Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts overexpressing udk

Overexpression of udk, an Escherichia coli gene encoding a uridine/cytidine kinase, interferes with T7 bacteriophage growth. We show here that inhibition of T7 phage growth by udk overexpression can be overcome by inhibition of host RNA polymerase. Overexpression of gene 2, whose product inhibits host RNA polymerase, restores T7 phage growth on hosts overexpressing udk. In addition, rifampicin, an inhibitor of host RNA polymerase, restores the burst size of T7 phage on udk-overexpressing hosts to normal. In agreement with these findings, suppressor mutants that overcome the inhibition arising from udk overexpression gain the ability to grow on hosts that are resistant to inhibition of RNA polymerase by gene 2 protein, and suppressor mutants that overcome a lack of gene 2 protein gain the ability to grow on hosts that overexpress udk. Mutations that eliminate or weaken strong promoters for host RNA polymerase in T7 DNA, and mutations in T7 gene 3.5 that affect its interaction with T7 RNA polymerase, also reduce the interference with T7 growth by host RNA polymerase. We propose a general model for the requirement of host RNA polymerase inhibition.     

Short-term effects of Al3+ on the osmotic behavior of red beet (Beta vulgaris L.) protoplasts

The short-term (less than 10 min) effects of Al³⁺ on the biophysical properties of plasma membranes were investigated by time-series image analysis of osmotically-induced volumetric and morphologic changes of red beet (Beta vulgaris L.) protoplasts. Exposure to Al³⁺ under hypotonic conditions reduced the volumetric expansion of protoplasts and their resultant burst: i.e. lysis of protoplasts in a concentration-dependent manner. Under hypertonic conditions, protoplasts exposed to Al³⁺ underwent an enhanced volumetric contraction in cross-sectional area, while maintaining higher protoplast roundness. The residual effects of Al³⁺ pre-treatment on subsequent osmotic behavior were also examined, and protoplasts pre-treated with Al³⁺ also exhibited less lysis during subsequent exposure to hypotonic conditions and enhanced volumetric contractions and higher roundness under subsequent hypertonic conditions. Under our experimental conditions, Al³⁺ consistently minimized protoplast surface area by inhibiting osmotic expansion or by enhancing osmotic contraction, as well as by maintaining higher protoplast roundness. These results suggested that the electrostatic property of Al³⁺ might have induced the binding and possible cross-linking of negatively-charged sites on the plasma membrane surface. This may be an important factor in understanding the mechanism of Al³⁺ phytotoxicity.     

Differential phosphorylation of PKR associates with deregulation of eIF-2α phosphorylation and altered growth characteristics in 3T3-F442A fibroblasts

Murine embryonic 3T3-F442A fibroblasts contain elevated levels of a factor (dRF) inhibitory to the phosphorylation of PKR, when cultured under differentiation restrictive (10% cat serum) as compared to permissive conditions (10% fetal bovine serum). Experiments were conducted with the objective of understanding the effect of altered PKR activity on the growth characteristics of 3T3-F442A fibroblasts. Analysis of the phosphoprotein pattern confirmed that the phosphorylation of PKR was reduced in cells cultured in cat serum during specific stages of growth. In a similar manner, evaluation of eIF-2 phosphorylation by vertical slab gel iso-electric focusing indicated that inactivation of PKR correlated with reduction of eIF-2 phosphorylation. The expression of PKR was confirmed by western blotting ruling out the possibility of diminished protein as the cause of loss of activity. In addition, the expression of dRF coincided with the inactivation of PKR as shown by immunoblotting and phosphorylation studies. The reduction in PKR activity and subsequent deregulation of eIF-2 phosphorylation was related to appearance of tumor-like cellular morphology and increased cell density as shown by cell counts and [³H]-thymidine uptake. Taken together, these results support a hypothesis that PKR functions to regulate the growth of 3T3-F442A cells. Furthermore, our findings raise the possibility that deregulation of PKR by endogenous inhibitory molecules, such as dRF, may alter normal growth and differentiation. Such a deregulation of PKR may also contribute to the proliferation of tumor cells.     

Determination of the true intraocular pressure and modulus of elasticity of the human cornea in vivo

The purpose of this study was to determine the true intraocular pressure and modulus of elasticity of the human cornea in vivo. The cornea was modeled as a shell, and the equations for the deformations of a shell due to applanating and intraocular pressures were combined to model the behavior of the cornea during applanation tonometry. At certain corneal dimensions called the calibration dimensions, the applanating and intraocular pressures are considered to be equal. This relationship was used to determine the modulus of elasticity of the cornea and the relationship between the applanating and intraocular pressures. The true intraocular pressure (IOPT) was found to be related to Goldmann’s applanating pressure (IOPG) as (IOPT = IOPG/K, where K is a correction factor. For the calibration corneal thickness of 0.52 mm, the modulus of elasticity E in MPa of the human cornea was found to be related to the true intraocular pressure IOPT in mmHg as E = 0.0229IOPT. The generalization of the Imbert—Fick law that takes into account the effect of corneal dimensions and stiffness was found to be given by IOPT = 73.5W/(K A), where W is the applanating weight in gf (gram force) and A is the applanated area in mm². The calculated true intraocular pressure and modulus of elasticity were found to agree with published experimental results. The mathematical model developed may therefore be used to improve results from applanation tonometry and to estimate the mechanical property of the cornea in vivo.     

Identification of the changes in phospholipase C isozymes in ischemic-reperfused rat heart

Phospholipase C (PLC) influences cardiac function. This study examined PLC isozymes of the cardiac sarcolemma (SL) membrane and in the cytosol compartment in isolated perfused rat hearts subjected to global ischemia for 30 min followed by up to 30 min of reperfusion. Although the total SL PLC activity was decreased in ischemia and increased upon reperfusion, differential changes in PLC isozymes were detected. PLC beta(1) mRNA and SL protein abundance and activity were increased in ischemia, with concomitant decreases in activity and protein level in the cytosol. On the other hand, upon reperfusion, PLC beta(1) activity was decreased, but remained higher than control values. Although no change in the PLC delta(1) mRNA level in ischemia was detected, SL PLC delta(1) activity and content were depressed. Furthermore, in the cytosol, PLC delta(1) activity was increased, but the protein level decreased. SL PLC gamma(1) activity was decreased, independent of gene expression and protein content; however, decreases in the activity and protein abundance were detected in the cytosol. Increases in PLC gamma(1) and delta(1) activities occurred upon reperfusion, but were not accounted for by altered mRNA and protein levels. The results indicate that ischemia-reperfusion induces differential changes in PLC isozymes.     

Depletion of CD8+ T cells exacerbates CD4+ Th cell-associated inflammatory lesions during murine mycoplasma respiratory disease

Mycoplasma infection is a leading cause of pneumonia worldwide and can lead to other respiratory complications. A component of mycoplasma respiratory diseases is immunopathologic, suggesting that lymphocyte activation is a key event in the progression of these chronic inflammatory diseases. The present study delineates the changes in T cell populations and their activation after mycoplasma infection and determines their association with the pathogenesis of murine Mycoplasma respiratory disease, due to Mycoplasma pulmonis infection. Increases in T cell population numbers in lungs and lower respiratory lymph nodes were associated with the development of mycoplasma respiratory disease. Although both pulmonary Th and CD8(+) T cells increased after mycoplasma infection, there was a preferential expansion of Th cells. Mycoplasma-specific Th2 responses were dominant in lower respiratory lymph nodes, while Th1 responses predominated in spleen. However, both mycoplasma-specific Th1 and Th2 cytokine (IL-4 and IFN-gamma) responses were present in the lungs, with Th1 cell activation as a major component of the pulmonary Th cell response. Although a smaller component of the T cell response, mycoplasma-specific CD8(+) T cells were also a significant component of pulmonary lymphoid responses. In vivo depletion of CD8(+) T cells resulted in dramatically more severe pulmonary disease, while depletion of CD4(+) T cells reduced its severity, but there was no change in mycoplasma numbers in lungs after cell depletion. Thus, mycoplasma-specific Th1 and CD8(+) T cell activation in the lung plays a critical regulatory role in development of immunopathologic reactions in Mycoplasma respiratory disease.     

Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement

A single copy of bacteriophage T7 DNA polymerase and DNA helicase advance the replication fork with a processivity greater than 17,000 nucleotides. Nonetheless, the polymerase transiently dissociates from the DNA without leaving the replisome. Ensemble and single-molecule techniques demonstrate that this dynamic processivity is made possible by two modes of DNA polymerase-helicase interaction. During DNA synthesis the polymerase and the helicase interact at a high-affinity site. In this polymerizing mode, the polymerase dissociates from the DNA approximately every 5000 bases. The polymerase, however, remains bound to the helicase via an electrostatic binding mode that involves the acidic C-terminal tail of the helicase and a basic region in the polymerase to which the processivity factor also binds. The polymerase transfers via the electrostatic interaction around the hexameric helicase in search of the primer-template.     

Norfloxacin-induced DNA cleavage occurs at the dif resolvase locus in Escherichia coli and is the result of interaction with topoisomerase IV

The dif locus is a site-specific recombination site located within the terminus region of the chromosome of Escherichia coli. Recombination at dif resolves circular dimer chromosomes to monomers, and this recombination requires the XerC, XerD and FtsK proteins, as well as cell division. In order to characterize other enzymes that interact at dif, we tested whether quinolone-induced cleavage occurs at this site. Quinolone drugs, such as norfloxacin, inhibit the type 2 topoisomerases, DNA gyrase and topoisomerase IV, and can cleave DNA at sites where these enzymes interact with the chromosome. Using strains in which either DNA gyrase or topoisomerase IV, or both, were resistant to norfloxacin, we determined that specific interactions between dif and topoisomerase IV caused cleavage at that site. This interaction required XerC and XerD, but did not require the C-terminal region of FtsK or cell division.     

Sex differences in global metabolomic profiles of COVID-19 patients

Coronavirus disease (COVID-19), caused by SARS-CoV-2, leads to symptoms ranging from asymptomatic disease to death. Although males are more susceptible to severe symptoms and higher mortality due to COVID-19, patient sex has rarely been examined. Sex-associated metabolic changes may implicate novel biomarkers and therapeutic targets to treat COVID-19. Here, using serum samples, we performed global metabolomic analyses of uninfected and SARS-CoV-2-positive male and female patients with severe COVID-19. Key metabolic pathways that demonstrated robust sex differences in COVID-19 groups, but not in controls, involved lipid metabolism, pentose pathway, bile acid metabolism, and microbiome-related metabolism of aromatic amino acids, including tryptophan and tyrosine. Unsupervised statistical analysis showed a profound sexual dimorphism in correlations between patient-specific clinical parameters and their global metabolic profiles. Identification of sex-specific metabolic changes in severe COVID-19 patients is an important knowledge source for researchers striving for development of potential sex-associated biomarkers and druggable targets for COVID-19 patients.Subject terms: Metabolomics, Immunological disorders