Molecular Mechanisms Associated with Aging Kidneys and Future Perspectives.

The rapid growth of the elderly population is making the need for extensive and advanced information about age-related organ dysfunction a crucial research area. The kidney is one of the organs most affected by aging. Aged kidneys undergo functional decline, characterized by a reduction in kidney size, decreased glomerular filtration rate, alterations in renal blood flow, and increased inflammation and fibrosis. This review offers a foundation for understanding the functional and molecular mechanisms of aging kidneys and for selecting identifying appropriate targets for future treatments of age-related kidney issues.

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase.

Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused-backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1.

Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3',5'-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo-cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the ß subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.

Isolated MCA disease in patients without significant atherosclerotic risk factors: a high-resolution magnetic resonance imaging study.

BACKGROUND AND PURPOSE: Diagnosis of intracranial artery atherosclerosis remains often uncertain. The high-resolution magnetic resonance imaging (HR-MRI) enables vessel wall assessment for more precise diagnoses. The aim of the present study was to investigate the etiologies of middle cerebral artery steno-occlusive disease in young adult patients with few atherosclerotic risk factors using HR-MRI. METHODS: We prospectively studied patients who visited a tertiary hospital in Seoul, Korea, and had (1) unilateral middle cerebral artery disease (≥50% stenosis or occlusion), (2) were ≤55 years old and had no or minimal (≤1) atherosclerotic risk factors. We excluded patients with a confirmed diagnosis of Moyamoya disease, vasculitis, or dissection and those having emboligenic sources. A presumptive diagnosis was made based on HR-MRI findings, and patients were categorized as HR-athero (atherosclerotic disease), HR-MMD (Moyamoya disease), HR-dissection, or HR-vasculitis. RESULTS: Among 95 patients analyzed, 26 (27.4%) had HR-athero who were more often male (P=0.004), smokers (P=0.018), and had focal stenosis (P=0.003) than others.As compared with the HR-athero patients, 29 HR-MMD patients were more often female (P<0.001) and more often had occlusive lesions (P=0.001) and nonfocal stenosis (P<0.001). The 22 HR-dissection patients tended to have hypertension less often, and the 13 HR-vasculitis patients were younger (P=0.004) and tended to have nonfocal stenosis. [corrected]. CONCLUSIONS: In our cohort of young patients with minimal risk factors, atherosclerosis seems to be an uncommon pathology of middle cerebral artery stenosis. HR-MRI aids us to make a more reliable diagnosis.

Phospholipase-catalyzed hydrolysis in an artificial cell membrane in the presence of melittin.

Biomimicry involves the use of the structure and function of biological systems as models for the design and engineering of materials and machines. An artificial cell membrane was developed using biomembrane components, and the membrane, formed by a lipid bilayer, was analyzed using surface plasmon resonance (SPR) to monitor hydrolysis by phospholipase (PL). The simultaneous atomic force microscope (AFM) images show that PL catalyzed the nanometer-scale hydrolysis of the artificial lipid biomembranes through enzymatic hydrolysis. In addition, it was confirmed that the combination of PL and melittin allowed the control of enzyme hydrolysis for the degradation of the lipid bilayer. Regarding the expected activating effect of melittin on hydrolysis, no difference with respect to the non-treated lipid membrane was observed in the AFM images. It is assumed that the partitioning of melittin into the membrane might prevent the binding or hydrolysis of Phospholipase A2 (PLA2). This study provides basic knowledge on a new approach for patterning biomimicking lipid membranes on a nano-scale.

Detection of single nucleotide polymorphisms using a biosensor-containing titanium-well array.

The rapid identification and verification of single nucleotide polymorphisms (SNPs) were demonstrated using a well array sensor containing anti-biofouling titanium (Ti). Probe single-stranded DNA (ssDNA) was immobilized inside a titanium-well array on amine-modified glass surfaces with anti-biofouling behavior via a streptavidin-biotin interaction. Fluorescence intensity changes originating from the hybridization of nucleic acids to protein-bound nucleic acids linked to Alexa Fluor (FL) 647 were observed. The protocol was highly sensitive and reproducible for the detection of DNA hybridization. Significant changes in fluorescence signals were observed when using target DNA with a single base mismatch, indicating that this method is applicable to SNP detection. The microarray technology for the detection of SNPs using anti-biofouling Ti and other methods can be used as a highly sensitive in vitro medical sensor, as highlighted by an increase in genotyping accuracy.

Single-molecule studies reveal the off-pathway elemental pause state as a target of streptolydigin inhibition of RNA polymerase and its dramatic enhancement by Gre factors.

Antibiotic streptolydigin (Stl) inhibits bacterial transcription by blocking the trigger loop folding in the active center of RNA polymerase (RNAP), which is essential for catalysis. We use acoustic force spectroscopy to characterize the dynamics of transcription elongation in ternary elongation complexes of RNAP (ECs) in the presence of Stl at a single-molecule level. We found that Stl induces long-lived stochastic pauses while the instantaneous velocity of transcription between the pauses is unaffected. Stl enhances the short-lived pauses associated with an off-pathway elemental paused state of the RNAP nucleotide addition cycle. Unexpectedly, we found that transcript cleavage factors GreA and GreB, which were thought to be Stl competitors, do not alleviate the streptolydigin-induced pausing; instead, they synergistically increase transcription inhibition by Stl. This is the first known instance of a transcriptional factor enhancing antibiotic activity. We propose a structural model of the EC-Gre-Stl complex that explains the observed Stl activities and provides insight into possible cooperative action of secondary channel factors and other antibiotics binding at the Stl-pocket. These results offer a new strategy for high-throughput screening for prospective antibacterial agents.

The clinical and prognostic value of antinuclear antibodies in NMO-IgG seropositive neuromyelitis optica spectrum disorder.

In neuromyelitis optica spectrum disorders (NMOSD), the clinical and long-term prognostic value of antinuclear antibodies (ANAs) is unclear. We analyzed registry data of NMO-IgG seropositive NMOSD patients (n = 74) according to ANA presence. The ANA-positive group (n = 32) demonstrated more frequent other autoantibodies (anti-SSA/Ro, anti-SSB/La, antiphospholipid, and anti-double stranded DNA antibodies) than did the ANA-negative group (n = 42). Clinically, annual relapse rates, and average lesion extents on MRI during attacks were comparable between the two groups (median follow-up of 7 years). The development of a poor outcome (walking with unilateral aid) also did not differ. In conclusion, although common, ANAs were not associated with a benign/malignant disease course in our NMOSD cohort.

Analysis of promoter targets for Escherichia coli transcription elongation factor GreA in vivo and in vitro.

Transcription elongation factor GreA induces nucleolytic activity of bacterial RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is unclear which of these functions is (are) most relevant in vivo. By comparing global gene expression profiles of Escherichia coli strains lacking Gre factors and strains expressing either the wild type (wt) or a functionally inactive GreA mutant, we identified genes that are potential targets of GreA action. Data analysis revealed that in the presence of chromosomally expressed GreA, 19 genes are upregulated; an additional 105 genes are activated upon overexpression of the wt but not the mutant GreA. Primer extension reactions with selected transcription units confirmed the gene array data. The most prominent stimulatory effect (threefold to about sixfold) of GreA was observed for genes of ribosomal protein operons and the tna operon, suggesting that transcript cleavage by GreA contributes to optimal expression levels of these genes in vivo. In vitro transcription assays indicated that the stimulatory effect of GreA upon the transcription of these genes is mostly due to increased RNAP recycling due to facilitated promoter escape. We propose that transcript cleavage during early stages of initiation is thus the main in vivo function of GreA. Surprisingly, the presence of the wt GreA also led to the decreased transcription of many genes. The mechanism of this effect is unknown and may be indirect.

Different types of pausing modes during transcription initiation.

In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape. The previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi et al. 1 and from Lerner, Chung et al. 2 In both studies, pausing is attributed to a lack of forward translocation of the nascent transcript during initiation. However, the two works report on different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we discuss these studies, together with additional experimental results from single-molecule FRET focusing on a specific distance within the transcription bubble. We show that the results of these studies are complementary to each other and are consistent with a model involving two types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer nascent transcript and a long-lived pause that occurs as a result of 1-2 nucleotide backtracking of a 7-mer transcript.

Bacterial RNA Polymerase-DNA Interaction-The Driving Force of Gene Expression and the Target for Drug Action.

DNA-dependent multisubunit RNA polymerase (RNAP) is the key enzyme of gene expression and a target of regulation in all kingdoms of life. It is a complex multifunctional molecular machine which, unlike other DNA-binding proteins, engages in extensive and dynamic interactions (both specific and nonspecific) with DNA, and maintains them over a distance. These interactions are controlled by DNA sequences, DNA topology, and a host of regulatory factors. Here, we summarize key recent structural and biochemical studies that elucidate the fine details of RNAP-DNA interactions during initiation. The findings of these studies help unravel the molecular mechanisms of promoter recognition and open complex formation, initiation of transcript synthesis and promoter escape. We also discuss most current advances in the studies of drugs that specifically target RNAP-DNA interactions during transcription initiation and elongation.

Clinical presentation, imaging findings, and prognosis of spinal dural arteriovenous fistula.

Spinal dural arteriovenous fistula (SDAVF) is a relatively common acquired vascular malformation of the spinal cord. Assessment of a SDAVF is often difficult because of non-specific findings on non-invasive imaging modalities. Diagnosis of a SDAVF is often delayed, and some patients receive unnecessary treatment and treatment delays, often resulting in a poor outcome. The aim of this study was to characterize the clinical presentation, typical imaging findings, and long-term outcome of SDAVF. Forty patients (13 women, 27 men; mean age 58.18 ± standard deviation 14.75 years) who were treated at our hospital from June 1992 to March 2014 were retrospectively reviewed. We investigated the baseline characteristics, clinical presentation, imaging findings, treatment modalities, and outcome of the patients. The most common clinical presentation was a sensory symptom (80%), followed by motor weakness (70%), and sphincter dysfunction (62.5%). Roughly one-third (32.5%) of patients had a stepwise progression of fluctuating weakness and sensory symptoms, but the most common presentation was chronic progressive myelopathic symptoms (47.5%). Thirty-four patients (85%) had T2 signal change on the spinal cord MRI, indicative of cord edema. Thirty-eight patients had typical perimedullary vessel flow voids on T2-weighted MRI. Twenty-eight patients were treated with endovascular embolization, five patients underwent surgery, and four patients underwent both. Clinical outcome was determined by severity of initial deficit (p=0.008), extent of cord edema (p=0.010), treatment failure (p=0.004), and a residual fistula (p=0.017). SDAVF causes a treatable myelopathy, so early diagnosis and intervention is essential.

Role of fatty acid composites in the toxicity of titanium dioxide nanoparticles used in cosmetic products.

It has been recognized that the use of nanoparticles (NPs) in the cosmetic industry results in products with better efficacy and functionality. However, recent advances in molecular toxicology have revealed that NP exposure can promote cytotoxicity and oxidative damage, which has raised health concerns in the use of NPs in personal care products. Nevertheless, the mechanistic basis for the toxicity and safety of cosmetic NPs is poorly understood. The goal of the study was to determine the cytotoxicity and intracellular distribution of titanium dioxide (TiO2) NPs containing fatty acid composites (palmitoleic acid, palmitic acid, stearic acid and oleic acid) commonly used in cosmetic products. Two types of cells, human fibroblast skin cells and adenocarcinoma lung cells, were exposed to either bare TiO2 NPs or TiO2 NPs mixed with fatty acids for up to 48 hr. NMR analysis confirmed that the fatty acid composites remained in the NPs after wash. The cytotoxicity of TiO2 NPs was determined by cell viability measurement using quantitative confocal microscopy, and the localization of two different forms of TiO2 NPs were assessed using electron spectroscopic imaging with transmission electron microscopy. TiO2 NPs containing fatty acids posed significantly reduced cytotoxicity (80-88% decreases) than bare NPs in both cell types. Furthermore, there was less intracellular penetration of the NPs containing fatty acid composites compared with bare NPs. These results provide important insights into the role of fatty acids in protecting the cells from possible toxicity caused by NPs used in the production of cosmetic products.

RNA polymerase structure and function at lac operon.

Transcription of E. coli lac operon by RNA polymerase (RNAP) is a classic example of how the basic functions of this enzyme, specifically the ability to recognize/bind promoters, melt the DNA and initiate RNA synthesis, is positively regulated by transcription activators, such as cyclic AMP-receptor protein, CRP, and negatively regulated by lac-repressor, LacI. In this review, we discuss the recent progress in structural and biochemical studies of RNAP and its binary and ternary complexes with CRP and lac promoter. With structural information now available for RNAP and models of binary and ternary elongation complexes, the interaction between these factors and RNAP can be modeled, and possible molecular mechanisms of their action can be inferred.

pH-dependent conformational switch activates the inhibitor of transcription elongation.

Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N-terminal coiled-coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C-terminal domain (CTD). We show that depending on pH, Gfh1-CTD exists in two alternative orientations. At pH above 7, it assumes an inactive 'flipped' orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1-CTD switches to an active 'Gre-like' orientation, which enables Gfh1 to bind to and inhibit RNAP.

Bacterial transcription elongation factors: new insights into molecular mechanism of action.

Like transcription initiation, the elongation and termination stages of transcription cycle serve as important targets for regulatory factors in prokaryotic cells. In this review, we discuss the recent progress in structural and biochemical studies of three evolutionarily conserved elongation factors, GreA, NusA and Mfd. These factors affect RNA polymerase (RNAP) processivity by modulating transcription pausing, arrest, termination or anti-termination. With structural information now available for RNAP and models of ternary elongation complexes, the interaction between these factors and RNAP can be modelled, and possible molecular mechanisms of their action can be inferred. The models suggest that these factors interact with RNAP at or near its three major, nucleic acid-binding channels: Mfd near the upstream opening of the primary (DNA-binding) channel, NusA in the vicinity of both the primary channel and the RNA exit channel, and GreA within the secondary (backtracked RNA-binding) channel, and support the view that these channels are involved in the maintenance of RNAP processivity.

Inhibition of bacterial RNA polymerase by streptolydigin: stabilization of a straight-bridge-helix active-center conformation.

We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to but not overlapping the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight-bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents.

Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase.

Prokaryotic transcription elongation factors GreA and GreB stimulate intrinsic nucleolytic activity of RNA polymerase (RNAP). The proposed biological role of Gre-induced RNA hydrolysis includes transcription proofreading, suppression of transcriptional pausing and arrest, and facilitation of RNAP transition from transcription initiation to transcription elongation. Using an array of biochemical and molecular genetic methods, we mapped the interaction interface between Gre and RNAP and identified the key residues in Gre responsible for induction of nucleolytic activity in RNAP. We propose a structural model in which the C-terminal globular domain of Gre binds near the opening of the RNAP secondary channel, the N-terminal coiled-coil domain (NTD) protrudes inside the RNAP channel, and the tip of the NTD is brought to the immediate vicinity of RNAP catalytic center. Two conserved acidic residues D41 and E44 located at the tip of the NTD assist RNAP by coordinating the Mg2+ ion and water molecule required for catalysis of RNA hydrolysis. If so, Gre would be the first transcription factor known to directly participate in the catalytic act of RNAP.

Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution.

In bacteria, the binding of a single protein, the initiation factor sigma, to a multi-subunit RNA polymerase core enzyme results in the formation of a holoenzyme, the active form of RNA polymerase essential for transcription initiation. Here we report the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6 A resolution. In the structure, two amino-terminal domains of the sigma subunit form a V-shaped structure near the opening of the upstream DNA-binding channel of the active site cleft. The carboxy-terminal domain of sigma is near the outlet of the RNA-exit channel, about 57 A from the N-terminal domains. The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N- and C-terminal domains. The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation.