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Modulation of E rosette forming activity of human lymphocytes by rosette inhibiting factor (RIF) ann rosette restoring factor (RRF).

The mechanism of action of RIF and RRF on spontaneous rosette formation by human T lymphocytes with SRBC was studied on cells isolated from healthy donors and from patients with active pulmonary tuberculosis. E rosette formation by lymphocytes from healthy donor is inhibited in vitro by RIF. The effect of RIF is reversible since the primary activity of lymphocytes can be restored by contact with RRF. On the other hand, E rosette formation by lymphocytes from tuberculous patients by means of RRF can be depressed by RIF to the starting level. It was found that prostaglandin synthetase inhibitors (indomethacin and RO 20-5720) prevent inhibition or restoration of E rosette activity by RIF and RRF, respectively.

Blood Donors

In vitro response to rosette inhibition factor (RIF) and rosette restoring factor (RRF) of human lymphocyte after fractionation on a discontinuous density albumin gradient.

In vitro studies on peripheral blood lymphocytes from tuberculous patients and healthy subjects are reported. Factors isolated from human serum used in the experiments included the Rosette Inhibition Factor (RIF) from serum of tuberculous patients and the Rosette Restoring Factor (RRF) from serum of healthy subjects. Lymphocytes were fractionated on a discontinuous BSA gradient. Lymphocytes from healthy subjects, sensitive to RIF, as well as lymphocytes from tuberculous patients, sensitive to RRF, were localized in the third fraction of the BSA gradient. A small percentage of bone marrow cells upon coming into contact with RRF spontaneously formed rosettes with SRBC and were also localized in the third fraction after centrifugation in discontinuous BSA gradient. The results indicate that RIF and RRF act on the same subpopulations, probably precursors of T cells.

Blood Donors

Extragenic suppression of two ribosomal protein cistrons lying near the rif locus in Escherichia coli.

The experiments reported here involve temperature-sensitive mutations in two of five cistrons encoding 50S ribosomal proteins that lie near the rif locus in Escherichia coli. I selected spontaneous TS+ mutants able to grow at elevated temperatures in which the TS+ event takes place outside this tract of cistrons near rif. Six distinct classes of extragenic suppressors were found, five of which have been mapped. Two of these suppressors lie near 64 min, a region known to be rich in cistrons ribosomal proteins (Dennis and Nomura, 1975). The remaining three extragenic suppressors were located near 16.5, 47, and 86 min.

Bacterial Proteins

Organization of genes for transcription and translation in the rif region of the Escherichia coli chromosome.

The lambdarifd18 transducing phage is known to carry several genes for components of transcriptional and translational machineries; these genes are clustered in the rif region at 88 min on the Escherichia coli genetic map. They include a set of genes for rRNA's (rrnB), a gene for spacer tRNA, tRNA2Glu (tgtB), one of the two genes for EF-Tu (tufB), genes for four ribosomal proteins (rplK, A, J, and L), genes for the beta and beta' subunits of RNA polymerase (rpoB and rpoC), and genes for three tRNA's (tyrU, gluT, and thrT). An additional tRNA gene (subsequently identified as thrU by Landy and his co-workers) and a gene for a protein (protein U) with unknown functions were found to be carried by lambdarif d18. We analyzed the organization of these genes by using various deletion and hybrid phages derived from lambdarif d18 and lambdarif d12, a phage related to lambdarif d18. The expression of various genes was examined in UV-irradiated cells infected with these transducing phages. Two main conclusions were obtained. First, the four tRNA genes are not cotranscribed with the genes in rrnB, even though these tRNA genes are located close to the distal end of rrnB. Second, the four ribosomal protein genes are organized into two separate transcriptional units; rplK and A are in one unit and rplJ and L are in the second unit. The first group of genes was shown to have a promoter separate from that for tufB or protein U. The second group of genes shares the promoter with rpoB and C, as described in a separate paper (M. Yamamoto and M. Nomura, Proc. Natl. Acad. Sci. U.S.A., 75:3891--3895). These and other results described in this paper show that the genes are organized in the following order: promoter, genes in rrnB; promoter, thrU, tyrU, (promoter?) glyT, thrT; (promoter?) tufB; promoter, a gene for protein U; promoter, rplK, rplA; promoter, rplJ, rplL, rpoB, rpoC.

Bacterial Proteins

Interaction between mutations of ribosomes and RNA polymerase: a pair of strA and rif mutants individually temperature-insensitive but temperature-sensitive in combination.

A temperature-sensitive lethal mutant of Escherichia coli has been constructed by combining two temperature-insensitive mutations: a rif180 mutation that modifies RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a strA24 mutation that modifies the ribosomal protein S12. The temperature sensitivity is a property of the combination of these two particular alleles; replacement of either of the alleles relieves the temperature sensitivity. An isogenic strain containing a different strA mutation (i.e., rif180 strA11) is not temperature sensitive. Evidently ribosomes modified by the particular strA24 polymerase altered by the rif180 mutation, which suggests that in vivo there may exist some interaction between structures of ribosomes and the RNA polymerase.

Bacterial Proteins

U.S. Public Health Service Cooperative trial of three rifampin-isoniazid regimens in treatment of pulmonary tuberculosis.

A total of 822 patients with newly diagnosed pulmonary tuberculosis were assigned randomly to one of 3 daily rifampin-isoniazid (RIF-INH) regimens: 450, 600, or 750 mg of RIF in combination with 300 mg of INH. After an initial 20 weeks of therapy with RIF-INH, patients recieved 300 mg of INH and 15 mg of ethambutol (EMB) per kg of body weight for either 12 or 18 months after their sputum cultures became negative. The rate of bacteriologic conversion of sputum among the 3 RIF-INH regimens was compared for 552 patients who completed the 20 weeks of RIF-INH therapy. Apporximately 60 per cent of these patients also completed their assigned INH-EMB therapy and were examined for relapse for at least one year after therapy was stopped. There was no significant difference in the rate of sputum conversion or rate of relapse between the group of patients who received 600 mg of RIF and those who received 750 mg of RIF. However, the 450-mg RIF regimen was significantly less effective than the other 2 regimens, as manifested by a lower rate of sputum conversion and a higher rate of treatment failures. Further analysis showed that RIF dosages of less than 9 mg per kg of body weight per day may be inadequate for treatment of pulmonary tuberculosis. The acceptability of these regimens was high, and the incidence of adverse reactions requiring discontinuation of RIF-INH therapy was quite low (3.3 per cent). A large proportion of patients (44 per cent) developed increased concentrations of transaminase during therapy with RIF-INH. These abnormalities were usually transient and, in most cases, of no clinical significance. In the relapse analysis, 12 months of chemotherapy after sputum conversion was shown to be as effective as 18 months of therapy after conversion of these RIF-containing regimens.

Adolescent

Lymphocyte E rosette inhibitory factor: a regulatory serum lipoprotein.

Rosette inhibitory factor, RIF, previously described in serum from patients with hepatitis B virus infection, has been isolated and identified as a minor species of beta-lipoprotein of the low-density (LDL) class. It is unrelated to hepatitis B virus proteins or particles. Although discrete by reference to charge and density (1.050 +/- 0.004 g/cm3), RIF appears to be a complex macromolecular structure containing apolipoproteins A, B, and C. Greater than 400% recovery is achieved upon 300,000-fold purification from RIF+ sera suggesting activation of a precursor form that is not present in normal serum. RIF inhibits E rosette function of T lymphocytes in vitro with a lag period of approximately 4 h and maximal effect at 24 h consistent with a metabolically-induced event. RIF is functionally active at concentrations of 1 X 10(-12) M or greater, rapidly binds to lymphocytes, and has a functionally effective half-life of approximately 1.5 h. Approximately 2,900 receptors for RIF appear to be present per cell and a high mutual affinity is apparent (k approximately to 9 X 10(10) liters/mol). RIF has no detectable effect on mitogen (PHA) responsiveness of lymphocytes, but inhibits the capacity of lymphocytes to respond to histoincompatible cells in vitro at concentrations greater than 10(-8) M. Equivalent RIF- lipoprotein fractions from normal serum are equally inhibitory in the mixed lymphocyte reaction suggesting that this effect is not directly attributable to RIF activity. These data indicate that RIF is a unique and functionally specific species of LDL that represents either an association complex of lipoproteins or a hybrid molecule of unusual composition. The association of this factor with viral-induced hepatocellular injury underscores the need to elucidate its structure and function in greater detail.

Adult

Comprehensive analysis of circRNA-miRNA-mRNA network related to angiogenesis in recurrent implantation failure.

BACKGROUND: Abnormal endometrial blood flow causes a decrease in endometrial receptivity and is considered a relatively independent risk factor for recurrent implantation failure (RIF). This study aimed to explore the potentially functional circRNA-miRNA-mRNA network in RIF, and further explore its mechanism. METHODS: Datasets were downloaded from the GEO database to identify differentially expressed circRNAs, miRNAs and mRNAs. The circRNA-miRNA-mRNA and PPI networks were constructed using Cytoscape 3.6.0 and the STRING database, the hub genes were identified with the cytoHubba plug-in, and a circRNA-miRNA-hub mRNA regulatory sub-network was constructed. Then, GO and KEGG pathway enrichment analyses of the hub genes were performed to comprehensively analyze the mechanism of hub mRNAs in RIF. Due to the results of circRNAs-miRNAs-hub mRNAs regulatory network, we verified the expression of circRNA_0001721, circRNA_0000714, miR-17-5p, miR-29b-3p, HIF1A and VEGFA in the RIF mouse model by qRT‒PCR and western blotting. RESULTS: We initially identified 175 DEmRNAs, 48 DEmiRNAs and 56 DEcircRNAs in RIF associated with angiogenesis and constructed a circRNA-miRNA‒mRNA network and PPI network. We further identified six hub genes in the acquired network. Based on these genes, functional enrichment analysis revealed that the HIF-1 signaling pathway plays a vital role in endometrial angiogenesis in RIF. In addition, the interaction networks of circRNA_0001721/miR-17-5p/HIF1A and the circRNA_0000714/miR-29b-3p/VEGFA axis were predicted. In the RIF mouse model, circRNA_0001721, circRNA_0000714, HIF1A and VEGFA were down-regulated, whereas miR-17-5p and miR-29b-3p were up-regulated according to qRT‒PCR and western blotting. CONCLUSION: This study revealed that the HIF-1 signaling pathway plays a vital role in endometrial angiogenesis in RIF. The circRNA_0001721/miR-17-5p/HIF1A and circRNA_0000714/miR-29b-3p/VEGFA axes might play a role in the pathogenesis of endometrial angiogenesis in RIF.

MicroRNAs

Genomic Insights Into Convergent Evolution: Adaptation to Rocky Habitats in Rock-Inhabiting Fungi.

Rock-inhabiting fungi (RIF), obligate colonizers of bare rocks, are primarily distributed across two major phylogenetic classes: Dothideomycetes and Eurotiomycetes. These fungi display striking convergence in morphology and physiology, characterized by meristematic growth, melanized cell walls, and extreme stress tolerance. However, the genomic underpinnings of this adaptive convergence remain poorly understood. Here, through comparative genomic analysis of 9 RIF and 18 non-RIF fungi, we revealed that RIF possess compact, gene-dense genomes marked by contraction of genes involved in nutrient uptake and secondary metabolism, alongside expansions in cell wall biosynthesis, lipid metabolism, and stress-responsive pathways. We identified two genes under positive selection across multiple RIF lineages: Ino80 ATPase (chromatin remodeling) and the ER chaperone BiP (protein folding). Further evidence of convergence was found in the mannosyltransferase Mnn9, a key enzyme in cell wall assembly, where two RIF-specific amino acid substitutions were predicted to enhance protein stability. Additionally, a unique Mnn9-like clade has expanded exclusively in RIF. RNAi-mediated knockdown of an Mnn9-like gene in Rachicladosporium sp. confirmed its role in cell wall mannosylation, osmotic stress response, and the transition from meristematic to filamentous growth. Our findings elucidate a set of common genomic adaptations and highlight the specialized evolution of the Mnn9 family in driving the convergent success of phylogenetically diverse RIF in rocky environments.

Phylogeny

Dissociation kinetics of complexes between the antibiotic rifamycin and DNA-dependent RNA polymerase from Escherichia coli.

Phosphocellulose chromatography was employed to measure the binding of 3-(2-[14C]acetamidoethyl)-thiorifamycin(abbreviated[14C]AcNHEtS-Rif) to RNA polymerase core enzyme. The technique yielded a correct value for the stoichiometry of interaction. The same method was successfully applied to an investigation of the dissociation kinetics of the [14C]AcNHEtS-Rif - core-polymerase complex. We observed biphasic dissociation kinetics not in agreement with the existence of one single first-order dissociation step. Assuming two independent dissociation reactions, rate constants have been evaluated differing roughly by 10-fold (1.7 X 10(-3) s-1 and 1.5 X 10(-4) s-1 at 25 degrees C). The ratio of the amplitudes of the biphasic dissociation kinetics changed with temperature. The kinetic data are interpreted as indirect evidence for the existence of two enzyme species differing in their dynamic properties with respect to the binding of the antibiotic rifamycin. Our data furthermore lend support to the assumption that the two enzyme forms are in equilibrium. The observed sigmoidal dependence of the dissociation rate on temperature could indicate a conformational transition of RNA core polymerase with a transition midpoint at around 20 degrees C. The dye, rose bengal, was found to be as effective as AcNHEtS-Rif itself as a competing agent. The dissociation kinetics of the [14C]-AcNHEtS-Rif - core-polymerase complex in the presence of an excess of the dye rose bengal were found to be very similar to those measured in the presence of AcNHEtS-Rif. This could mean that rose bengal and AcNHEtS-Rif compete for the same site at RNA core polymerase. The dissociation of the ternary complex [14C]AcNHEtS-Rif - core-polymerase - poly[d(A-T)] was followed by gel filtration. Up to the extent of dissociation measured, the reaction appeared to follow first-order kinetics. The dissociation rate constant was calculated to be 1.7 X 10(-4) s-1. Experiments to determine the effect of the antibiotic streptolydigin on the dissociation kinetics of the [14C]AcNHEtS-Rif - core-polymerase complex have not led to conclusive results.

Binding Sites

Genotyping of natural killer cell immunoglobulin-like receptors in human early reproductive losses.

Genotyping of killer cell immunoglobulin-like receptors (KIR) of NK-cells was performed in 634 women with early pregnancy losses (EPL), including 158 women with recurrent implantation failure (RIF) after at least three IVF cycles, and 110 women with at least two intrauterine pregnancy losses characterized by clinically confirmed retention of a non-viable fetus in the uterus without spontaneous miscarriage, defined as recurrent pregnancy loss (RPL). The control group consisted of 431 women with at least two healthy children. In the RIF, a significant shift in the frequency of KIR-genotypes was observed compared with both the control and the RPL groups. A significantly higher frequency of the KIR-AA genotype was observed in the RIF group compared to the control group (&#x3c7;&#xb2; = 26.78; p&#x202f;<&#x202f;0.0001; OR = 2.7) and the RPL group (&#x3c7;&#xb2; = 15.83; p&#x202f;<&#x202f;0.0001; OR = 2.86). Analysis of the frequency of full-length/deletion alleles of KIR2DS4 among AA genotype carriers showed an increased frequency of the 2DS4-del in the RIF compared with the RPL and control groups. A significantly higher frequency of the cenAA was also observed in RIF compared with the control (&#x3c7;&#xb2; = 20.10; p&#x202f;<&#x202f;0.0001) and the RPL(&#x3c7;&#xb2; = 12.05; p&#x202f;<&#x202f;0.005). The significantly increased frequency of the KIR-AA genotype with predominance of deletion KIR2DS4 alleles, along with the elevated frequency of the cenAA in RIF, may indicate insufficient NK-cell activation at the stage of embryo implantation. These findings suggest different etiological mechanisms for RIF and RPL.

Humans

Enhanced transformability with heterospecific deoxyribonucleic acid upon removal of nascent ribonucleic acid from the Streptococcus sanguis genome.

Treatment of Streptococcus sanguis recipient cells with rifampin (RIF) at the time of deoxyribonucleic acid (DNA) addition was an effective means of reducing discrimination, that is, of causing an increase in the number of transformants induced by irreversibly bound heterospecific DNA without significantly changing the number induced by bound homospecific DNA. RIF was unable to reduce discrimination when the recipient cells were RIF resistant due to an altered ribonucleic acid (RNA) polymerase. When recipient cells were treated at the time of DNA addition with concentrations of streptolydigin (STG) as inhibitory of RNA synthesis as RIF, discrimination was not reduced. The kinetics of RNA synthesis inhibition with these inhibitors indicated that, as reported for other bacterial species, RIF inhibited the initiation of transcription by RNA polymerase, whereas STG inhibited the progression of RNA polymerase at any point. Pulse-labeling of RNA immediately before STG addition showed that, if cells were incubated under STG inhibition for 10 to 15 min, their nascent RNA was degraded. Genome-bound RNA polymerase was not released under these conditions. When recipient cells were incubated with STG until nascent RNA was degraded and then exposed to transforming DNA, STG was as effective as RIF in reducing discrimination. The presence of nascent RNA was thereby implicated in the transforming inefficiency of incompletely homologous DNA.

Aminoglycosides