The origin,diversity, and structure function relationships of insect luciferases

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. Whereas the oxidative mechanism which leads to light emission is similar for most luciferases, these enzymes and their substrates are evolutionarily unrelated. Among all bioluminescent groups, insects constitute one of the most diverse in terms of biochemistry. In the fungus-gnats (Mycetophilidae: Diptera), for example, bioluminescence is generated by two biochemically distinct systems. Despite the diversity, investigations on insect luciferases and biochemistry have been conducted mostly with fireflies. The luciferases from the related phengodid beetles, which can produce green to red bioluminescence using the same chemistry as firefly luciferases, have been recently investigated. Beetle luciferases originated from ancestral acyl-CoA ligases. Present data suggest that conserved motifs among this class of ligases are involved in substrate adenylation. The three-dimensional structure of firefly luciferase was recently solved and mutagenesis studies have been performed identifying putative residues involved in luciferin binding and bioluminescence color determination in several beetle luciferases. The knowledge gained through these studies is helping in the development of useful reporter gene tools for biotechnological and biomedical purposes. Received 4 March 2002; received after revision 13 May 2002; accepted 21 May 2002     

Split Bregman迭代算法生物发光断层成像

生物发光断层成像重建中,发光光源在生物体内稀疏分布,基于压缩感知思想,将?1范数正则化的稀疏重建应用于生物发光断层成像,并采用Split Bregman迭代算法求解?1范数目标函数,以获得快速、稳定的重建.三维数字鼠模型数值实验结果表明,该算法应用于生物发光断层成像重建,在没有使用任何光源可行区域先验和多光谱测量信息的条件下,仍能获得准确的定位和定量重建结果,算法对噪声具有较好的鲁棒性.     

Imaging of oxygen and hypoxia in cell and tissue samples

Molecular oxygen (O₂) is a key player in cell mitochondrial function, redox balance and oxidative stress, normal tissue function and many common disease states. Various chemical, physical and biological methods have been proposed for measurement, real-time monitoring and imaging of O₂ concentration, state of decreased O₂ (hypoxia) and related parameters in cells and tissue. Here, we review the established and emerging optical microscopy techniques allowing to visualize O₂ levels in cells and tissue samples, mostly under in vitro and ex vivo, but also under in vivo settings. Particular examples include fluorescent hypoxia stains, fluorescent protein reporter systems, phosphorescent probes and nanosensors of different types. These techniques allow high-resolution mapping of O₂ gradients in live or post-mortem tissue, in 2D or 3D, qualitatively or quantitatively. They enable control and monitoring of oxygenation conditions and their correlation with other biomarkers of cell and tissue function. Comparison of these techniques and corresponding imaging setups, their analytical capabilities and typical applications are given.     

Super-high sensitivity systems for detection and spectral analysis of ultraweak photon emission from biological cells and tissues

In this paper we summarize and discuss the modern technology and systems, studied and established by our research group, for performing the detection and special analysis incorporated with the super-high sensitivity photon counting method for the study of ultraweak photon emission; for example, extra-weak bioluminescence and chemiluminescence from living cells and tissues, closely related to biochemical and biophysical processes and activities. An excellent sensitivity of the basic photon counting system, making it possible to achieve count rates in the very low range of one photoelectron per second to one per minute, allowed us to carry out in vivo as well as in vitro measurements, and analyses of ultraweak bioluminescence and chemiluminescence. Recent results concerning ultraweak photon emission from blood samples and organ homogenates of rats are presented and reviewed as one of the interesting and valuable applications of our modern technology for studying ultraweak cell and tissue radiation.     

Super-high sensitivity systems for detection and spectral analysis of ultraweak photon emission from biological cell cells and tissues

Summary In this paper we summarize and discuss the modern technology and systems, studied and established by our research group, for performing the detection and special analysis incorporated with the super-high senstivity photon counting method for the study of ultraweak photon emission; for example, extra-weak bioluminescence and chemiluminescence from living cells and tissues, closely related to biochemical and biophysical processes and activities. An excellent sensitivity of the basic photon counting system, making it possible to achieve count rates in the very low range of one photoelectron per second to one per minute, allowed us to carry out in vivo as well as in vitro measurements, and analyses of ultraweak bioluminescence and chemiluminescence. Recent results concerning ultraweak photon emission from blood samples and organ homogenates of rats are presented and reviewed as one of the interesting and valuable applications of our modern technology for studying ultraweak cell and tissue radiation.     

Synphilin-1 inhibits alpha-synuclein degradation by the proteasome

Intracellular deposits of aggregated alpha-synuclein are a hallmark of Parkinson’s disease. Protein–protein interactions are critical in the regulation of cell proteostasis. Synphilin-1 interacts both in vitro and in vivo with alpha-synuclein promoting its aggregation. We report here that synphilin-1 specifically inhibits the degradation of alpha-synuclein wild-type and its missense mutants by the 20S proteasome due at least in part by the interaction of the ankyrin and coiled-coil domains of synphilin-1 (amino acids 331–555) with the N-terminal region (amino acids 1–60) of alpha-synuclein. Co-expression of synphilin-1 and alpha-synuclein wild-type in HeLa and N2A cells produces a specific increase in the half-life of alpha-synuclein, as degradation of unstable fluorescent reporters is not affected. Synphilin-1 inhibition can be relieved by co-expression of Siah-1 that targets synphilin-1 to degradation. Synphilin-1 inhibition of the proteasomal pathway of degradation of alpha-synuclein may help to understand the pathophysiological changes occurring in PD and other synucleinopathies.     

Functional investigations of exercising muscle: a noninvasive magnetic resonance spectroscopy-magnetic resonance imaging approach

Muscle fatigue, which is defined as the decline in muscle performance during exercise, may occur at different sites along the pathway from the central nervous system through to the intramuscular contractile machinery. Historically, both impairment of neuromuscular transmission and peripheral alterations within the muscle have been proposed as causative factors of fatigue development. However, according to more recent studies, muscle energetics play a key role in this process. Intramyoplasmic accumulation of inorganic phosphate (P_i) and limitation in ATP availability have been frequently evoked as the main mechanisms leading to fatigue. Although attractive, these hypotheses have been elaborated on the basis of experimental results obtained in vitro, and their physiological relevance has never been clearly demonstrated in vivo. In that context, noninvasive methods such as 31-phosphorus magnetic resonance spectroscopy and surface electromyography have been employed to understand both metabolic and electrical aspects of muscle fatigue under physiological conditions. Mapping of muscles activated during exercise is another interesting issue which can be addressed using magnetic resonance imaging (MRI). Exercise-induced T2 changes have been used in order to locate activated muscles and also as a quantitative index of exercise intensity. The main results related to both issues, i.e. the metabolic and electrical aspects of fatigue and the MRI functional investigation of exercising muscle, are discussed in the present review.Received 4 September 2003; received after revision 4 December 2003; accepted 22 December 2003     

Visualizing odor representation in the brain: a review of imaging techniques for the mapping of sensory activity in the olfactory glomeruli

The brain transforms clues from the external world, the sensory stimuli, into activities in neuroglial networks. These circuits are activated in specialized sensory cortices where specific functional modules are responsible for the spatiotemporal coding of the stimulus. A major challenge in the neuroscience field has been to image the spatial distribution and follow the temporal dynamics of the activation of such large populations in vivo. Functional imaging techniques developed in the last 30 years have enabled researchers to solve this critical issue, and are reviewed here. These techniques utilize sources of contrast of radioisotopic, magnetic and optical origins and exploit two major families of signals to image sensory activity: the first class uses sources linked to cellular energy metabolism and hemodynamics, while the second involves exogenous indicators of neuronal activity. The whole panel of imaging techniques has fostered the functional exploration of the olfactory bulb which is one of the most studied sensory structures. We summarize the major results obtained using these techniques that describe the spatial and temporal activity patterns in the olfactory glomeruli, the first relay of olfactory information processing in the main olfactory bulb. We conclude this review by describing promising technical developments in optical imaging and future directions in the study of olfactory spatiotemporal coding.     

Molecular enigma of multicolor bioluminescence of firefly luciferase

Firefly luciferase-catalyzed reaction proceeds via the initial formation of an enzyme-bound luciferyl adenylate intermediate. The chemical origin of the color modulation in firefly bioluminescence has not been understood until recently. The presence of the same luciferin molecule, in combination with various mutated forms of luciferase, can emit light at slightly different wavelengths, ranging from red to yellow to green. A historical perspective of development in understanding of color emission mechanism is presented. To explain the variation in the color of the bioluminescence, different factors have been discussed and five hypotheses proposed for firefly bioluminescence color. On the basis of recent results, light-color modulation mechanism of firefly luciferase propose that the light emitter is the excited singlet state of OL⁻ [¹(OL⁻)*], and light emission from ¹(OL⁻)* is modulated by the polarity of the active-site environment at the phenol/phenolate terminal of the benzothiazole fragment in oxyluciferin.     

New frontiers of primary antibody deficiencies

Primary antibody deficiencies (PAD) form the largest group of inherited disorders of the immune system. They are characterized by a marked reduction or absence of serum immunoglobulins (Ig) due to disturbed B cell differentiation and by a poor response to vaccination. PAD can be divided into agammaglobulinemia, Ig class switch recombination deficiencies, and idiopathic hypogammaglobulinemia. Over the past 20 years, defects have been identified in 18 different genes, but in many PAD patients the underlying gene defects have not been found. Diagnosis of known PAD and discovery of new PAD is important for good patient care. In this review, we present the effects of genetic defects in the context of normal B cell differentiation, and we discuss how new technical developments can support understanding and discovering new genetic defects in PAD.     

In vivo imaging of therapy-induced anti-cancer immune responses in humans

Immunotherapy aims to re-engage and revitalize the immune system in the fight against cancer. Research over the past decades has shown that the relationship between the immune system and human cancer is complex, highly dynamic, and variable between individuals. Considering the complexity, enormous effort and costs involved in optimizing immunotherapeutic approaches, clinically applicable tools to monitor therapy-induced immune responses in vivo are most warranted. However, the development of such tools is complicated by the fact that a developing immune response encompasses several body compartments, e.g., peripheral tissues, lymph nodes, lymphatic and vascular systems, as well as the tumor site itself. Moreover, the cells that comprise the immune system are not static but constantly circulate through the vascular and lymphatic system. Molecular imaging is considered the favorite candidate to fulfill this task. The progress in imaging technologies and modalities has provided a versatile toolbox to address these issues. This review focuses on the detection of therapy-induced anticancer immune responses in vivo and provides a comprehensive overview of clinically available imaging techniques as well as perspectives on future developments. In the discussion, we will focus on issues that specifically relate to imaging of the immune system and we will discuss the strengths and limitations of the current clinical imaging techniques. The last section provides future directions that we envision to be crucial for further development.     

Tumor cell migration in complex microenvironments

Tumor cell migration is essential for invasion and dissemination from primary solid tumors and for the establishment of lethal secondary metastases at distant organs. In vivo and in vitro models enabled identification of different factors in the tumor microenvironment that regulate tumor progression and metastasis. However, the mechanisms by which tumor cells integrate these chemical and mechanical signals from multiple sources to navigate the complex microenvironment remain poorly understood. In this review, we discuss the factors that influence tumor cell migration with a focus on the migration of transformed carcinoma cells. We provide an overview of the experimental and computational methods that allow the investigation of tumor cell migration, and we highlight the benefits and shortcomings of the various assays. We emphasize that the chemical and mechanical stimulus paradigms are not independent and that crosstalk between them motivates the development of new assays capable of applying multiple, simultaneous stimuli and imaging the cellular migratory response in real-time. These next-generation assays will more closely mimic the in vivo microenvironment to provide new insights into tumor progression, inform techniques to control tumor cell migration, and render cancer more treatable.     

Inhibition of firefly bioluminescence by scavengers of singlet oxygen,superoxide radicals and hydroxyl radicals

Summary The participation of highly energetic oxygen species in the ATP-induced bioluminescence of a firefly-extract has been investigated. The inhibition of light emission by a variety of specific scavengers suggests that singlet oxygen, superoxide radicals and hydroxyl radicals are important intermediates in the firefly bioluminescence reaction.Acknowledgments. I thank Prof. R. Bachofen, Institut für Allgemeine Botanik, Abteilung Mikrobiologie, Universität Zürich, Switzerland, in whose laboratory most of these studies have been performed, for his cooperativity. Financial support by the Deutsche Studienstiftung is gratefully acknowledged.     

Maternal eating behavior is a major synchronizer of fetal and postnatal peripheral clocks in mice

Most living organisms show circadian rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. To study peripheral clocks in vivo, we developed an original model, the Rev-Luc mouse to follow noninvasively and longitudinally Rev-Luc oscillations in peripheral clocks using in vivo bioluminescence imaging. We found in vitro and in vivo a robust diurnal rhythm of Rev-Luc, mainly in liver, intestine, kidney and adipose tissues. We further confirmed in vivo that Rev-Luc peripheral tissues are food-entrainable oscillators, not affected by age or sex. These data strongly support the relevance of the Rev-Luc model for circadian studies, especially to investigate in vivo the establishment and the entrainment of the rhythm throughout ontogenesis. We then showed that Rev-Luc expression develops dynamically and gradually, both in amplitude and in phase, during fetal and postnatal development. We also demonstrate for the first time that the immature peripheral circadian system of offspring in utero is mainly entrained by maternal cues from feeding regimen. The prenatal entrainment will also differentially determine the Rev-Luc expression in pups before weaning underlining the importance of the maternal chrononutrition on the circadian system entrainment of the offspring.     

Human motor neuron generation from embryonic stem cells and induced pluripotent stem cells

Motor neuron diseases (MNDs) are a group of neurological disorders that selectively affect motor neurons. There are currently no cures or efficacious treatments for these diseases. In recent years, significant developments in stem cell research have been applied to MNDs, particularly regarding neuroprotection and cell replacement. However, a consistent source of motor neurons for cell replacement is required. Human embryonic stem cells (hESCs) could provide an inexhaustible supply of differentiated cell types, including motor neurons that could be used for MND therapies. Recently, it has been demonstrated that induced pluripotent stem (iPS) cells may serve as an alternative source of motor neurons, since they share ES characteristics, self-renewal, and the potential to differentiate into any somatic cell type. In this review, we discuss several reproducible methods by which hESCs or iPS cells are efficiently isolated and differentiated into functional motor neurons, and possible clinical applications.     

Memory

The molecular mechanisms underlying the induction and maintenance of memory are highly dynamic and comprise distinct phases covering a time window from seconds to even a lifetime. Neuronal networks, which contribute to these processes, have been extensively characterized on various levels of analysis, and imaging techniques allow monitoring of both gross brain activity as well as functional changes in defined brain areas during the time course of memory formation. New techniques developed in honeybees and fruit flies even allow for manipulation of neuronal networks and molecular cascades in a short temporal domain while a living animal under observation acquires new associative memories. These advantages make honeybees and flies ideal organisms to study transient molecular events underlying dynamic memory processing in vivo. In this review we will focus on the temporal features of molecular processes in learning and memory formation, summarize recent knowledge and present an outlook on future developments.     

Alkaline phosphatase isozymes in cultured human cancer cells

Alkaline phosphatase, an ubiquitous enzyme is known to exist in several isozymic forms. At least three different isozymes have now been identified in humans. Alkaline phosphatase isozymes are among the substances synthesized ectopically by a variety of human tumors and many continuous cell lines derived from different cancers have retained the capacity to produce these membrane-located glycoproteins. This paper reviews the identification of alkaline phosphatase isozymes in cultured tumor cells and relates these findings with recent developments concerning these cell membrane located glycoproteins.     

Alkaline phosphatase isozymes in cultured human cancer cells

Summary Alkaline phosphatase, an ubiquitous enzyme is known to exist in several isozymic forms. At least three different isozymes have now been identified in humans. Alkaline phosphatase isozymes are among the substances synthesized ectopically by a variety of human tumors and many continuous cell lines derived from different cancers have retained the capacity to produce these membrane-located glycoproteins. This paper reviews the identification of alkaline phosphatase isozymes in cultured tumor cells and relates these finding with recent developments concerning these cell membrane located glycoproteins.     

Analysis of <Emphasis Type="Italic">Dictyostelium</Emphasis> TACC reveals differential interactions with CP224 and unusual dynamics of <Emphasis Type="Italic">Dictyostelium</Emphasis> microtubules

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-α-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.