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PubMed1975

Determination of secondary structure in rabbit globin messenger RNA by thermal denaturation.

The secondary structure of highly purified globin messenger RNA has been investigated by alkaline hydrolysis, nuclease digestion, and thermal denaturation. The thermal denaturation properties of globin messenger have been compared to poly(U), poly (A), and a synthetic random sequence RNA copolymer. From these studies it is concluded that globin mRNA contains considerable secondary structure and that the amount of helical structure is greater than that which occurs with a random sequence polyribonucleotide. Globin mRNA contains, by comparison to the secondary structures of native DNA, tRNAs, or 18S rRNA, helices with involve 55-62% of the bases or 58-68% if a correction is made for the 3'-terminal poly(A) segment. The helices of globin mRNA appear to be unique as differences in the NaCl stabilization of this RNA have been noted when compared to other naturally ooccurring and synthetic RNAs. Comparison of the hyperchromicity maxima, obtained at 260 and 280 nm for globin mRNA and 18S rRNA, indicates that the helices of the two RNAs contain similar numbers of G-C base pairs. Differential analysis of NaCl stabilization curves indicate three discrete thermally denaturable helix types in globin mRNA.

Animals↗
PubMed1975

Gastro-oesophageal acid reflux. Method for 12-hour continuous recording of oesophageal pH with analysis of records.

Description of method for continuous recording of oesophageal pH and automatic, electronic analysis of records. Stability of apparatus has been tested over 12 hours in the laboratory with different buffer solutions and gastric secretions with and without bile, and on 36 patients with oesophageal disorders. Drift of apparatus max. 0.2 pH units. The analyser, dividing the pH range into arbitrary intervals, performs a rapid and reliable analysis of single examinations from variable criteria. The temporal distribution of pH values into the intervals is expressed in percentages of the total time of recording. Reliability of the analyser was tested by analysis of a simulated pH curve on which changes in amplitude corresponded to the most rapid changes seen in practice. Deviation below one per cent. Repeated analyses of the same records showed a coefficient of variation for the various analytical interval to be 10-0.01 per cent. The method allows recording of the number of reflux episodes, duration of each episode and total duration of pH within chosen limits.

Adult↗
PubMed1976

Mapping of mitochondrial genes in Saccharomyces cerevisiae. Populations and pedigree analysis of retention or loss of four genetic markers in Rho-cells.

1. Retention or loss of mitochondrial markers CR321, OR1, PR454, TR (gene loci RIB1, OLI1, PAR1, TSM1 respectively has been analysed in a large number of ethidium bromide induced primary rho-clones. Retention of one or more of the four markers with a single clone was observed frequently, only 20 to 25% of clones were found to be (TOCOOOPO). Primary clones retaining two or more of the four markers were found to be mixed, i.e. the primary rho- cell contained a heterogeneous population of variously deleted mitDNA molecules which segregated into different cell lines in the corresponding primary clone. 2. A representative sample of the population of ethidium bromide induced rho- mutants has been analysed by a first subcloning performed after some 30 cell generations of vegetative multiplication in the abscence of the drug. At this level the heterogeneous population of mitDNA molecules, generated by the mutagenic treatment in the primary cell, has been sorted out. The cells forming secondary clones are thus essentially homoplasmic. In contrast to primary clones, genotypes of secondary clones therefore could be determined unambiguously, and the frequency of cell types can be regarded as a faithful representation of the frequency of mitDNA molecules. Retention of markers was low, in less than 2% of secondary clones one or several markers have been found. This observation has been interpreted as indicating that induction of rho-mutants by ethidium bromide is accompanied by deletion of very large sequences of mitDNA in a very large fraction of mitDNA molecules. 3. Five individual rho-clones retaining the four markers TRCRORPR have been isolated and analysed for spontaneous deletion of one or several of these markers during successive subclonings (pedigree analysis). High genetic stability (98-99.5% per cell generation) has been observed in these clones. 4. A method has been developed allowing an unambiguous determination of the order of the four markers on a circular map. It is based on the concomitant loss of two markers and retention of the other two markers (double loss/double retention analysis). The results of four out of five pedigrees of individual rho-clones analysed (spontaneous deletion) and the results of the analysis of populations of secondary rho-clones (ethidium bromide induced deletion) were in full agreement and the order of genes has been determined as being P-T-C-O-P. In the fifth pedigree results suggest an inversion of the T and C markers. 5. Relative distances between pairs of markers have been derived from the frequencies of separation of markers by deletion and were found to be C-T less than C-O less than T-O less than T-P less than C-P less than O-P. Linkage of the four markers could be established, and distances calculated are additive. 6. The general relevance of this approach of mapping by deletion and the methods used for the determination of order and distances of mitochondrial genes has been discussed. (ABSTRACT TRUNCATED)

DNA, Mitochondrial↗
PubMed2024

Causal effect of the age at first birth with depression: a mendelian randomization study.

BACKGROUND: This study aimed to explore the causal relationship between age at first birth (AFB) and depression. METHODS: Using the univariable Mendelian randomization (UVMR) and multivariable Mendelian randomization (MVMR) methods to examine the potential correlation between age at first birth (AFB) and major depressive disorder and postpartum depression. A public database was used to obtain the genome-wide association studies (GWAS) summary data. We put inverse-variance-weighted (IVW) as the primary method in Mendelian randomization (MR) analysis and used sensitivity analysis to confirm the robustness of our result. RESULTS: We found a significant causal association between AFB and major depressive disorder by using the IVW algorithm (odd ratio [OR] 0.826; 95% confidence interval [CI] 0.793&#x2009;-&#x2009;0.861; P&#x2009;=&#x2009;4.51&#x2009;&#xd7;&#x2009;10-&#x2009;20). MR-Egger, weighted median, simple mode and weighted mode method concluded the same result (P&#x2009;<&#x2009;0.05). During the sensitivity analysis, the heterogeneity test (Q-value&#x2009;=&#x2009;55.061, df&#x2009;=&#x2009;48, P&#x2009;=&#x2009;2.81&#x2009;&#xd7;&#x2009;10-&#x2009;01, I2&#x2009;=&#x2009;12.82%) and the leave-one-out plot analysis confirmed the stability of the results. The outcomes of the pleiotropy test (MR-Egger intercept&#x2009;=&#x2009;8.932&#x2009;&#xd7;&#x2009;10-&#x2009;3. SE&#x2009;=&#x2009;6.909&#x2009;&#xd7;&#x2009;10-&#x2009;3. P&#x2009;=&#x2009;2.02&#x2009;&#xd7;&#x2009;10-&#x2009;01) and MR_PRESSO global test (P&#x2009;=&#x2009;2.03&#x2009;&#xd7;&#x2009;10-&#x2009;01) indicated there is no pleiotropy. CONCLUSION: There is solid evidence that a higher age at first birth is associated with a lower risk of major depressive disorder.

Humans↗
PubMed2026

Quantitative RNA modification mapping by mass spectrometry with isobaric tags and nucleobase fragment analysis.

RNA modifications regulate RNA stability, translation, stress responses, and disease processes, yet their function remains poorly understood due to technical limitations in sequence analysis. Here, we present an RNA-specific isobaric tandem mass tagging (RMT) platform for omic-scale quantitative mapping of RNA modifications. The platform combines RNA-specific tags adapted from proteomics with an end-to-end workflow spanning sample preparation through data processing. Validation using synthetic oligonucleotides and total tRNA from Pseudomonas aeruginosa yielded reproducible quantification, with coefficients of variation below 5%. Together with nucleobase fragment analysis, we identified and quantified 24 RNA modifications in PA14 tRNAs, including previously undescribed m2A38 and Gm/Cm39, and assigned their corresponding writer enzymes. Further analyses of tRNAs from writer knockout strains and stressed cells revealed dynamic modification patterns, modification interdependencies, and their potential roles in stress adaptation. This method provides a robust, cost-effective platform for quantitative RNA modification mapping, enabling deeper biological insights.

RNA, Transfer↗
PubMed2026

Deficient arsenic methylation and global proteomic reprogramming in human keratinocytes during arsenic-induced skin carcinogenesis.

Chronic inorganic arsenic (iAs) exposure affects&#x2009;>&#x2009;220&#xa0;million people worldwide and skin cancer is a hallmark of long-term iAs exposure. Limited information exists regarding arsenic methylation by human keratinocytes and how methylation influences skin carcinogenesis. Inorganic arsenite (iAsIII) and its methylated metabolites disrupt diverse zinc finger proteins, leading to differential toxicity patterns. We examined arsenic methylation capacity in non-malignant human keratinocytes and interrogated proteomic remodeling across three stages of iAsIII induced malignant transformation using the well-established preclinical HaCaT model. Arsenic methylation was assessed by hydride generation cryotrapping inductively coupled-mass spectrometry and global proteomic changes were analyzed by tandem-mass tagging liquid chromatography-tandem mass spectrometry. Primary, hTERT-immortalized and HaCaT human keratinocytes exhibited negligible arsenic methylation, with iAsIII comprising at least 98.5% of total intracellular arsenic, attributable to minimal expression of arsenite methyltransferase. Proteomic profiling identified over 275 differentially expressed proteins at each stage of transformation, including multiple zinc finger proteins implicated in cell cycle control, RNA metabolism, and genome stability. Ingenuity&#xae; Pathway Analysis revealed progressive, coordinated disruption of cancer-associated pathways and regulatory networks over the transformation timeline, including zinc-coordinating upstream regulators that may explain widespread pathway dysregulation. Collectively, our findings suggest that iAsIII promotes skin carcinogenesis by disrupting C3H1- and C4-type zinc finger protein-centered regulatory networks that coordinate cancer-associated signaling and metabolic pathways in human keratinocytes, highlighting key candidates for future mechanistic studies.

Arsenic↗
PubMed1976

Plasma membrane phosphorylation by endogenous phosphate donors in human blood platelets. Selectivity of the action of dibutyryl cyclic AMP.

Incubation of platelet-rich plasma with 32Pi leads to cellular uptake of the isotope and covalent incorporation into several cell constituents. Plasma membranes isolated from intact labelled platelets, delipidated and solubilized in sodium dodecyl sulfate, show, upon gel electorphoretic analysis, three main peaks of radioactivity: two in the molecular weight range 100 000-30 000 and an additional very slow migrating component strong positive by the peirodic acid-Schiff reaction. Treatment of the cells with dibutyryl cyclic AMP under conditions just sufficient to completely inhibit platelet aggregation leads to an increased isotope incorporation. Electrophoretic analysis of membranes isolated from dibutyryl cyclic AMP-treated cell reveals: (a) no change in the general pattern of distribution of the isotope, (b) no difference in the isotope incorporation to the two components of lower mol. wt. and (c) a marked increase (greater than 100%) in isotope incorporation in the slow migrating material as compared to membranes isolated from control cells. This material can be extracted from platelet plasma membranes after treatment of the membranes for 5 h with Triton X-100, at a detergent-to-protein ratio of 7.5. When the membrane material extracted with Triton X-100 is subjected to gel chromatography in Agarose (Biogel A-15m), the phosphorylated material that corresponds to the slow migrating band in polyacrylanide gel electrophoresis is eluted with or very close to the void volume of the column. Isoelectric focussing of this fraction, shows a single radioactive peak corresponding to an isolectric point of 3.78. The isolated component is pronase-sensitive, contains 52% of carbohydrate and 15% sialic acid. Analysis of the stability of the bound phosphate suggests that about 43% of it is bound as acyl-phosphate. The results reported, obtained through an approach that closely resembles physiological conditions are compatible with the participation of this membrane phosphoglycoprotein in the phenomena of platelet aggregation.

Blood Platelets↗
PubMed1977

Fatty acid synthetase complex from the insect Ceratitis capitata.

Fatty acid synthesis capacity of the insect Ceratitis capitata has been investigated in vitro from [1-14C]acetyl-CoA using homogenates at different stages of development. A maximum activity was observed after 5--6 days of larval development. But homogenates of the pharate adult insect did not show synthetic capacity of fatty acids. Fatty acid synthetase complex has been isolated from the particle-free supernatant fraction of homogenates from the 6-day C. capitata larvae. The enzyme complex was purified 182-fold with respect to the protein contained in the crude extract. The complex was homogeneous when analysed by gel filtration and by polyacrylamide-gel electrophoresis. The molecular weight was 5.2X10(5). The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants (V and Km) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42+/-1-SH residues and one phosphopatetheine moiety per 5.2X10(5). Activity was dependent on the presence of NADPH; FMN strongly inhibited the enzyme activity promoted by NADPH. The enzyme complex synthesized a range of fatty acid (10:0--18:0), palmitate being the predominant end product. The proportions of fatty acids synthesized varied with substrate concentrations. Fatty acids released from the complex were almost completely in the free form.

Amino Acids↗
PubMed1978

[The affected medullary segment in paraplegics. Relation to sexual function in men (author's transl)].

The authors made a detailed clinical analysis of 119 stabilized total paraplegics in order to define, as precisely as possible, the limits of the medullary segment which, for each patient, had lost all its functional value. The relationships between the affected segments and the sexual capabilities of these patients show that the segment must: --exclude the sacral spinal cord if reflex erections are to occur; --underly Th. IX at least, or better still Th. XI, for psychogenic turgescence to appear; the efferent pathways seem therefore, to emerge from the cord quite close to the level which receives testicular afferent pathways; --underly or overly Th. XII, L I, and L II for at least the greater part, for emission of sperm to occur; a true abrupt ejaculation probably requires the complementary integrity of the sacral spinal cord.

Adult↗
PubMed1977

Metabolic properties of the products of mitochondrial protein synthesis in HeLa cells.

The metabolic behavior of the mitochondrial protein synthesis products has been investigated in HeLa cells. Particular attention was given to the four major electrophoretic components (designated as Nos. 2, 3, 5, and 8) of the 10 previously identified as organelle-specific products. Inhibition of cytoplasmic protein synthesis with emetine or cycloheximide causes a rapid decline in the rate of mitochondrial protein synthesis, with an estimated half-life of 1 to 2 h, affecting in a parallel way all the discrete components. About 30% of the original synthetic activity appears to be resistant to emetine treatment for at least 24 h; however, all the polypeptides synthesized after the first 4 h of cell exposure to emetine are metabolically unstable, possibly because of lack of integration into the inner mitochondrial membrane. An analysis of the stability of newly synthesized products of mitochondrial protein synthesis pulse-labeled in the presence of cycloheximide and then chased in the absence of the drug (i.e. under conditions of resumed cytoplasmic protein synthesis) has revealed marked differences among the various discrete components. In particular, about three-fourths of the radioactivity associated with components 3 and 5 decays within 4 h of chase, the remainder being substantailly stable afterwards; by contrast, the radioactivity in components 2 and 8 shows only a slow decline during a 3-day chase. If the chase is carried out under conditions of a persistent block of cytoplasmic protein synthesis, as is the situation after a pulse labeling in the presence of emetine, all newly synthesized components appear to be destablized in various degrees, with the exception of component 5, which is to a great extent stabilized. Inhibition of mitochondrial protein synthesis with chloramphenicol has a progressive stabilizing effect on most of the discrete components newly synthesized after removal of the drug; this effect is especially striking in the case of component 5 which, in experiments of continuous labeling in the presence of emetine after prolonged chloramphenicol treatment, becomes, after 24 h of labeling or more, the only recognizable peak in the electrophoretic pattern of the sodium dodecyl sulfate-lysed mitochondrial fraction. The results of the kinetic experiments described here are interpreted in terms of two roles of cytoplasmically synthesized proteins, one required for the synthesis of polypeptides within the organelles, the other necessary for the stabilization of the mitochondrial products.

Cell Division↗
PubMed1978

Fenoprofen: drug form selection and preformulation stability studies.

Several fenoprofen salts were prepared to obtain the most acceptable form for an oral dosage formulation. Thermal analysis techniques were used to compare stabilities of the water of hydration in different salt forms and to assess the effects of the water of hydration on compatibility with propoxyphene and codeine salts. Photodegradation products of fenoprofen were isolated and identified, and their relevance to product formulation was evaluated.

Amines↗
PubMed1978

[Adaptation of an entirely enzymatic triglyceride assay to the GSA II Greiner. Application to the study of the stability of triglycerides, and factors causing interference in the analysis (author's transl)].

The assay of triglycerides by an entirely enzymatic pathway was adapted for use on the GSA II Greiner. The method requires only 20 microliters of biological material. The reaction time is short: 8 min at 37 degrees C. The coefficient of variation does not exceed 6% over a period of one month. Linearity of the measurements was verified up to a level of 4 mmol/l. It was shown that the free glycerol level should be systematically measured and deducted from that of the total glycerol to assure accurate results. The stability of the plasma triglycerides was investigated as a function of the length and temperature of storage (+4 degrees C and -196 degrees C). The free glycerol increases as a function of time, whereas the level of triglycerides stays constant. Haemoglobin and various medicaments, in particular, hypolipemiants and hypoglycemiants, did not interfere.

Autoanalysis↗
PubMed2025

KDM3B Regulates Postradiation Fibrotic Responses in Prostate Stroma via N6-methyladenosine Modification of LOX.

PURPOSE: Genome-wide association studies have uncovered single-nucleotide polymorphisms (SNPs) linked to radiation therapy (RT)-induced toxicities in patients with prostate cancer. SNP rs17599026, located in intron 21 of the KDM3B gene, has been associated with late-onset urinary toxicity, with an increased frequency of urination observed 2 years post-RT compared with pretreatment conditions. This study aimed to explore the underlying mechanisms driving this association. METHODS AND MATERIALS: A clustered regularly interspaced short palindromic repeats-dead Cas9 prime editing system was used to mimic KDM3B genetic variants in prostate stromal cell lines. Murine models with wild-type and heterozygous Kdm3b genotypes were used to assess fibrosis following radiation. RNA immunoprecipitation, transcript stability assays, and protein analysis elucidated the role of N6-methyladenosine (m6A) modification in regulating lysyl oxidase (LOX) expression. &#x3b1;-ketoglutarate (&#x3b1;-KG) supplementation was tested for its effects on KDM3B protein stability, LOX expression, and fibrosis mitigation. RESULTS: The rs17599026 SNP reduced KDM3B protein expression via circular RNA and microRNA-mediated mechanisms, leading to decreased m6A modification and increased stability of LOX messenger RNA. Elevated LOX expression promoted collagen cross-linking and fibrosis in prostate stroma. &#x3b1;-KG supplementation restored KDM3B protein levels, reduced LOX expression, and mitigated fibrosis in vitro and in vivo. CONCLUSIONS: KDM3B genetic variations influence radiation-induced fibrosis through posttranscriptional regulation of LOX. Dietary &#x3b1;-KG supplementation may serve as a mechanism-based strategy to alleviate radiation toxicity in patients with prostate cancer, offering a potential therapeutic pathway to improve treatment outcomes.

Male↗
PubMed2025

COG6 is an essential host factor for influenza A virus infection.

Influenza A virus (IAV) relies on the host cellular machinery to support its replication. Understanding these host dependencies can inform the development of novel antiviral strategies. In this study, we identified conserved oligomeric Golgi complex subunit 6 (COG6) as a novel host factor critical for IAV replication through a genome-wide clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) knockout screen. Disruption of COG6 significantly impaired viral replication. Mechanistically, COG6 supports IAV replication via two distinct means. First, consistent with the role of the COG complex in Golgi homeostasis, COG6 is required for the proper presentation of surface sialic acids, the primary receptor for IAV entry. Second, COG6 deficiency unexpectedly led to lysosome-dependent degradation of viral proteins. Notably, lysosomal activity was also upregulated in IAV-infected wild-type cells, albeit to a lesser extent than in COG6-deficient cells. Treatment with lysosomal inhibitors rescued viral protein stability in COG6 knockout cells. Protein interaction analysis further demonstrated that COG6-mediated stabilization of viral proteins did not rely on viral protein-COG6 interaction, refuting the hypothesis that COG6 acts as a shield factor to protect viral protein from lysosomal degradation. Moreover, knockout of other COG subunits produced similar antiviral effects, suggesting that an intact COG complex is required for IAV replication. Together, these findings uncover a critical role of the COG complex in regulating IAV replication and highlight a previously unappreciated functional link between the Golgi and lysosomes that could be exploited for treating IAV infections.IMPORTANCEDespite advances in virology, numerous host determinants facilitating influenza A virus (IAV) pathogenesis remain uncharacterized. Our study establishes conserved oligomeric Golgi complex subunit 6 (COG6) as a critical host factor promoting IAV infection through complementary mechanisms: receptor modulation and viral protein stabilization. This represents the first demonstration that the COG complex regulates viral pathogenesis through proteostasis mechanisms, fundamentally expanding our understanding of host-virus interactions at the organelle interface. These findings not only provide new perspectives on viral exploitation of Golgi trafficking networks but also identify potential therapeutic targets against evolving influenza strains.

Influenza A virus↗
PubMed1978

Time-dependent increase in the stability of collagen fibrils formed in vitro. I. Effects of pH and salt concentration on the dissolution of the fibrils.

The time-dependent increase in stability, as measured in terms of the rate of dissolution, of collagen fibrils formed in vitro from pepsin-treated collagen was significantly affected only by temperature, and not by either ionic strength or pH. This is in contrast with collagen fibril formation, a process which is greatly affected by ionic strength and pH. Within the range of temperature 29-37 degrees C, lower temperature caused slower fibril formation and faster fibril stabilization. These results suggest that the intermolecular interactions involved in stabilizing collagen fibrils are entirely different from those involved in fibril formation. Based on kinetic analysis of the dissolution and stabilization of the fibrils, it is proposed that collagen molecules first form unstable fibrils which become gradually stabilized on prolonged incubation, without necessarily introducing covalent cross-links.

Animals↗
PubMed1977

[New methods of coagulation analysis using chromogenic substrates].

The available assay systems for the determination of coagulation factors based on clotting methods are unsatisfactory, despite efforts to stabilize and to standardize them. Analysis by means of chromogenic peptide substrates offers new possiblities for routine diagnostics, and for fundamental research. The development of these new methods, however, is at an early stage.

Blood Coagulation Factors↗