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Results for “Generational stabilization”

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At least 91 records · Page 5Linked to original sources

PubMed1978

Transfer of genetic information via isolated amphibian metaphase chromosomes.

The metaphase chromosome transfer system of McBride and Ozer (1973) has been adapted to a haploid, euploid, frog cell line. Genes coding for a deoxypyrimidine kinase and an enzyme responsible for a thymidine-specific saturable transport system have each been transferred at frequencies between 10(-6) and 10(-5) transferents per cell treated. Revertants for each of these two genes were observed at frequencies between 10(-8) and 10(-7) revertants per cell tested. Selfing controls showed no transferents. Two colonies were obtained in which cotransfer of both genes may have occurred. Activities of the transferred genes were assayed by incorporation of [3H]thymidine into alkali-stable, acid-precipitable material. Growth properties of 13 transferents in various media were also determined and presence of the appropriate enzymes inferred. These transferents were tested for stability early (25 generations) after transfer and were found to be stable. All 13 transferents possess the normal haploid number of chromosomes (n = 13) with no cytologically detectable chromosomal fragments.

Animals↗

PubMed1992

Round table on epidemiology and control of fox rabies.

The current epizootic of rabies in Europe has as its main host the fox. Oral vaccination of the fox population has proven to be particularly effective. It is clear that the major components for a successful vaccination programme are a potent and stable vaccine, and an effective baiting system; the latter should attract the target animal but no non-target species. Recently, vaccines of increased stability have been generated; amongst these is a vaccinia recombinant virus which expresses rabies virus glycoprotein. Consequently, both attenuated live virus vaccines and a recombinant vaccine are available for routine field vaccination of the fox population.

Animals↗

PubMed1985

Genetic transformation of the phytopathogenic bacteria, Erwinia chrysanthemi.

Erwinia chrysanthemi is an enterobacterium whose phytopathogenicity is due to its pectinolytic and cellulolytic activities. The CaCl2 mediated transformation procedure was successfully applied to two E. chrysanthemi wild type strains. The highest efficiency of transformation of E. chrysanthemi with pBR322 was found using 0.1 M CaCl2, 0.1 M MgCl2 treated cells and a heat pulse at 30 degrees C for 6 min. This yielded about 600 transformants per microgram of pBR322 DNA and 2.3 X 10(-6) per viable cell. Plasmid stability after twenty generations was lower than in E. coli: only 40-60% of the cells retained the plasmid in the absence of selective pressure. Based on this result, cloning in E. chrysanthemi with pBR322 vectors should therefore be possible, making it a potential host for cloning any gene for biomedical or industrial purposes.

Calcium Chloride↗

PubMed2025

Chemoselective Tagging of Protein Methacrylation.

Protein lysine methacrylation (Kmea) is a recently identified post-translational modification whose biofunction remains poorly understood. Until now, there has been no chemical labeling method for Kmea modification, which has severely hindered the discovery and functional studies of methacrylated proteins. Here, we developed a photocatalytic thia-Michael reaction system for the chemoselective labeling of protein methacrylation. By exploiting the dual effect of steric hindrance and the stability of the generated C-center radical, the reaction interference of the structural isomer crotonylation can be efficiently avoided. Based on this reaction, a multifunctional water-soluble benzenethiol-azide probe azDSH was designed and synthesized, and a workflow for the specific labeling, enrichment, and identification of Kmea proteins was developed. Proteomic identification of histone and nuclear protein extracts and whole-cell lysate revealed a number of novel Kmea proteins and modification sites besides histones, such as HMGB1, TdIF2, UHRF1, HNRPD, BRWD1, TAF1, TACC1, and SETD3, providing new targets for the study of epigenetic regulation. This study provides an effective method for the analysis of protein methacrylation modifications in biological systems.

Humans↗

PubMed1983

Crystallographic studies on apocarboxypeptidase A and the complex with glycyl-L-tyrosine.

The crystal structures of zinc-free carboxypeptidase A (apocarboxypeptidase A) and the complex of glycyl-L-tyrosine with apocarboxypeptidase A are described and compared to the corresponding structures of the zinc-containing enzyme. Only small conformational changes in the zinc ligands accompany removal of the metal. Interactions between the tyrosine residue of glycyl-L-tyrosine and apocarboxypeptidase A are similar to those observed in the complex with the holoenzyme. However, in the absence of zinc, the carbonyl oxygen of the glycyl moiety now receives a hydrogen bond from the side chain of arginine-127. Although not as yet observed, a similar shift of the carbonyl oxygen of a susceptible bond from the zinc to arginine-127 could stabilize tetrahedral intermediates generated during the hydrolysis of substrates by carboxypeptidase.

Apoenzymes↗

PubMed1991

Familial transmission of alcohol use: IV. A seventeen-year follow-up on the relationships between parent and adult offspring alcohol use; Tecumseh, Michigan.

The sample for this study comprised 190 three member sets of father, mother, and adult son or daughter who were first interviewed in 1960 as part of Cardiovascular Examination I of the Tecumseh Community Health Study. At that time, alcohol data were collected from self-reports for both parents and offspring. In 1977 alcohol data were again collected for the adult offspring only, from self-reports. In 1960 offspring's drinking was significantly related to both fathers' and mothers' drinking and remained so in 1977 despite shifts over time in the proportions of offspring across five alcohol use categories. The majority of offspring did not fall into their parents' category, but the mode by and large reflected the parent category. The majority of offspring of non-drinkers did drink, but at low and not high volume; the majority of offspring of high volume drinkers drank at lower levels than their parents, and were not likely to be life-long abstainers, although they may have become past drinkers. Two non-drinking parents reinforced non-drinking in their adult offspring and two drinking parents reinforced the early onset and persistence of high volume drinking. Among offspring who changed their drinking level between 1960 and 1977, those who had drunk less than their parents were the most likely to have increased their drinking, and those who had drunk more than their parents were the most likely to have decreased their drinking. Patterns observed in this study may be viewed as intrafamilial sources of drinking stability transmitted across generations in a single community.

Adolescent↗

PubMed1989

Monitoring and selection of resistance to pyrethroids in the Australian sheep blowfly, Lucilia cuprina.

Field and laboratory populations of the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Calliphoridae), were surveyed by bioassay for possible resistance to the synthetic pyrethroids, a group of insecticides under development for blowfly control. A normal distribution of LC50 values was found using deltamethrin, the test pyrethroid, with no indication of specific resistance despite widespread use of deltamethrin on sheep to control the sheep body louse, Damalinia ovis (Schrank) (Trichodectidae). There was no cross-resistance to deltamethrin from existing organophosphate (OP) resistance nor from previous use of DDT. Selection with deltamethrin on a combined field strain, CSF85, increased the LC50 gradually over the first twenty generations until it stabilized at approximately 25x that of the unselected CSF85. This laboratory-induced resistance extended to other pyrethroids, cypermethrin (16x), cyhalothrin (25x) and cycloprothrin (10x), and increased the existing resistance of CFS85 to the OP diazinon (11x) and the carbamate, butacarb (83x).

Animals↗

PubMed1992

Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein.

To investigate the role of binding sites for Rep initiation protein in the replication of pSC101, a series of plasmids was constructed which carried different combinations of mutations in three binding sites within the minimal origin of replication. Mutation of all three sites reduced the affinity of purified Rep protein for the origin by 100-fold, as measured by a competition binding assay. Mutations in individual binding sites prevented binding of Rep protein to the mutant site but not to adjacent wild-type sites. Transformation efficiency, copy number, and stability over 150 generations were measured for each of the mutant plasmids. Unlike other similar plasmids related to pSC101, the Rep binding sites were found not to be equivalent. A mutation in the site RS1, proximal to repeated sequences which serve as DnaB helicase entry sites in oriC, had a severe effect on replication activity. A similar mutation in the distal site RS3 caused a reduction in copy number, but the mutant plasmid was stably maintained despite a broadened distribution of copy number within the population. A mutation in the middle RS2 site had no significant effect on pSC101 replication.

Bacterial Proteins↗

PubMedDate not supplied

Protective action by methylprednisolone, allopurinol and indomethacin against stroke-induced damage to adenylate cyclase in gerbil cerebral cortex.

Adenylate cyclase activity was investigated in either homogenate or particulate fractions from the frontal cerebral cortex of the gerbil following five experimental conditions of bilateral ischemia. After periods of 15 min ischemia, 15 min ischemia plus 15 min of recirculation or 60 min ischemia the enzyme generally displayed enhanced responses to GTP, norepinephrine (NE), dopamine (DA), NE + GTP and DA + GTP. Pretreatment of the gerbils with methylprednisolone, allopurinol or indomethacin did not significantly influence the outcome of these findings. When the animals were subjected to 60 min ischemia plus 15 min of reflow, enzyme responses to the stimulatory agents including forskolin and NaF were all reduced. Pretreatment with methylprednisolone, allopurinol or indomethacin prevented the damage to adenylate cyclase in the 60 min ischemia plus 15 min reflow animals. When animals were made ischemic for 15 min followed by one week of recovery, enzyme sensitivity to GTP, calmodulin-Ca++, NE, combinations thereof and forskolin were reduced in only the particulate fractions. Enzyme damage was reversed following methylprednisolone. Enzyme damage may result from generation of free radicals during reflow and drugs that either inhibit synthesis pathways generating free radicals, stabilize cell membranes or act as free radical scavengers may be therapeutically beneficial under specific conditions of stroke.

Adenylyl Cyclases↗

PubMed2025

Characterization and purification of Pseudomonas aeruginosa phages for the treatment of canine infections.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in both human and veterinary medicine, presenting significant challenges in treatment because of biofilm production and its intrinsic resistance. This problem is exacerbated by the increase in acquired antimicrobial resistance. Bacteriophage (phage) therapy has emerged as a promising alternative for treating infection classically treated with antibiotics, offering a targeted approach to combat this infection. This study aimed to evaluate the therapeutic potential of 7 phages, focusing on their suitability for treating canine infections, as well as their purification and safety analysis for therapeutic use. RESULTS: Two self-isolated phages and five provided phages were analysed. All tested phages reduced bacterial load in vitro; however, their efficacy varied across different concentrations. The host range analysis revealed a spectrum between 9.8 and 68.6% of canine clinical P. aeruginosa isolates. In our in vitro tests 3 out of 7 phages were able to significantly reduce the biofilm biomass, achieving reductions up to 93.38%. The sequence analysis did not discover known virulence factors and genes connected to antimicrobial resistance mechanisms. The self-isolated phages were classified as lysogenic, whereas the other phages had a lytic infection cycle. Through the purification of the phages, high-titre phage preparations (> 1011 PFU/ml) were generated with high stability for at least 1.5 years. The tested endotoxin units are below the regulatory limits. CONCLUSION: Investigating phages as alternative treatment option seems promising with lytic phages covering a broad host range and a genomic potential for biofilm degradation. These findings support the development of phage cocktails as a targeted alternative for treating canine P. aeruginosa infections, particularly in cases of antibiotic resistance, and highlight the importance of selecting well-characterized lytic phages for therapeutic efficacy and safety.

Pseudomonas aeruginosa↗

PubMed1976

[The battery-state control. A new possibility for the control of pacemakers. Preliminary report (author's transl)].

After a brief examination of the technical problems of current pacemakers, with special reference to the battery state control, the authors present a new technique for such control. A power stabilizer able to generate a standard pulse of 4.3 in every condition of battery charge has been fitted on the power supply of a fixed rate pacemaker of normal production. By activating (through an external magnet) such apparatus, it is possible with an easy oscilloscopic measurement to obtain the real value of the battery voltage. Thus the comparison with previously taken measurements and the errors connected with the repetition of such measurements can be avoided. The stimulation rate and the pulse duration corresponding to the discharged state of the batteries can also be known in advance. The data obtained from the first two controls are reported.

Arrhythmias, Cardiac↗

PubMed1991

Nonradical oxidants of the phagocyte type induce the activation of plasmatic single chain- urokinase.

Single chain- urokinase (scu-PA) is the proenzyme of the plasminogen activator urokinase (tcu-PA). In human blood scu-PA is of great stability. Activated phagocytes generate large amounts of single chain- urokinase and of reactive oxidants (chloramines and HOCl). Since these cells participate in physiologic fibrinolysis, we were interested in the interaction between plasmatic scu-PA and chloramines. The oxidants dose dependently induce the activation of plasmatic scu-PA. Optimal activation of scu-PA occurs at about 3-5 mmol/l of chloramine-T. The findings suggest a control mechanism of scu-PA stability/activity by oxidatively modifiable plasma proteins, such as alpha-2-antiplasmin. The oxidation mechanism seems to be mediated by singlet molecular oxygen, an excited oxygen species. Basing on this scu-PA/oxidant synergism a sensitive and fast functional assay of scu-PA in human plasma is presented. Plasmatic inhibitors normally interfering with functional scu-PA measurements are inactivated by addition of chloramine-T, imitating the physiological oxidants generated by activated phagocytes. The scu-PA concentration in plasma of n = 36 healthy individuals has been determined to be 5.8 +/- 1.6 ng/ml. The lower detection limit of plasma scu-PA by the procedure described is about 1.5 ng/ml of plasma. By means of this technique scu-PA concentration during thrombolytic therapy can be measured within minutes in undiluted (direct) plasma samples, allowing adjustments of the scu-PA dosage. The present study gives further credence for a role of singlet molecular oxygen, possibly a new type of locally acting hormones (autacoid), in the regulation of the fibrinolytic pathway.

Adult↗

PubMed1991

[Establishment of transplantable HL-60 xenograft lines in athymic nude mice].

In vivo models of HL-60 cells were successfully established as ascites and/or solid forms in nude mice (BALB/c, nu/nu). HL-60 ascites and solid tumor cells produced 100% lethal takes after their 7th transplant generation and were stabilized at serial transplant intervals of 14 and 40 days respectively. Morphological studies demonstrated that the ascites and solid tumor cells exhibited morphological characteristics similar to those of HL-60 cells in vitro. The establishment of transplantable HL-60 xenograft lines provides a potential tool for the screening of effective differentiation inducers.

Animals↗

PubMedDate not supplied

[Photochemical electron transfer in reaction centers of photosynthesis].

Recent works of the Laboratory are reviewed in terms of modern concepts on the nature of primary photoprocesses in reaction centers of photosynthesis. The main feature of the primary charge separation in reaction centers is the fast (within several picoseconds) electron transfer between the pigments of chlorophyllous nature followed by electron transfer stage to nonchlorophyllous electron carriers for several hundreds of picoseconds The primary charge separation is determined by the ability of excited chlorophyll and its analogues to be reversibly oxidized or reduced as well as by peculiar molecular arrangement of pigment system in reaction centers. The stabilization of photochemically generated electron and the hole is realized at the expense of its spatial separation and some loss of energy in the course of electron transfer.

Bacterial Proteins↗

PubMed1984

NADPH-generating system: influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions.

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisole O-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and beta-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures. In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD). Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH. At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D7 strain of Saccharomyces cerevisiae.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1985

Cleavage within an RNase III site can control mRNA stability and protein synthesis in vivo.

We report that processing at a cloned bacteriophage T7 RNase III site results in strong stabilization of the mRNA relative to the full-length transcript. In contrast, processing by RNase III of the bacteriophage lambda int transcript leads to rapid degradation of the messenger. It is proposed that the mode of cleavage within the RNase III site determines mRNA stability. Single cleavage leaves part of the phage T7 RNase III site in a folded structure at the generated 3' end and stabilizes the upstream mRNA whereas double cleavage at the lambda int site removes the folded structure and accelerates degradation. In addition, the processed transcript is as active a messenger as the unprocessed one and can direct protein synthesis for longer times. This increased efficiency is accompanied by a proportional (3-4 fold) increase in protein levels. In contrast, processing at the lambda int site reduces Int synthesis. Thus, processing may either stabilize mRNA and stimulate gene expression or destabilize a messenger and prevent protein synthesis. The end result appears to be determined by the mode of cleavage within the RNase III site.

DNA Restriction Enzymes↗

PubMed1989

Codon usage determines translation rate in Escherichia coli.

We wish to determine whether differences in translation rate are correlated with differences in codon usage or with differences in mRNA secondary structure. We therefore inserted a small DNA fragment in the lacZ gene either directly or flanked by a few frame-shifting bases, leaving the reading frame of the lacZ gene unchanged. The fragment was chosen to have "infrequent" codons in one reading frame and "common" codons in the other. The insert in these constructs does not seem to give mRNAs that are able to form extensive secondary structures. The translation time for these modified lacZ mRNAs was measured with a reproducibility better than plus or minus one second. We found that the mRNA with infrequent codons inserted has an approximately three-seconds longer translation time than the one with common codons. In another set of experiments we constructed two almost identical lacZ genes in which the lacZ mRNAs have the potential to generate stem structures with stabilities of about -75 kcal/mol. In this way we could investigate the influence of mRNA structure on translation rate. This type of modified gene was generated in two reading frames with either common or infrequent codons similar to our first experiments. We find that the yield of protein from these mRNAs is reduced, probably due to the action in vivo of an RNase. Nevertheless, the data do not indicate that there is any effect of mRNA secondary structure on translation rate. In contrast, our data persuade us that there is a difference in translation rate between infrequent codons and common codons that is of the order of sixfold.

Bacterial Proteins↗

PubMed1991

Cell wall assembly in Bacillus megaterium: incorporation of new peptidoglycan by a monomer addition process.

The pattern of cross-linking in the peptidoglycan of Bacillus megaterium has been studied by the pulsed addition of radiolabeled diaminopimelic acid. The distribution of label in muropeptides, generated by digestion with Chalaropsis muramidase and separated by high-performance liquid chromatography, stabilized after 0.15 of a generation time. The proportion of label in the acceptor and donor positions of isolated muropeptide dimers stabilized over the same period of time. The results have led to the formulation a new model for the assembly of peptidoglycan into the cylindrical wall of B. megaterium by a monomer addition process. Single nascent glycan peptide strands form cross-linkages only with material at the inner surface of the wall. Maturation is a direct consequence of subsequent incorporation of further new glycan peptide strands, and there is no secondary cross-linking process. The initial distribution of muropeptides is constant. It follows that the final pattern of cross-linking in the wall is determined solely by, and can be forecast from, this repetitive pattern of incorporation. In a modified form, this model can also be applied to assembly of cell walls in rod-shaped gram-negative bacteria.

Bacillus megaterium↗