Biomolecules, Volume 5, Issue 2 (June 2015) – 45 articles , Pages 282-1194

Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac⁺ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac⁺ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac⁺ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes. Full article

Oxidative stress is now a well-researched area with thousands of new articles appearing every year. We want to give the reader here an overview of the topics in biomedical and basic oxidative stress research which are covered by the authors of this thematic issue. We also want to give the newcomer a short introduction into some of the basic concepts, definitions and analytical procedures used in this field. Full article

Post-transcriptional processing, involving cleavage of precursor messenger RNA (pre mRNA), and further incorporation of poly(A) tail to the 3' end is a key step in the expression of genetic information. Alternative polyadenylation (APA) serves as an important check point for the regulation of gene expression. Recent studies have shown widespread prevalence of APA in diverse systems. A considerable amount of research has been done in characterizing different subunits of so-called Cleavage and Polyadenylation Specificity Factor (CPSF). In plants, CPSF30, an ortholog of the 30 kD subunit of mammalian CPSF is a key polyadenylation factor. CPSF30 in the model plant Arabidopsis thaliana was reported to possess unique biochemical properties. It was also demonstrated that poly(A) site choice in a vast majority of genes in Arabidopsis are CPSF30 dependent, suggesting a pivotal role of this gene in APA and subsequent regulation of gene expression. There are also indications of this gene being involved in oxidative stress and defense responses and in cellular signaling, suggesting a role of CPSF30 in connecting physiological processes and APA. This review will summarize the biochemical features of CPSF30, its role in regulating APA, and possible links with cellular signaling and stress response modules. Full article

Metabolic disturbances in white adipose tissue in obese individuals contribute to the pathogenesis of insulin resistance and the development of type 2 diabetes mellitus. Impaired insulin action in adipocytes is associated with elevated lipolysis and increased free fatty acids leading to ectopic fat deposition in liver and skeletal muscle. Chronic adipose tissue hypoxia has been suggested to be part of pathomechanisms causing dysfunction of adipocytes. Hypoxia can provoke oxidative stress in human and animal adipocytes and reduce the production of beneficial adipokines, such as adiponectin. However, time-dose responses to hypoxia relativize the effects of hypoxic stress. Long-term exposure of fat cells to hypoxia can lead to the production of beneficial substances such as leptin. Knowledge of time-dose responses of hypoxia on white adipose tissue and the time course of generation of oxidative stress in adipocytes is still scarce. This paper reviews the potential links between adipose tissue hypoxia, oxidative stress, mitochondrial dysfunction, and low-grade inflammation caused by adipocyte hypertrophy, macrophage infiltration and production of inflammatory mediators. Full article

Parkinson’s disease (PD) is a very common neurodegenerative disorder characterized by the accumulation of α-synuclein (α-syn) into Lewy body (LB) inclusions and the loss of neuronmelanin (NM) containing dopamine (DA) neurons in the substantia nigra (SN). Pathological α-syn and NM are two prominent hallmarks in this selective and progressive neurodegenerative disease. Pathological α-syn can induce dopaminergic neuron death by various mechanisms, such as inducing oxidative stress and inhibiting protein degradation systems. Therefore, to explore the factors that trigger α-syn to convert from a non-toxic protein to toxic one is a pivotal question to clarify the mechanisms of PD pathogenesis. Many triggers for pathological α-syn aggregation have been identified, including missense mutations in the α-syn gene, higher concentration, and posttranslational modifications of α-Syn. Recently, the role of NM in inducing α-syn expression and aggregation has been suggested as a mechanism for this pigment to modulate neuronal vulnerability in PD. NM may be responsible for PD and age-associated increase and aggregation in α-syn. Here, we reviewed our previous study and other recent findings in the area of interaction between NM and α-syn. Full article

Alcohol is a simple and consumable biomolecule yet its excessive consumption disturbs numerous biological pathways damaging nearly all organs of the human body. One of the essential biological processes affected by the harmful effects of alcohol is proteostasis, which regulates the balance between biogenesis and turnover of proteins within and outside the cell. A significant amount of published evidence indicates that alcohol and its metabolites directly or indirectly interfere with protein homeostasis in the endoplasmic reticulum (ER) causing an accumulation of unfolded or misfolded proteins, which triggers the unfolded protein response (UPR) leading to either restoration of homeostasis or cell death, inflammation and other pathologies under severe and chronic alcohol conditions. The UPR senses the abnormal protein accumulation and activates transcription factors that regulate nuclear transcription of genes related to ER function. Similarly, this kind of protein stress response can occur in other cellular organelles, which is an evolving field of interest. Here, I review recent advances in the alcohol-induced ER stress response as well as discuss new concepts on alcohol-induced mitochondrial, Golgi and lysosomal stress responses and injuries. Full article

Haskap (Lonicera caerulea L.) berries have long been used for their health promoting properties against chronic conditions. The current study investigated the effect of Canadian haskap berry extracts on pro-inflammatory cytokines using a human monocytic cell line THP-1 derived macrophages stimulated by lipopolysaccharide. Methanol extracts of haskap from different growing locations in Canada were prepared and characterized for their total phenolic profile using colorimetric assays and liquid chromatography—Mass spectrometry (UPLC-MS/MS). Human THP-1 monocytes were seeded in 24-well plates (5 × 10⁵/well) and treated with phorbol 12-myristate 13-acetate (PMA, 0.1 μg/mL) for 48 h to induce macrophage differentiation. After 48 h, the differentiated macrophages were washed with Hank’s buffer and treated with various concentrations of test compounds for 4 h, followed by the lipopolysaccharide (LPS)-stimulation (18 h). Borealis cultivar showed the highest phenolic content, flavonoid content and anthocyanin content (p < 0.05). A negative correlation existed between the polyphenol concentration of the extracts and pro-inflammatory cytokines: Interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), prostaglandin (PGE₂), and cyclooxygenase-2 (COX-2) enzyme. Borealis exhibited comparable anti-inflammatory effects to COX inhibitory drug, diclofenac. The results showed that haskap berry polyphenols has the potential to act as an effective inflammation inhibitor. Full article

This article will review our current understanding of transcription elongation and termination in E. coli. We discuss why transcription elongation complexes pause at certain template sites and how auxiliary host and phage transcription factors affect elongation and termination. The connection between translation and transcription elongation is described. Finally we present an overview indicating where progress has been made and where it has not. Full article

Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the −10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis. Full article

Enhanced expression of chemotactic cytokines (aka chemokines) within pancreatic islets likely contributes to islet inflammation by regulating the recruitment and activation of various leukocyte populations, including macrophages, neutrophils, and T-lymphocytes. Because of the powerful actions of these chemokines, precise transcriptional control is required. In this review, we highlight what is known about the signals and mechanisms that govern the transcription of genes encoding specific chemokine proteins in pancreatic islet β-cells, which include contributions from the NF-κB and STAT1 pathways. We further discuss increased chemokine expression in pancreatic islets during autoimmune-mediated and obesity-related development of diabetes. Full article

Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression. We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed. Full article

Axonal swellings are histological hallmarks of axonopathies in various types of disorders in the central nervous system, including neurodegenerative diseases. Given the pivotal role of axonopathies during the early phase of neurodegenerative process, axonal swellings may be good models which may provide some clues for early pathogenesis of α-synucleinopathies, including Parkinson’s disease and dementia with Lewy bodies (DLB). In this mini-review, such a possibility is discussed based on our recent studies as well as other accumulating studies. Consistent with the current view that dysfunction in the autophagy-lysosomal system may play a major role in the formation of axonal swellings, our studies showed globule, small axonal swellings, derived from transgenic mice expressing either human wild-type α-synuclein (αS-globule) or DLB-linked P123H β-synuclein (βS-globule), contained autophagosome-like membranes. However, other pathological features, such as abnormal mitochondria, enhanced oxidative stress and LRRK2 accumulation, were observed in the αS-globules, but not in the βS-globules. Collectively, it is predicted that αS and βS may be involved in axonopathies through similar but distinct mechanisms, and thus, contribute to diverse axonal pathologies. Further studies of the axonal swellings may lead to elucidating the pathogenic mechanism of early α-synucleinopathies and illuminating a strategy for a disease-modifying therapy against these devastating disorders. Full article

The peptidyl-prolyl cis-trans isomerases (PPIases) that include immunophilins (cyclophilins and FKBPs) and parvulins (Pin1, Par14, Par17) participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo. Full article

The 70-kDa heat shock protein (Hsp70), one of the major stress-inducible molecular chaperones, is localized not only in the cytosol, but also in extracellular milieu in mammals. Hsp70 interacts with various cell surface glycolipids including sulfatide (3'-sulfogalactosphingolipid). However, the molecular mechanism, as well as the biological relevance, underlying the glycolipid-Hsp70 interaction is unknown. Here we report that sulfatide promotes Hsp70 oligomerization through the N-terminal ATPase domain, which stabilizes the binding of Hsp70 to unfolded protein in vitro. We find that the Hsp70 oligomer has apparent molecular masses ranging from 440 kDa to greater than 669 kDa. The C-terminal peptide-binding domain is dispensable for the sulfatide-induced oligomer formation. The oligomer formation is impaired in the presence of ATP, while the Hsp70 oligomer, once formed, is unable to bind to ATP. These results suggest that sulfatide locks Hsp70 in a high-affinity state to unfolded proteins by clustering the peptide-binding domain and blocking the binding to ATP that induces the dissociation of Hsp70 from protein substrates. Full article

The high mobility group protein A1 (HMGA1) is a master regulator of chromatin structure mediating its major gene regulatory activity by direct interactions with A/T-rich DNA sequences located in the promoter and enhancer regions of a large variety of genes. HMGA1 DNA-binding through three AT-hook motifs results in an open chromatin structure and subsequently leads to changes in gene expression. Apart from its significant expression during development, HMGA1 is over-expressed in virtually every cancer, where HMGA1 expression levels correlate with tumor malignancy. The exogenous overexpression of HMGA1 can lead to malignant cell transformation, assigning the protein a key role during cancerogenesis. Recent studies have unveiled highly specific competitive interactions of HMGA1 with cellular and viral RNAs also through an AT-hook domain of the protein, significantly impacting the HMGA1-dependent gene expression. In this review, we discuss the structure and function of HMGA1-RNA complexes during transcription and epigenomic regulation and their implications in HMGA1-related diseases. Full article

Plants of the Lauraceae family are widely used in traditional medicine and are sources of various classes of secondary metabolites. Two genera of this family, Beilschmiedia and Endiandra, have been the subject of numerous investigations over the past decades because of their application in traditional medicine. They are the only source of bioactive endiandric acid derivatives. Noteworthy is that their biosynthesis contains two consecutive non-enzymatic electrocyclic reactions. Several interesting biological activities for this specific class of secondary metabolites and other constituents of the two genera have been reported, including antimicrobial, enzymes inhibitory and cytotoxic properties. This review compiles information on the structures of the compounds described between January 1960 and March 2015, their biological activities and information on endiandric acid biosynthesis, with 104 references being cited. Full article

Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions. Full article

Mutations and duplication/triplication of the alpha-synuclein (αSyn)-coding gene have been found to cause familial Parkinson’s disease (PD), while genetic polymorphisms in the region controlling the expression level and stability of αSyn have been identified as risk factors for idiopathic PD, pointing to the importance of wild-type (wt) αSyn dosage in the disease. Evidence that αSyn is present in the cerebrospinal fluid and interstitial brain tissue and that healthy neuronal grafts transplanted into PD patients often degenerate suggests that extracellularly-released αSyn plays a role in triggering the neurodegenerative process. αSyn’s role in neurotransmission has been shown in various cell culture models in which the protein was upregulated or deleted and in knock out and transgenic animal, with different results on αSyn’s effect on synaptic vesicle pool size and mobilization, αSyn being proposed as a negative or positive regulator of neurotransmitter release. In this review, we discuss the effect of αSyn on pre- and post-synaptic compartments in terms of synaptic vesicle trafficking, calcium entry and channel activity, and we focus on the process of exocytosis and internalization of αSyn and on the spreading of αSyn-driven effects due to the presence of the protein in the extracellular milieu. Full article

Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477–42485), an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank), describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription. Full article

Iron and oxygen share a delicate partnership since both are indispensable for survival, but if the partnership becomes inadequate, this may rapidly terminate life. Virtually all cell components are directly or indirectly affected by cellular iron metabolism, which represents a complex, redox-based machinery that is controlled by, and essential to, metabolic requirements. Under conditions of increased oxidative stress—i.e., enhanced formation of reactive oxygen species (ROS)—however, this machinery may turn into a potential threat, the continued requirement for iron promoting adverse reactions such as the iron/H₂O₂-based formation of hydroxyl radicals, which exacerbate the initial pro-oxidant condition. This review will discuss the multifaceted homeodynamics of cellular iron management under normal conditions as well as in the context of oxidative stress. Full article

This study investigates the regulation of hepcidin, the key iron-regulatory molecule, by alcohol and hydrogen peroxide (H₂O₂) in glutathione peroxidase-1 (gpx-1^−/−) and catalase (catalase^−/−) knockout mice. For alcohol studies, 10% ethanol was administered in the drinking water for 7 days. Gpx-1^−/− displayed significantly higher hepatic H₂O₂ levels than catalase^−/− compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The basal level of liver hepcidin expression was attenuated in gpx-1^−/− mice. Alcohol increased H₂O₂ production in catalase^−/− and wild-type, but not gpx-1^−/−, mice. Hepcidin expression was inhibited in alcohol-fed catalase^−/− and wild-type mice. In contrast, alcohol elevated hepcidin expression in gpx-1^−/− mice. Gpx-1^−/− mice also displayed higher level of basal liver CHOP protein expression than catalase^−/− mice. Alcohol induced CHOP and to a lesser extent GRP78/BiP expression, but not XBP1 splicing or binding of CREBH to hepcidin gene promoter, in gpx-1^−/− mice. The up-regulation of hepatic ATF4 mRNA levels, which was observed in gpx-1^−/− mice, was attenuated by alcohol. In conclusion, our findings strongly suggest that H₂O₂ inhibits hepcidin expression in vivo. Synergistic induction of CHOP by alcohol and H₂O_2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH. Full article

Adenosine, an immunomodulatory biomolecule, is produced by the ecto-enzymes CD39 (nucleoside triphosphate dephosphorylase) and CD73 (ecto-5'-nucleotidase) by dephosphorylation of extracellular ATP. CD73 is expressed by many cell types during injury, infection and during steady-state conditions. Besides host cells, many bacteria also have CD39-CD73-like machinery, which helps the pathogen subvert the host inflammatory response. The major function for adenosine is anti-inflammatory, and most recent research has focused on adenosine’s control of inflammatory mechanisms underlying various autoimmune diseases (e.g., colitis, arthritis). Although adenosine generated through CD73 provides a feedback to control tissue damage mediated by a host immune response, it can also contribute to immunosuppression. Thus, inflammation can be a double-edged sword: it may harm the host but eventually helps by killing the invading pathogen. The role of adenosine in dampening inflammation has been an area of active research, but the relevance of the CD39/CD73-axis and adenosine receptor signaling in host defense against infection has received less attention. Here, we review our recent knowledge regarding CD73 expression during murine Salmonellosis and Helicobacter-induced gastric infection and its role in disease pathogenesis and bacterial persistence. We also explored a possible role for the CD73/adenosine pathway in regulating innate host defense function during infection. Full article

The human papillomavirus (HPV) life cycle is strictly linked to the differentiation program of the infected mucosal epithelial cell. In the basal and lower levels of the epithelium, early genes coding for pro-mitotic proteins and viral replication factors are expressed, while terminal cell differentiation is required for activation of late gene expression and production of viral particles at the very top of the epithelium. Such productive infections are normally cleared within 18–24 months. In rare cases, the HPV infection is stuck in the early stage of the infection. Such infections may give rise to cervical lesions that can progress to cancer, primarily cancer of the uterine cervix. Since cancer progression is strictly linked to HPV gene expression, it is of interest to understand how HPV gene expression is regulated. Cis-acting HPV RNA elements and cellular RNA-binding proteins control HPV mRNA splicing and polyadenylation. These interactions are believed to play a particularly important role in the switch from early to late gene expression, thereby contributing to the pathogenesis of HPV. Indeed, it has been shown that the levels of various RNA binding proteins change in response to differentiation and in response to HPV induced cervical lesions and cancer. Here we have compiled published data on RNA binding proteins involved in the regulation of HPV gene expression. Full article

Insoluble and fibrillar forms of α-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. α-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. α-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for α-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins’s role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from α-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of α-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies. Full article

Many neurodegenerative diseases, including Huntington’s, Alzheimer’s and Parkinson’s diseases, are characterized by protein misfolding and aggregation. The capability of trehalose to interfere with protein misfolding and aggregation has been recently evaluated by several research groups. In the present work, we studied, by means of synchrotron radiation circular dichroism (SRCD) spectroscopy, the dose-effect of trehalose on α-synuclein conformation and/or stability to probe the capability of this osmolyte to interfere with α-synuclein’s aggregation. Our study indicated that a low trehalose concentration stabilized α-synuclein folding much better than at high concentration by blocking in vitro α-synuclein’s polymerisation. These results suggested that trehalose could be associated with other drugs leading to a new approach for treating Parkinson’s and other brain-related diseases. Full article

Extracellular traps (ETs) are reticulate structures of extracellular DNA associated with antimicrobial molecules. Their formation by phagocytes (mainly by neutrophils: NETs) has been identified as an essential element of vertebrate innate immune defense. However, as ETs are also toxic to host cells and potent triggers of autoimmunity, their role between pathogen defense and human pathogenesis is ambiguous, and they contribute to a variety of acute and chronic inflammatory diseases. Since the discovery of ET formation (ETosis) a decade ago, evidence has accumulated that most reaction cascades leading to ET release involve ROS. An important new facet was added when it became apparent that ETosis might be directly linked to, or be a variant of, the autophagy cell death pathway. The present review analyzes the evidence to date on the interplay between ROS, autophagy and ETosis, and highlights and discusses several further aspects of the ROS-ET relationship that are incompletely understood. These aspects include the role of NADPH oxidase-derived ROS, the molecular requirements of NADPH oxidase-dependent ETosis, the roles of NADPH oxidase subtypes, extracellular ROS and of ROS from sources other than NADPH oxidase, and the present evidence for ROS-independent ETosis. We conclude that ROS interact with ETosis in a multidimensional manner, with influence on whether ETosis shows beneficial or detrimental effects. Full article

Heme oxygenase (HO), in conjunction with biliverdin reductase, degrades heme to carbon monoxide, ferrous iron and bilirubin (BR); the latter is a potent antioxidant. The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress. The mechanisms by which HO mediates the described effects are not completely clear. However, the degradation of heme, a strong pro-oxidant, and the generation of BR are considered to play key roles. The aim of this study was to determine the effects of BR on vital functions of hepatocytes focusing on mitochondria and the endoplasmic reticulum (ER). The affinity of BR to proteins is a known challenge for its exact quantification. We consider two major consequences of this affinity, namely possible analytical errors in the determination of HO activity, and biological effects of BR due to direct interaction with protein function. In order to overcome analytical bias we applied a polynomial correction accounting for the loss of BR due to its adsorption to proteins. To identify potential intracellular targets of BR we used an in vitro approach involving hepatocytes and isolated mitochondria. After verification that the hepatocytes possess HO activity at a similar level as liver tissue by using our improved post-extraction spectroscopic assay, we elucidated the effects of increased HO activity and the formed BR on mitochondrial function and the ER stress response. Our data show that BR may compromise cellular metabolism and proliferation via induction of ER stress. ER and mitochondria respond differently to elevated levels of BR and HO-activity. Mitochondria are susceptible to hemin, but active HO protects them against hemin-induced toxicity. BR at slightly elevated levels induces a stress response at the ER, resulting in a decreased proliferative and metabolic activity of hepatocytes. However, the proteins that are targeted by BR still have to be identified. Full article

In the process of transcription initiation, the bacterial RNA polymerase binds double-stranded (ds) promoter DNA and subsequently effects strand separation of 12 to 14 base pairs (bp), including the start site of transcription, to form the so-called “open complex” (also referred to as RP_o). This complex is competent to initiate RNA synthesis. Here we will review the role of σ⁷⁰ and its homologs in the strand separation process, and evidence that strand separation is initiated at the −11A (the A of the non-template strand that is 11 bp upstream from the transcription start site) of the promoter. By using the fluorescent adenine analog, 2-aminopurine, it was demonstrated that the −11A on the non-template strand flips out of the DNA helix and into a hydrophobic pocket where it stacks with tyrosine 430 of σ⁷⁰. Open complexes are remarkably stable, even though in vivo, and under most experimental conditions in vitro, dsDNA is much more stable than its strand-separated form. Subsequent structural studies of other researchers have confirmed that in the open complex the −11A has flipped into a hydrophobic pocket of σ⁷⁰. It was also revealed that RP_o was stabilized by three additional bases of the non-template strand being flipped out of the helix and into hydrophobic pockets, further preventing re-annealing of the two complementary DNA strands. Full article

Bacteriophage N4 regulates the temporal expression of its genome through the activity of three distinct RNA polymerases (RNAP). Expression of the early genes is carried out by a phage-encoded, virion-encapsidated RNAP (vRNAP) that is injected into the host at the onset of infection and transcribes the early genes. These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs. Both N4 RNAPs belong to the T7-like “single-subunit” family of polymerases. Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle. Full article

N-acetyl-l-histidine (NAH) is a prominent biomolecule in brain, retina and lens of poikilothermic vertebrates. In fish lens, NAH exhibits an unusual compartmentalized metabolism. It is synthesized from L-histidine (His) and acetyl Co-enzyme A. However, NAH cannot be catabolized by lens cells. For its hydrolysis, NAH is exported to ocular fluid where a specific acylase cleaves His which is then actively taken up by lens and re-synthesized into NAH. This energy-dependent cycling suggested a pump mechanism operating at the lens/ocular fluid interface. Additional studies led to the hypothesis that NAH functioned as a molecular water pump (MWP) to maintain a highly dehydrated lens and avoid cataract formation. In this process, each NAH molecule released to ocular fluid down its gradient carries with it 33 molecules of bound water, effectively transporting the water against a water gradient. In ocular fluid the bound water is released for removal from the eye by the action of NAH acylase. In this paper, we demonstrate for the first time the identification of NAH in fish brain using proton magnetic resonance spectroscopy (MRS) and describe recent evidence supporting the NAH MWP hypothesis. Using MRS, we also document a phylogenetic transition in brain metabolism between poikilothermic and homeothermic vertebrates. Full article