kinematic, electromyographic (EMG), and kinetic responses that occurred when cats stepped over an obstacle placed in their path. Analyses of the kinematics as each of the forelimbs stepped over the obstacle showed that changes in joint angles were most pronounced at the elbow of the first (lead) limb, and at the shoulder of the second (trailing) limb. In the hindlimbs, there was a pronounced change in the knee joint angle in both the leading and trailing limbs. Examination of the horizontal and vertical velocities of the tip of the forepaw suggests that the movements can be divided into two phases: one in which the limb is rapidly lifted above and over the obstacle, and a slower one during which the limb is carefully repositioned on the floor. On the basis of the velocity profiles, we suggest that the repositioning of the paw on the support surface is more critically controlled for the forelimb than for the hindlimb. Analysis of the changes in the ground reaction forces in the supporting limbs during these gait modifications showed that there were two major increases in vertical reaction force. One of these occurred as the two forelimbs were straddling the obstacle, the other when the two hindlimbs were straddling it. There was also a net increase in the anteroposterior force that resulted in a small increase in propulsion as the cat stepped over the obstacle. Each change in the vertical ground reaction force was paralleled by a similar change in the amplitude of the EMG recorded from the respective extensor muscles. An analysis of the vertical displacement of the scapula and of the pelvis showed that there was a slight increase in the height of the scapula in the support limb just prior to and during the swing phase of the trailing forelimb, and a more pronounced and progressive change in the height of the pelvis prior to and during the passage of both hindlimbs over the obstacle. We suggest that the increases in vertical ground reaction force raise the height of the body sufficiently to allow, respectively, passage of the trail forelimb and each of the hindlimbs over the obstacle. The results are discussed with respect to both the biomechanical changes underlying these gait modifications and the neuronal mechanisms implicated in their control.

Animals↗
PubMed1991

Ventriculojugular shunt against the direction of blood flow. IV. Technical modifications and policy for treatment.

During evolution of the surgical procedure to implant ventriculojugular (VJ) shunts against the direction of blood flow for the treatment of hydrocephalus, various modifications were made in the shunting catheter and in the implantation technique with the aim of improving the results. Shunt implantation was difficult in infants and young children with compressible craniums and the modifications failed to improve the results. Accordingly, they were not considered suitable for this method of cerebrospinal fluid (CSF) shunting. Our present policy is to implant VJ shunts in hydrocephalic patients with rigid craniums and ventriculoperitoneal (VP) shunts in infants and young children to tide them over a period until they become suitable for VJ shunting. During the past 5 years, 110 hydrocephalic patients with rigid craniums were treated by implanting VJ shunts without need for further modifications. The shunt was revised in one patient during the early postoperative period due to CSF leakage under the scalp. There were no complications related to CSF overdrainage in any of the cases.

Cerebrospinal Fluid Shunts↗
PubMed1980

The ral gene of phage lambda. I. Identification of a non-essential gene that modulates restriction and modification in E. coli.

Host controlled restriction in Escherichia coli can be relieved by pre-infecting restricting cells with modified lambda helper phages. This process, in which intact unmodified phage genomes are allowed to escape restriction attack, is mediated by a newly identified lambda function called ral. The ral gene has been located by deletion mapping between cIII and N. Efficient expression of the ral gene requires the product of the regulator gene N. Polyacrylamide gel analysis of the lambda proteins specified by the cIII-N region failed to reveal the product of the ral gene, but demonstrated that protein Ea10 is encoded by a gene located immediately to the left of ral. From these results the map order cIII-Ea10-ral-TL1-N was deduced. Ral specifically alleviates restriction in E. coli K and E. coli B, but does not affect restriction systems EcoRI, EcoRII and EcoP1. In addition, ral enhances the modification activity of the EcoK and EcoB restriction enzymes: we observed that efficient modification of progeny phages obtained by propagating unmodified lambda phages in r-m+ hosts, is dependent upon the presence of ral. We thus conclude that the ral gene product acts by modulating the restriction and modification activities of the type I restriction systems in E. coli, and the possible mechanisms will be discussed.

Bacteriophage lambda↗