β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome

Staphylococci have two mechanisms for resistance to β-lactam antibiotics. One is the production of β-lactamases, enzymes that hydrolytically destroy β-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by β-lactam antibiotics. Strains of S. aureus exhibiting either β-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism. Received 2 April 2005; received after revision 15 July 2005; accepted 25 July 2005     

Diversity of Cl− Channels

Cl⁻ channels are widely found anion pores that are regulated by a variety of signals and that play various roles. On the basis of molecular biologic findings, ligand-gated Cl⁻ channels in synapses, cystic fibrosis transmembrane conductors (CFTRs) and ClC channel types have been established, followed by bestrophin and possibly by tweety, which encode Ca²⁺-activated Cl⁻ channels. The ClC family has been shown to possess a variety of functions, including stabilization of membrane potential, excitation, cellvolume regulation, fluid transport, protein degradation in endosomal vesicles and possibly cell growth. The molecular structure of Cl⁻ channel types varies from 1 to 12 transmembrane segments. By means of computer-based prediction, functional Cl⁻ channels have been synthesized artificially, revealing that many possible ion pores are hidden in channel, transporter or unidentified hydrophobic membrane proteins. Thus, novel Cl⁻-conducting pores may be occasionally discovered, and evidence from molecular biologic studies will clarify their physiologic and pathophysiologic roles. Received 28 July 2005; received after revision 25 August 2005; accepted 21 September 2005     

Indole-3-carbinol enhances ultraviolet B-induced apoptosis by sensitizing human melanoma cells

Indole-3-carbinol (I3C) has been found to act against several types of cancer, while ultraviolet B (UVB) is known to induce the apoptosis of human melanoma cells. Here, we investigated whether I3C can sensitize G361 human melanoma cells to UVB-induced apoptosis. We examined the effects of combined I3C and UVB (I3C/UVB) at various dosages. I3C (200 μM)/UVB (50 mJ/cm²) synergistically reduced melanoma cell viability, whereas I3C (200 μM) or UVB (50 mJ/cm²), separately, had little effect on cell viability. DNA fragmentation assays indicated that I3C/UVB induced apoptosis. Further results show that I3C/UVB activates caspase-8, −3, and Bid and causes the cleavage of poly(ADP-ribose) polymerase. Moreover, I3C decreased the expression of the anti-apoptotic protein, Bcl-2, whereas UVB increased the translocation of Bax to mitochondria. Thus, an increased Bax/Bcl-2 ratio by I3C/UVB may result in melanoma apoptosis. In conclusion, our study demonstrated that I3C sensitizes human melanoma cells by down-regulating Bcl-2. Received 5 July 2006; received after revision 25 August 2006; accepted 11 September 2006     

TNF-α upregulates adenosine 2b (A2b) receptor expression and signaling in intestinal epithelial cells: a basis for A2bR overexpression in colitis

Adenosine is an endogenous signaling molecule upregulated during inflammatory conditions. Acting through the A2b receptor (A2bR), the predominant adenosine receptor in human colonic epithelia, adenosine has been directly implicated in immune and inflammatory responses in the intestine. Little is known about expression and regulation of A2bR during inflammation. Tumor necrosis factor alpha (TNF-α) is highly upregulated during chronic and acute inflammatory diseases. This study examined the expression of A2bR during colitis and studied effects of TNF-α on A2bR expression, signaling and function. Results demonstrated that A2bR expression increases during active colitis. TNF-α pretreatment of intestinal epithelial cells increased A2bR messenger RNA and protein expression. TNF-α significantly increased adenosine-induced membrane recruitment of A2bR and cyclic adenosine monophosphate downstream signaling. Further, TNF-α potentiated adenosine-induced shortcircuit current and fibronectin secretion. In conclusion, we demonstrated that TNF-α is an important regulator of A2bR, and during inflammation, upregulation of TNF-α may potentiate adenosine-mediated responses. Received 21 July 2005; received after revision 22 August 2005; accepted 19 September 2005 †These authors contributed equally to this work.     

Understanding the importance of selenium and selenoproteins in muscle function

Selenium is an essential trace element. In cattle, selenium deficiency causes dysfunction of various organs, including skeletal and cardiac muscles. In humans as well, lack of selenium is associated with many disorders, but despite accumulation of clinical reports, muscle diseases are not generally considered on the list. The goal of this review is to establish the connection between clinical observations and the most recent advances obtained in selenium biology. Recent results about a possible role of selenium-containing proteins in muscle formation and repair have been collected. Selenoprotein N is the first selenoprotein linked to genetic disorders consisting of different forms of congenital muscular dystrophies. Understanding the muscle disorders associated with selenium deficiency or selenoprotein N dysfunction is an essential step in defining the causes of the disease and obtaining a better comprehension of the mechanisms involved in muscle formation and maintenance. Received 13 July 2005; received after revision 9 September 2005; accepted 4 October 2005     

Polyphenolic phytochemicals – just antioxidants or much more?

Polyphenolic phytochemicals are ubiquitous in plants, in which they function in various protective roles. A ‘recommended’ human diet contains significant quantities of polyphenolics, as they have long been assumed to be ‘antioxidants’ that scavenge excessive, damaging, free radicals arising from normal metabolic processes. There is recent evidence that polyphenolics also have ‘indirect’ antioxidant effects through induction of endogenous protective enzymes. There is also increasing evidence for many potential benefits through polyphenolic-mediated regulation of cellular processes such as inflammation. Inductive or signalling effects may occur at concentrations much lower than required for effective radical scavenging. Over the last 2 – 3 years, there have been many exciting new developments in the elucidation of the in vivo mechanisms of the health benefits of polyphenolics. We summarise the current knowledge of the intake, bio-availability and metabolism of polyphenolics, their antioxidant effects, regulatory effects on signalling pathways, neuro-protective effects and regulatory effects on energy metabolism and gut health. Received 14 May 2007; received after revision 27 June 2007; accepted 24 July 2007     

Selenium and selenoproteins in mammals: extraordinary,essential, enigmatic

Selenium (Se), once known only for its potential toxicity, is now well established as an essential trace element for mammals. Insufficient Se intake predisposes to and manifests in a variety of diseases. Recent studies have proven that it is the synthesis of selenocysteine (Sec)-containing proteins, designated selenoproteins, which represents an essential prerequisite for regular development and a long and healthy life. New transgenic mouse models analysing those selenoproteins with proven enzymatic functions displayed particular phenotypes and highlighted essential Se-dependent processes in development, growth or against specific challenges. While there is a growing molecular understanding of and general agreement on the importance of sufficiently high Se intake and undisturbed selenoprotein biosynthesis, many of the recently identified selenoproteins are still uncharacterised, and the effects and consequences of supra-physiological Se dosages are not biochemically understood. With the recent definition of the human and mouse selenoproteomes and a growing number of available tools, the Se field is now geared for a great leap forward. Se biology has already broadened our knowledge about the genetic code and about protein translation. It now holds great promises also for a better understanding of some key aspects of cancer, inflammation, fertility and prevention of age-associated diseases.Received 17 March 2004; received after revision 29 April 2004; accepted 27 May 2004     

Chromogranin A: a novel susceptibility gene for essential hypertension

Chromogranin A (CHGA) is ubiquitously expressed in secretory cells of the endocrine, neuroendocrine, and neuronal tissues. Although this protein has long been known as a marker for neuroendocrine tumors, its role in cardiovascular disease states including essential hypertension (EH) has only recently been recognized. It acts as a prohormone giving rise to bioactive peptides such as vasostatin-I (human CHGA_1–76) and catestatin (human CHGA_352–372) that exhibit several cardiovascular regulatory functions. CHGA is over-expressed but catestatin is diminished in EH. Moreover, genetic variants in the promoter, catestatin, and 3′-untranslated regions of the human CHGA gene alter autonomic activity and blood pressure. Consistent with these findings, targeted ablation of this gene causes severe arterial hypertension and ventricular hypertrophy in mice. Transgenic expression of the human CHGA gene or exogenous administration of catestatin restores blood pressure in these mice. Thus, the accumulated evidence establishes CHGA as a novel susceptibility gene for EH.     

Selenoproteins and oxidative stress-induced inflammatory tumorigenesis in the gut

Selenium is an essential micronutrient that is incorporated into at least 25 selenoproteins encoded by the human genome, many of which serve antioxidant functions. Because patients with inflammatory bowel disease (IBD) demonstrate nutritional deficiencies and are at increased risk for colon cancer due to heightened inflammation and oxidative stress, selenoprotein dysfunction may contribute to disease progression. Over the years, numerous studies have analyzed the effects of selenoprotein loss and shown that they are important mediators of intestinal inflammation and carcinogenesis. In particular, recent work has focused on the role of selenoprotein P (SEPP1), a major selenium transport protein which also has endogenous antioxidant function. These experiments determined SEPP1 loss altered immune and epithelial cellular function in a murine model of colitis-associated carcinoma. Here, we discuss the current knowledge of SEPP1 and selenoprotein function in the setting of IBD, colitis, and inflammatory tumorigenesis.     

The human selenoproteome: recent insights into functions and regulation

Selenium (Se) is a nutritional trace mineral essential for various aspects of human health that exerts its effects mainly through its incorporation into selenoproteins as the amino acid, selenocysteine. Twenty-five selenoprotein genes have been identified in humans and several selenoproteins are broadly classified as antioxidant enzymes. As progress is made on characterizing the individual members of this protein family, however, it is becoming clear that their properties and functions are quite diverse. This review summarizes recent insights into properties of individual selenoproteins such as tissue distribution, subcellular localization, and regulation of expression. Also discussed are potential roles the different selenoproteins play in human health and disease.     

Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27–31, EGSLQ), but not the des (27–31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca²⁺]_i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca²⁺]_i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca²⁺]_i response, and those with Ala at positions 2–5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca²⁺]_i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na⁺,K⁺ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca²⁺]_i via G-protein-coupled pathways, giving downstream enzyme effects. Received 13 May 2002; accepted 16 May 2002     

Inhibition of hepatitis C virus internal ribosome entry site-mediated translation by an RNA targeting the conserved IIIf domain

Hepatitis C virus (HCV) translation initiation depends on an internal ribosome entry site (IRES). We previously identified an RNA molecule (HH363–10) able to bind and cleave the HCV IRES region. This paper characterizes its capacity to interfere with IRES function. Inhibition assays showed that it blocks IRES activity both in vitro and in a human hepatoma cell line. Although nucleotides involved in binding and cleavage reside in separate regions of the inhibitor HH363–10, further analysis demonstrated the strongest effect to be an intrinsic feature of the entire molecule; the abolishment of either of the two activities resulted in a reduction in its function. Probing assays demonstrate that HH363–10 specifically interacts with the conserved IIIf domain of the pseudoknot structure in the IRES, leading to the inhibition of the formation of translationally competent 80S particles. The combination of two inhibitory activities targeting different sequences in a chimeric molecule may be a good strategy to avoid the emergence of resistant viral variants. Received 26 July 2007; received after revision 24 September 2007; accepted 26 September 2007     

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions

Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27 – 30 detectable by the 3F4 antibody against human PrP109 – 112. We recently identified a new PK-resistant PrP species, designated PrP^*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97 – 108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP^*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP^*20 but also a small amount of 3F4-detected PK-resistant PrP27 – 30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP^*20 to the occurrence of the 3F4-detected PrP27 – 30 was observed. Our study suggests that an increase in the level of PrP^*20 characterizes the early stages of prion diseases. Received 17 October 2007; received after revision 5 December 2007; accepted 14 December 2007     

Neue mehrfache Untersuchungen am gleichen Kammerwasser menschlicher Augen

Summary The normal human aequous has a volume of 0.12–0.20 cm³ and its protein content is from 10 to 20 mg%. We ascertained the specific weight by the falling drop method in a mixture of xylol and brombenzene. In order to estimate the protein content, we placed a series of drops from the human aequous on filter paper with the Agla pipette; they were then tinted with amido-black and the optical density of the eluted spots was read in the Beckman spectrophotometer. The remnants of 2–3 specimens of human aequous were then brought together and concentrated there until the protein concentration is just sufficient to allow a separation into the various fractions by paper-electrophoresis.      

The structure of human interferon-β: implications for activity

Interferons (IFNs) are potent extracellular protein mediators of host defence and homoeostasis. This article reviews the structure of human IFN-β (HuIFN-β), in particular in relation to its activity. The recently determined crystal structure of HuIFN-β provides a framework for understanding of the mechanism of differentiation of type I IFNs by their common receptor. Insights are generated by comparison with the structures of other type I IFNs and from the interpretation of existing mutagenesis data. The details of the observed carbohydrate structure, together with biochemical data, implicate the glycosylation of HuIFN-β, which is uncommon among type I IFNs, as an important factor in the solubility, stability and, consequently, activity of the protein. Finally, these structural implications are discussed in the context of the clinical use of HuIFN-β. Received 12 June 1998; received after revision 16 July 1998; accepted 16 July 1998     

Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway. Received 28 May 1998; received after revision 6 July 1998; accepted 6 July 1998     

In pre-sterol carrier protein 2 (SCP2) in solution the leader peptide 1 – 20 is flexibly disordered, and residues 21 – 143 adopt the same globular fold as in mature SCP2

The preform of the rabbit sterol carrier protein 2 (pre-rSCP2) was cloned, the uniformly ¹⁵N-labelled protein expressed in Escherichia coli and studied by three-dimensional ¹⁵N-resolved nuclear magnetic resonance spectroscopy. In spite of its low solubility in aqueous solution of only ∼0.3 mM, sequential ¹⁵N and ¹H backbone resonance assignments were obtained for 105 out of the 143 residues. From comparison of the sequential and medium-range nuclear Overhauser effects (NOEs) in the two proteins, all regular secondary structures previously determined in mature human SCP2 (hSCP2) [Szyperski et al. (1993) FEBS Lett. 335: 18–26] were also identified in pre-rSCP2. Near-identity of the backbone ¹⁵N and ¹H chemical shifts and 1 : 1 correspondence of 24 long-range NOEs to backbone amide groups in the two proteins show that the residues 21 – 143 adopt the same globular fold in pre-rSCP2 and mature hSCP2. The N-terminal 20-residue leader peptide of pre-rSCP2 is flexibly disordered in solution and does not observably affect the conformation of the polypeptide segment 21 – 143. Received 11 May 1998; accepted 15 May 1998     

Overexpression of HAb18G/CD147 promotes invasion and metastasis via α3β1 integrin mediated FAK-paxillin and FAK-PI3K-Ca2+ pathways

Mechanism of HAb18G/CD147 underlying the metastasis process of human hepatoma cells has not been determined. In the present study, we found that integrin α3β1 colocalizes with HAb18G/CD147 in human 7721 hepatoma cells. The enhancing effect of HAb18G/CD147 on adhesion, invasion capacities and matrix metalloproteinases (MMPs) secretion was decreased by integrin α3β1 antibodies (p<0.01). The expressions of integrin downstream molecules including focal adhesion kinase (FAK), phospho-FAK (p-FAK), paxillin, and phospho-paxillin (p-paxillin) were increased in human hepatoma cells overexpressing HAb18G/CD147. Deletion of HAb18G/CD147 reduces the quantity of focal adhesions and rearranges cytoskeleton. Wortmannin and LY294002, specific phosphatidylinositol kinase (PI3K) inhibitors, reversed the effect of HAb18G/CD147 on the regulation of intracellular Ca²⁺ mobilization, significantly reducing cell adhesion, invasion and MMPs secretion potential (p<0.01). Together, these results suggest that HAb18G/CD147 enhances the invasion and metastatic potentials of human hepatoma cells via integrin α3β1-mediated FAK-paxillin and FAKPI3K-Ca²⁺ signal pathways. Received 5 June 2008; received after revision 16 July 2008; accepted 23 July 2008     

Introduction: the selenium conundrum

Selenium was first suspected of being an essential dietary trace element in the 1950s. We now know that indeed it is an essential biological element that serves as an integral component of several enzymes, including those in the families of deiodinases and glutathione peroxidases as well as selenoproteins P and W. The multi-author review that follows this introduction concentrates on the important biological role of selenium in enzymes as well as some of the physiological aspects of selenium as either a potential anticarcinogenic agent or insulin mimetic. What should become clear from these contributed articles is the complex and dynamic role that selenium plays in many biological processes and that the investigations in these areas are at the edge of exciting new frontiers.