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At least 109 records · Page 6Linked to original sources
PubMed1986

Different characteristics of allogeneic and trinitrophenyl-modified H-2-restricted cell-mediated lympholysis: analysis of differences in interleukin-2 dependency using an ontogenical approach.

Ontogenical development of in vitro allogeneic cell-mediated lympholysis (CML) and in vitro trinitrophenyl (TNP)-modified CML was studied mainly in correlation with helper cell activity, using C3H/HeN and C57BL/6 mice. Allogeneic and TNP-modified CML were not detected in the spleen of these mice at 1 week after birth. Allogeneic CML was detectable, in parallel with increases in age. This activity in 5-week-old mice was much the same as in the 8-week-old mice but the TNP-modified CML did not appear until 8 weeks after birth. Exogenous interleukin-2 (IL-2) induced sufficient activity of TNP-modified CML, even in spleen cells from 1-week-old mice, while the same treatment had a weak but significant effect on the induction of allogeneic CML in these same cells. Experiments on the mixed lymphocyte reaction (MLR) in the presence of exogenous IL-2 showed that lymphocyte proliferation in response to TNP-modified cells was higher than that in response to allogeneic cells. These results suggest different dependencies on IL-2 between allogeneic and TNP-modified killer precursor cells. Endogenous IL-2 production and proliferative response in MLR showed that helper cells contributing to the TNP-modified CML matured later, compared to allogeneic CML. Different sensitivities to IL-2 in two types of CML, in addition to different ontogenical developments, suggest that cytotoxic T lymphocytes to allogeneic cells differ quantitatively and qualitatively from TNP-modified H-2-restricted killer cells.

Aging↗
PubMedDate not supplied

Mutational and recombinational events in carcinogen-modified plasmid DNA. Influence of host-cell repair genes.

A plasmid containing the STR operon has been modified in vitro (i) by irradiation with UV light, (ii) by reaction with ethyl methanesulphonate (EMS), (iii) by reaction with N-acetoxy-2-acetylaminofluorene (AcO-AAF), (iv) by reaction with (+/-)trans-benzo[a]pyrene-7, 8-dihydrodiol-9,10-epoxide (BPDE), and (v) by heating at 70 degrees C to produce apurinic sites. Suitably modified plasmid DNA was then used to transform both repair-proficient and repair-deficient strains of Escherichia coli, and the mutation frequency in the plasmid-encoded rspL+ gene measured. The influence of host mutations in the uvrB+, recA+, umuC+ and lexA+, genes on the mutation frequency have been investigated. Transformation into a uvrB strain significantly decreased survival and increased the level of mutations observed for UV- and AcO-AAF-modified plasmid DNA, while only a small increase in mutation frequency was seen with EMS-modified DNA and no increase in mutation frequency with plasmid DNA containing apurinic sites. Mutagenesis in UV- and BPDE-modified DNA (and probably also DNA containing apurinic sites) was totally dependent on he recA+ gene product, while EMS and AcO-AAF induced mutagenesis was only partially independent on the recA+ gene. Transformation of UV- or BPDE-modified DNA into a umuC or lexA strain, on the other hand, showed no change in mutation frequency from that observed with wild-type strain. Pre-irradiation of the wild-type host with UV light before transformation led to a significant increase in mutation frequency for UV- and BPDE-modified plasmid DNA. These results are discussed in terms of mutational or recombinational pathways which may be available to act on modified plasmid DNA, and suggest that the majority of the mutational events measured in this system are due to recombination between homologous regions on the plasmid and chromosomal DNA.

Carcinogens↗
PubMed1987

The Blalock-Taussig shunt in infants: standard versus modified.

In recent years, the modified Blalock-Taussig shunt--a polytetrafluoroethylene graft from the subclavian artery to the pulmonary artery--has been preferred over the standard shunt by some surgeons because (1) it requires less dissection and (2) length of native vessels is not critical. From January, 1979, to June, 1985, we operated on 51 infants less than 1 year of age, including 26 less than 1 week of age, to palliate severe complex cyanotic congenital cardiac malformations. Twenty-four modified Blalock-Taussig shunts and 29 standard Blalock-Taussig shunts were created. The groups were concurrent. We reviewed all available cineangiograms and measured branch pulmonary and subclavian arteries. Pulmonary artery index was not different preoperatively in patients given a modified versus a standard Blalock-Taussig shunt (144 +/- 118 and 118 +/- 59 mm2/m2, respectively), but it was greater postoperatively in patients with a modified shunt (431 +/- 188 and 189 +/- 106 mm2/m2) (p = 0.07). Distortion of the pulmonary artery occurred less often after a modified Blalock-Taussig shunt (4/11) than a standard Blalock-Taussig shunt (6/8) (p = 0.06), though none of the distortions was severe. Early and late shunt failure occurred less often with a modified shunt (5/24) than with a standard shunt (15/29) (p less than 0.05). The modified Blalock-Taussig shunt had advantages over the standard Blalock-Taussig shunt in our series: pulmonary artery growth was greater, distortion of pulmonary arteries was less commonly seen, and shunt failure occurred less often. Thus, in infants, we believe the modified Blalock-Taussig shunt should be considered a reasonable alternative to the standard Blalock-Taussig shunt.

Blood Vessel Prosthesis↗
PubMed1990

Genetic variation in a modifier system affecting the expression of bare mutant of Drosophila subobscura.

Genetic variation affecting the expression of Bare (Ba), a morphological mutant of Drosophila subobscura that reduces the number of bristles and is located on the O chromosome of this species, is reported. Our results show that O chromosomes extracted from a natural population (El Pedroso, Santiago de Compostela, Spain) show considerable genetic variation in modifier effect upon Ba expression. The amount of modifier variability is dependent on the chromosomal arrangement (OST and O3 + 4 + 7), since modifier variation is higher in OST than in O3 + 4 + 7 chromosomes. Investigations of the genetic architecture of this modifier system on the O chromosome carried out using biometrical methods indicate that a relatively small number of genetic "factors" can explain the differences in modifier effect between a wild O chromosome of high modifier effect and a marker chromosome of low score. In addition, the modifier effects show a non-uniform distribution along the O chromosome and some indication of clustering of modifier "factors" around the major locus (Ba) is also observed.

Analysis of Variance↗
PubMed1974

A kinetic and fluorimetric investigation of papain modified at tryptophan-69 and -177 by N-bromosuccinimide.

A systematic study of the modification of papain (its thiol group protected as a disulphide with mercaptoethanol) by N-bromosuccinimide, showed that 2 molar equiv. modified tryptophan-69 and 4 molar equiv. modified tryptophan-69 and -177. The Michaelis parameters for the catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester by these modified enzymes were determined. The enzymic activity of the modified enzymes was not seriously impaired, but modification of tryptophan-177 raised the apparent pK(a) of the acidic limb of the pH profile by more than 1 pH unit for both k(cat.) and k(cat.)/K(m). The fluorescence spectra (excitation at 288nm) of the modified enzymes showed that tryptophan-69 contributed about 8% to the fluorescence intensity, whereas tryptophan-177 contributed about 46% at neutral pH. However, the contribution of tryptophan-177 was quenched at low pH and its fluorescence intensity showed sigmoidal pH-dependence, with an apparent pK(a) of 4.2. Histidine-159, which is in close contact with tryptophan-177, is considered to be the residue responsible for the fluorescence quenching. When tryptophan-177 was modified, presumably generating a less hydrophobic micro-environment, the apparent pK(a) determined kinetically was raised to about 5.4. By comparing the Michaelis parameters of native papain, papain modified at tryptophan-69 and papain modified at tryptophan-69 and -177 with N-benzyloxycarbonylglycylglycine amide and N-benzyloxycarbonylglycyltryptophan amide, tryptophan-69 and tryptophan-177 were shown to be structural features of the S(2) and S(1)' subsites respectively.

Amino Acid Sequence↗
PubMed1972

The evolutionary development of modifier genes.

The main findings of a study of the evolution of modifier gene frequencies in models of deterministic population genetics are presented. A wide variety of random mating systems are subject to selection with modifiers operating, in different cases, on mutation rates, dominance, migration between subpopulations, and linkage between other loci. In all these instances the modifier frequencies evolve in such a way as to maximize the mean fitness of the population at equilibrium. This is remarkable since, except for the dominance modifier, the modifier genes are selectively neutral in the sense that they do not affect the fitness of their individual carriers. In nonrandom mating systems the mean fitness concept is not well defined, and there does not appear to be such a simple principle governing the evolution of modifier frequencies. In assortative mating systems modifiers favoring reduced assortment propensities tend to increase. In contrast, for selfing-outcrossing systems modifiers favoring increased selfing tend to increase.

Alleles↗
PubMed1975

Bifunctional major histocompatibility-linked genetic regulation of cell-mediated lympholysis to trinitrophenyl-modified autologous lymphocytes.

Murine thymus-derived lymphocytes can be sensitized in vitro to trinitrophenyl (TNP)-modified autologous spleen cells (1, 2). Cytotoxic effector cells were generated which were specific for TNP-modified target cells expressing the same H-2K and H-2D serological regions as the modified stimulator cells (3, 7). Spleen cells from two C57BL/10 congenic strains of mice sharing common I-C, S, and D regions, but differing at K, I-A, and I-B regions, generated different levels of lytic responses to the shared modified H-2Dd products upon sensitization with auto logous TNP-modified cells. Lymphocytes from an F1 between responder and nonresponder strain generated a level of cytolysis toward the H-2Dd modified specificity which was of the same order of magnitude as that obtained with the high responder, irrespective of whether F 1 or either parental strain of modified stimulator cell was used. These results suggest that the modification of H-2Dd products resulted in formation of new antigenic determinants in both parental strains. However, the difference observed in responsiveness appeared to be due to a gene or genes mapping in the K, I-A, or I-B region which influenced the ability of the responding lymphocytes to react to these modified H-2Dd products. Responsiveness was expressed as a dominant trait in the F1.

Alleles↗
PubMed1976

Multiple H-2 linked immune response gene control of H-2 D-associated T-cell-mediated lympholysis to trinitrophenyl-modified autologous cells: Ir-like genes mapping to the left of I-A and within the I region.

One of the more recent associations of the murine H-2 major histocompatibility complex (MHC) with immune function has been the finding that cytotoxic T-effector cells generated by sensitization with viral-infected (1-6), chemically modified (7-9), or weak transplantation antigen-associated (10,11) syngeneic cells can efficiently lyse target cells which express the same viral, chemical, or weak antigenic agent, and which share the H-2K and/or H-2D regions of the MHC with the responding and/or stimulating cells. Furthermore, an additional contribution of a gene(s) within the H-2 complex has been demonstrated which controls immune response potential (Ir genes) in the generation of cytotoxic effector cells to trinitrophenyl (TNP)-modified self components (12,13). In such studies it was found that certain B10 congenic strains generated good cytotoxic responses to both TNP- modified H-2K and H-2D region products, whereas other B10 congenic strains exhibited preferential or exclusive reactivity against TNP-modified H-2K region products. Some of these recombinant strains differing in response potential to TNP- modified H-2D products expressed the same haplotype at the D end, but differed at the K end of H-2. The low responsiveness observed in the B10.A strain to TNP-modified H-2D(d) when compared to B10.D2 and (B10.A x B10.D2)F(1) for the same specificity, suggested a role of dominant Ir genes which map in K, I-A, I-B, I-J, and/or I-E (12, 14). In the present report an attemnpt was made to further map within the MHC the Ir gene(s) controlling cell-mediated lympholysis (CML) to TNP-modified H-2D(d), by using recombinant mouse strains on the A and B10 backgrounds. Irrespective of the genetic background, the s and k haplotypes at the K end generated high and low cytotoxic responses, respectively, to H-2D(d)-TNP. The intermediate responder and low responder status of the A.TL and A.AL strains, respectively, indicated that a gene mapping in the K region of H-2 influences response potential. Furthermore, the differences in the levels of cytotoxicity detected in the A.TH and A.TL strains suggested an additional I region influence. Taken together these findings raise the possibility that multiple genes mapping within different regions of the MHC control the level of T-cell-mediated cytotoxicity to chemically modified autologous cells.

Animals↗
PubMed1987

The binding efficiency of polyclonal and monoclonal antibodies to DNA modified with benzo[a]pyrene diol epoxide is dependent on the level of modification. Implications for quantitation of benzo[a]pyrene-DNA adducts in vivo.

A number of polyclonal antibodies specific for DNA modified with (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) were obtained from the sera of New Zealand white rabbits immunized with BPDE-DNA, complexed with methylated bovine serum albumin (mBSA). Monoclonal antibodies were developed by fusion of mouse myeloma cells with spleen cells isolated from BALB/c mice immunized with the same complex of BPDE-DNA and mBSA. These antibodies have been characterized for specificity in a highly sensitive, enzyme-linked immunosorbent assay (ELISA). All antibodies showed a very high affinity for single-stranded BPDE-DNA, but had lower affinity towards native BPDE-DNA. The affinity for the free mononucleoside BPDE-dG was at least 100-fold lower than that for BPDE-DNA, and no affinity was detected for BP tetrols or DNA modified with N-acetoxy-N-acetyl-2-aminofluorene. A high cross reactivity was observed with DNA modified with (+/-)-trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene++ +. Using five different antibodies, monoclonal or polyclonal, we observed that the antibody affinity for BPDE-DNA was dependent on the level of modification; in the competitive ELISA as little as 4 fmol BPDE-DNA (50 pmol/micrograms) was sufficient for 50% inhibition with our best antisera, but 17 fmol of the adduct was required when [3H]BPDE-DNA of low modification (1-10 fmol/micrograms) was used as inhibitor. When samples of [3H]BP-DNA isolated from the livers of mice, treated i.p. with different doses of [3H]BP were examined by competitive ELISA and calibrated with [3H]BPDE-DNA of low modification (1-10 fmol/micrograms), binding values calculated from the immunoassay were in good agreement with those obtained from radioactivity measurements. In contrast, when this DNA was quantitated in competitive ELISA using highly modified BPDE-DNA as standards, values by ELISA were 20-40% of those obtained by radioactivity. These results indicate that the use of serially diluted BPDE-DNA of high modification as standard competitor in the ELISA will lead to erroneous results in the measurement of adducts in DNAs modified to a low extent (biological samples). The property of antisera specific for BP-DNA, recognizing highly modified DNA more efficiently than DNA modified to a low extent, may be common to all antisera elicited against highly modified DNA immunogens. Therefore we conclude that antibody affinity must be tested also with DNA samples of low modification, obtained either in vitro or in vivo.

2-Acetylaminofluorene↗
PubMed1991

opaque-2 modifiers increase gamma-zein synthesis and alter its spatial distribution in maize endosperm.

Through the action of opaque-2 modifier genes, the soft, floury endosperm of opaque-2 mutants is converted to a vitreous phenotype. This change in endosperm texture is associated with a twofold to threefold increase in gamma-zein content. To investigate the effect of opaque-2 modifiers on the expression of gamma-zein genes, we analyzed the synthesis and distribution of gamma-zein protein and the level of gamma-zein mRNAs in developing endosperms of the inbreds W64A and W64Ao2, a modified opaque-2 mutant Pool 34 QPM, and their reciprocal F1 hybrids. We also characterized the number and organization of gamma-zein genes in these and related maize genotypes. Our studies show that opaque-2 modifiers are semidominant genes, resulting in a twofold to threefold increase in gamma-zein gene expression in both opaque-2 and normal genetic backgrounds. The increase in gene expression appears to be a consequence of enhanced mRNA transcription or stability rather than gene amplification because gamma-zein genes occur in one or two copies in modified as well as nonmodified genetic backgrounds. Ultrastructural studies showed that gamma-zein occurs in high concentrations in the first few subaleurone cells of nonmodified endosperms, but high concentrations of gamma-zein occur in the subaleurone and central endosperm cells of modified opaque-2 mutants. The increased concentration and distribution of gamma-zein in modified endosperms are highly correlated with the activity of opaque-2 modifier genes.

Microscopy, Immunoelectron↗
PubMed1992

Oxidatively modified HDLs are potent inhibitors of cholesterol biosynthesis in human skin fibroblasts.

Several biological properties of lipoproteins are modified by oxidative reactions. Modified lipoproteins are rapidly degraded by macrophages, and this is likely to be a major pathway for the formation of foam cells in the early phases of atherosclerosis. The effect of modification on other aspects of cholesterol homeostasis has, however, received lesser attention. In this study, the influence of copper ion- as well as rat aortic smooth muscle cell-oxidation-modified high density lipoprotein (HDL) on cholesterol biosynthesis in human skin fibroblasts has been investigated. Modified lipoproteins eluted at higher ionic strength than did control HDL on a Mono-Q 5/5 anion-exchange column. However, only copper ion-modified HDLs displayed greater electrophoretic mobility than did control lipoproteins on agarose gel electrophoresis. Both control and modified HDLs decreased cholesterol esterification in fibroblasts. On the other hand, whereas control HDLs were virtually ineffective in modulating cholesterol biosynthesis, modified HDLs had a significant suppressing effect. This was observed in normal as well as low density lipoprotein (LDL) receptor-defective fibroblasts, which are unresponsive to the LDL-mediated downregulation of cholesterol synthesis. These results are consistent with the concept that oxidative modification of HDLs drastically alters their effect on cholesterol homeostasis in fibroblasts. The data furthermore suggest the existence of a lipoprotein pathway for cholesterol biosynthesis regulation that is independent of the LDL receptor-mediated pathway. Downregulation of cholesterol biosynthesis would be a new function for oxidatively modified lipoproteins.

Cells, Cultured↗
PubMed1991

Videoendoscopic evaluation of patients with dysphagia: an adjunct to the modified barium swallow.

The modified barium swallow is currently the most comprehensive, widely available, and easily interpreted technique for the evaluation of patients with dysphagia by the head and neck surgeon. However, it requires the facilities, personnel, and use of a radiology suite, a trained speech pathologist, and exposure of the patient to radiation. It would therefore be helpful to have an adjunctive, physician based, nonradiographic method of examination that could provide information similar to and possibly even more complete than that supplied by the modified barium swallow. Such an adjunctive method could help otolaryngologist-head and neck surgeons confronted by a new patient with swallowing difficulties to orient themselves to the nature and severity of the problem while waiting for the modified barium swallow to be scheduled, performed, and reviewed. It could also be a helpful tool for management of patients with cancer of the head and neck, whose swallowing function may change rapidly in the early postoperative period. In such cases, intervals between modified barium swallow examinations (dictated by concern over radiation exposure) may be too far apart to allow up-to-the-minute decisions on case management. Finally, some patients who may be too ill to travel to the radiology suite might benefit from a bedside procedure that would yield information about swallowing function similar to that provided by the modified barium swallow. Videoendoscopic evaluation of dysphagia (VEED) is a protocol I developed and have used regularly since 1984. Experience with this method of dysphagia evaluation has shown that it answers the needs outlined above. Its usefulness also goes beyond that of the modified barium swallow by providing a more detailed understanding of the component anatomic and functional deficits that comprise a given patient's swallowing problem, information about upper aerodigestive tract sensory deficits, and a means for visual feedback training of pharyngeal and laryngeal musculature. The protocol is reviewed here. Case reports illustrating the clinical usefulness of VEED as an adjunct to the modified barium swallow are also presented, and the relative strengths and weaknesses of VEED and the modified barium swallow are compared.

Aged↗
PubMed2026

Combinatorial multiomic analysis from a pedigree of Sox10Dom Hirschsprung mice identifies multiple high confidence candidate modifiers of Enteric Nervous System development.

Hirschsprung disease (HSCR) is characterized by absence of enteric ganglia (aganglionosis) along variable lengths of the distal intestine. This disorder results from deficient colonization of fetal intestine by enteric neural crest-derived cells (ENCDCs). HSCR exhibits complex, multifactorial inheritance with penetrance and severity varying widely even within families. SOX10 is among causal genes that predispose to aganglionosis. Yet, how gene interactions influence severity of HSCR aganglionosis is not understood. Prior mapping of aganglionosis modifiers was achieved in a standard F1-intercross utilizing the Sox10Dom HSCR mouse model. Here we deploy a novel strategy of genotyping an extended pedigree pedigree of Sox10Dom mice on a mixed genetic background. GWAS in this pedigree points to novel aganglionosis modifier intervals with replication and refinement of prior modifier regions. Complementary omics analysis of the developing Enteric Nervous System (ENS) enabled identification of multiple high-priority candidate genes within these modifier intervals based on gene expression, chromatin accessibility, and presence of conserved SOX10 binding motifs. We implemented a prioritization pipeline for ranking potential modifiers that generated candidate lists including several well-known for effects on ENS development as well as multiple novel genes. Among the novel genes, Dach1 ranked as a top priority candidate gene for modifying migration of ENCDCs and thus influencing aganglionosis severity. The results identify genome intervals with intrinsic genes that are logical candidates for modifying Sox10Dom aganglionosis severity. We also note that several human orthologs to aganglionosis modifier candidate genes are within linkage disequilibrium blocks containing genetic variants associated with human gut motility disorders, which offers opportunity for gaining biological insight into human HSCR severity.

Animals↗
PubMed1991

[Extracorporeal shock wave lithotripsy using Dornier modified HM3 lithotripter comparison with the results by Dornier HM3 lithotripter].

Ninety-four kidneys with renal stones less than or equal to 20 mm in diameter were treated by extracorporeal shock wave litotripsy (ESWL) using a Dornier modified HM3 lithotripter and the results were compared with those of 98 kidneys with similar size stones treated with a Dornier HM3 lithotripter. The Dornier modified HM3 lithotripter is equipped with a new type of shock wave generator with a reduced capacity for 30% less pressure peakes at the same voltage. It has an enlarged ellipsoid leading to a smaller focus and a reduced pressure per area at the shock wave entry into the skin. All treatments of modified HM3 litotripter series were performed under only intravenous analgosedation, without epidural anesthesia. The number of shock waves in the modified HM3 series ranged 900 to 6000, with the mean values of 2863 +/- 1234, which was 1.55 times as that in the Dornier HM3 series. Complete disintegration was achieved in 94 of 94 modified HM3 series kidneys and 98 of 98 kidneys of HM3 series. Complete removal of the stone was done at 72.6% in the modified HM3 series and at 70.4% in the HM3 series 3 months after ESWL. There were no severe complications in both modified HM3 series and HM3 series. Renal damage caused by ESWL was monitored by the level of urinary enzyme, N-acetyl-beta-glucosaminidase (NAG) and beta 2 microglobulin (beta 2MG) and the level of urinary protein. The levels of NAG, beta 2MG and urinary protein in the HM3 series were higher than those of the modified HM3 series.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1985

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk.

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.

Animals↗
PubMed1985

Modification of histidines in human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin.

Diethyl pyrocarbonate (ethoxyformic anhydride) was used to modify histidyl residues in prothrombin. Diethyl pyrocarbonate inactivated the potential fibrinogen-clotting activity of prothrombin with a second-order rate constant of 70 M-1 min-1 at pH 6.0 and 25 degrees C. The difference spectrum of the modified protein had a maximum absorption at 240 nm which is characteristic of N-carbethoxyhistidine. The pH dependence for inactivation suggested the participation of a residue with a pKa of 6.2. Addition of hydroxylamine to ethoxyformylated prothrombin reversed the loss of fibrinogen-clotting activity. No structural differences were detected between the native and modified proteins using fluorescence emission and high-performance size-exclusion chromatography. The tyrosine and tryptophan content was not altered, but approximately 1-2 amino groups were modified. Statistical analysis of residual enzyme activity and extent of modification indicates that among 7 histidyl residues modified per molecule, there is 1 essential histidine (not in the active site) involved in the potential fibrinogen-clotting activity of prothrombin. To further examine its properties, the modified prothrombin was activated to thrombin using Echis carinatus venom protease. There was no difference in the catalytic activity of thrombin obtained from either native or ethoxyformylated prothrombin, as measured by H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-Phe-Pip-Arg-NA) hydrolysis. However, thrombin produced from the modified protein showed a loss of fibrinogen-clotting activity but had a comparable apparent Ki value (about 20 microM) to thrombin from native prothrombin when fibrinogen was used as a competitive inhibitor during D-Phe-Pip-Arg-NA hydrolysis. The similarity in Ki values indicated that thrombin derived from diethyl pyrocarbonate-modified prothrombin does not have an altered fibrinogen-binding site. Although the histidyl residue involved during inactivation has not been identified, the results suggest that a histidyl residue in the thrombin portion of prothrombin is essential for interaction with fibrinogen.

Diethyl Pyrocarbonate↗
PubMed1980

Behavior of vesicular stomatitis virus glycoprotein in mouse LM cells with modified membrane-phospholipids.

LM cells in which the membrane phospholipids had been modified with choline analogues were infected with vesicular stomatitis virus. The choline analogues tested were choline, N,N'-dimethylethanolamine, N-monomethylethanolamine and ethanolamine. These modifications per se did not affect the syntheses of individual viral proteins. The viral glycoprotein was detected in the plasma membranes of all the modified cells by pronase digestion in pulse-chase experiments, but the amount of glycoprotein susceptible to proteolysis varied, decreasing in these modified cells in the following order: N,N'-dimethylethanolamine- greater than choline- greater than N-monomethylethanolamine- greater than ethanolamine-treated cells. After a 4-h chase, glycoprotein was mainly distributed in the plasma membranes of cells modified with N,N'-dimethylethanolamine, whereas it was found in both the microsomes and plasma membranes of cells modified with other analogues. Fairly large amounts of glycoprotein were also found in the soluble fraction of ethanolamine-treated cells, but not in that of choline- or N,N'-dimethylethanolamine-treated cells. More precise experiments on the behaviour of glycoprotein with a short period of chase strongly suggested that migration of glycoprotein from the microsomes to the plasma membranes was fastest in cells modified with N,N'-dimethylethanolamine and slowest in cells modified with ethanolamine. Membrane lipid modifications also resulted in release of different numbers of progeny virions from the cells, release of virions from the cells decreasing in the following order: N,N'-dimethylethanolamine- greater than choline- greather N-monomethylethanolamine- greater than ethanolamine-treated cells. These results indicate that modification of membrane phospholipids influences not only the insertion of glycoprotein into the microsomes and its migration to the plasma membranes, but also the production of progeny virions.

Animals↗
PubMed1984

Polyethylene glycol-modified catalase exhibits unexpectedly high activity in benzene.

Bovine liver catalase with molecular weight of 248,000, which consists of four subunits, was modified with 2,4-bis(o-methoxypolyethylene glycol)-6-chloro-s-triazine(activated PEG2). The modified catalase became soluble in organic solvents such as benzene by increasing the degree of modification of amino groups in the enzyme with activated PEG2. The enzymic activity of the modified catalase in benzene, in which 42% of the total amino groups were coupled with the modifier, was unexpectedly high in comparison with the activity of non-modified catalase in aqueous system. The absorption spectrum of the modified catalase in benzene showed the characteristic pattern of a haem protein with Soret band at 405 nm. The temperature-activity profile of the modified catalase in benzene was clarified and its activation energy was estimated to be 1900 cal/mol.

Animals↗