Human lactoferrin exerts bi-directional actions on PC12 cell survival via ERK1/2 pathway

Human lactoferrin (hLF) is a member of the transferrin family and is found in most body fluids of human. Recent study showed that hLF played some roles in the regulation of cell growth. However, the biological function of hLF in the central nervous system and neuronal cells is still unclear. The MTT was used to assay cell viability, ELISA tests were used to assay caspase activities, and TUNEL staining was used to test the cytotoxicity of hLF to the cells. Our result showed that 700 microg/ml hLF significantly reduced the cell viability and increased the caspase 3 and 8 activities in PC12 neuronal cells. TUNEL staining further showed that 700 microg/ml hLF was cytotoxic to the PC12 through apoptosis-mediated pathway. In addition, 700 microg/ml hLF significantly decreased the protein expressions of phosphorylated extracellular-signal-regulated kinase 1/2 (ERK1/2) and Bcl-2 in PC12 cells, whereas 50 microg/ml hLF significantly increased the phosphorylation of ERK1/2 which could be specifically inhibited by PD98059. Furthermore, 50 microg/ml hLF could not only up-regulate the Bcl-2 expression but also protect PC12 cells from FasL-induced apoptosis. In conclusion, hLF plays a crucial role in the regulation of apoptosis and anti-apoptosis in PC12 neuronal cells via ERK1/2 phosphorylation pathway.     

Comprehensive characterization of the site-specific N-glycosylation of wild-type and recombinant human lactoferrin expressed in the milk of transgenic cloned cattle

The glycosylation profile of a recombinant protein is important because glycan moieties can play a significant role in the biological properties of the glycoprotein. Here we determined the site-specific N-glycosylation profile of human lactoferrin (hLF) and recombinant human lactoferrin (rhLF) expressed in the milk of transgenic cloned cattle. We used combined approaches of monosaccharide composition analysis, lectin blot, glycan permethylation and sequential exoglycosidase digestion and analyzed samples using high-performance ion chromatography and mass spectrometry (MS). N-glycans from hLF are comprised entirely of highly branched, highly sialylated and highly fucosylated complex-type structures, and many contain Lewis(x) epitopes. Six of these structures are reported here for the first time. However, N-glycans from rhLF are of the high mannose-, hybrid- and complex-type structures, with less N-acetylneuraminic acid and fucose. Some contain a terminal N-acetylgalactosamine-N-acetylglucosamine (LacdiNAc) disaccharide sequence. Monosaccharide composition analysis of rhLF revealed small amounts of N-glycolylneuraminic acid, which were not detected by MS. hLF and rhLF appear to be glycosylated at the same two sites: Asn138 and Asn479. The third putative glycosylation site, at Asn624, is unglycosylated in both hLF and rhLF. The relative abundance of each N-glycan at each site was also determined. The different N-glycosylation profile of rhLF when compared with that of hLF is in consistent with the widely held view that glycosylation is species- and tissue/cell-specific. These data provide an important foundation for further studies of glycan structure/function relationships for hLF and rhLF and help to better understand the glycosylation mechanism in bovine mammary epithelial cells.     

BIOCOMPUTATION: Some history and prospects

At first glance, biology and computer science are diametrically opposed sciences. Biology deals with carbon based life forms shaped by evolution and natural selection. Computer Science deals with electronic machines designed by engineers and guided by mathematical algorithms. In this brief paper, we review biologically inspired computing. We discuss several models of computation which have arisen from various biological studies. We show what these have in common, and conjecture how biology can still suggest answers and models for the next generation of computing problems. We discuss computation and argue that these biologically inspired models do not extend the theoretical limits on computation. We suggest that, in practice, biological models may give more succinct representations of various problems, and we mention a few cases in which biological models have proved useful. We also discuss the reciprocal impact of computer science on biology and cite a few significant contributions to biological science.     

Probabilistic discovery of overlapping cellular processes and their regulation.

In this paper, we explore modeling overlapping biological processes. We discuss a probabilistic model of overlapping biological processes, gene membership in those processes, and an addition to that model that identifies regulatory mechanisms controlling process activation. A key feature of our approach is that we allow genes to participate in multiple processes, thus providing a more biologically plausible model for the process of gene regulation. We present algorithms to learn each model automatically from data, using only genomewide measurements of gene expression as input. We compare our results to those obtained by other approaches and show that significant benefits can be gained by modeling both the organization of genes into overlapping cellular processes and the regulatory programs of these processes. Moreover, our method successfully grouped genes known to function together, recovered many regulatory relationships that are known in the literature, and suggested novel hypotheses regarding the regulatory role of previously uncharacterized proteins.     

Immune surveillance properties of human NK cell-derived exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56(+) and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.     

Trypanosoma cruzi and its components as exogenous mediators of inflammation recognized through Toll-like receptors

Trypanosoma cruzi is the etiologic agent of Chagas' disease, a parasitic disease of enormous importance in Latin America. Herein we review the studies that revealed the receptors from innate immunity that are involved in the recognition of this protozoan parasite. We showed that the recognition of T. cruzi and its components occurs through Toll-like receptors (TLR) 2/CD14. Further, we showed in vivo the importance of the myeloid differentiation factor (MyD88), an adapter protein essential for the function of TLRs, in determining the parasitemia and mortality rate of mice infected with T. cruzi. We also discuss the implications of these findings in the pathophysiology of Chagas' disease.     

An inducible expression system for the production of human lactoferrin in Aspergillus nidulans.

The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described. The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (alcA) promoter. Recombinant hLF (re-hLF) is produced at levels up to 5 micrograms/ml. Approximately 30% of the re-hLF produced in this system is secreted into the growth medium. The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity. Furthermore, re-hLF is functional by the criterion of iron-binding capacity. The A. nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.     

Long pentraxin 3: A novel multifaceted player in cancer

Since its discovery in 1992, long pentraxin 3 (PTX3) has been characterized as soluble patter recognition receptor, a key player of the innate immunity arm with non-redundant functions in pathogen recognition and inflammatory responses. As a component of the extra-cellular matrix milieu, PTX3 has been implicated also in wound healing/tissue remodeling, cardiovascular diseases, fertility, and infectious diseases. Consequently, PTX3 levels in biological fluids have been proposed as a fluid-phase biomarker in different pathological conditions.In the last decade, experimental evidences have shown that PTX3 may exert a significant impact also on different aspects of cancer biology, including tumor onset, angiogenesis, metastatic dissemination and immune-modulation. However, it remains unclear whether PTX3 acts as a good cop or bad cop in cancer. In this review, we will summarize and discuss the scientific literature data focusing on the role of PTX3 in experimental and human tumors, including its putative translational implications.     

IL-13 is pivotal in the fibro-obliterative process of bronchiolitis obliterans syndrome

Acute allograft rejection is considered to be a predominately type 1 immune mediated response to the donor alloantigen. However, the type 2 immune mediated response has been implicated in multiple fibroproliferative diseases. Based on the fibro-obliterative lesion found during bronchiolitis obliterans syndrome (BOS), we hypothesized that the type 2 immune mediated response is involved in chronic lung allograft rejection. Specifically, whereas acute rejection is, in part, a type 1 immune response, chronic rejection is, in part, a type 2 immune response. We found the type 2 cytokine, IL-13, to be elevated and biologically active in human bronchoalveolar lavage fluid during BOS. Translational studies using a murine model of BOS demonstrated increased expression of IL-13 and its receptors that paralleled fibro-obliteration. In addition, in vivo neutralization of IL-13 reduced airway allograft matrix deposition and murine BOS, by a mechanism that was independent of IL-4. Furthermore, using IL-13Ralpha2(-/-) mice, we found increased fibro-obliteration. Moreover, anti-IL-13 therapy in combination with cyclosporin A had profound effects on reducing murine BOS. This supports the notion that IL-13 biological axis plays an important role during the pathogenesis of BOS independent of the IL-4 biological axis.     

Casein kinase 2, circadian clocks, and the flight from mutagenic light

Circadian clocks play a fundamental role in biology and disease. Much has been learned about the molecular underpinnings of these biological clocks from genetic studies in model organisms, such as the fruit fly, Drosophila melanogaster. Here we review the literature from our lab and others that establish a role for the protein kinase CK2 in Drosophila clock timing. Among the clock genes described thus far, CK2 is unique in its involvement in plant, fungal, as well as animal circadian clocks. We propose that this reflects an ancient, conserved function for CK2 in circadian clocks. CK2 and other clock genes have been implicated in cellular responses to DNA damage, particularly those induced by ultraviolet (UV) light. The finding of a dual function of CK2 in clocks and in UV responses supports the notion that clocks evolved to assist organisms in avoiding the mutagenic effects of daily sunlight.     

A structure-based approach to designing non-natural peptides that can activate anti-melanoma cytotoxic T cells

Tumor antigens presented by major histocompatibility complex (MHC) class I molecules and recognized by CD8(+) cytotoxic T lymphocytes (CTLs) may generate an efficient antitumor immune response after appropriate immunization. Antigenic peptides can be used in vivo to induce antitumor or antiviral immunity. The efficiency of naked peptides may be greatly limited by their degradation in the biological fluids. We present a rational, structure-based approach to design structurally modified, peptidase-resistant and biologically active analogues of human tumor antigen MAGE-1.A1. This approach is based on our understanding of the peptide interaction with the MHC and the T cell receptor and its precise degradation pathway. Knowledge of these mechanisms led to the design of a non-natural, minimally modified analogue of MAGE-1.A1, [Aib2, NMe-Ser8]MAGE-1.A1, which was highly peptidase-resistant and bound to MHC and activated MAGE-1.A1-specific anti-melanoma CTLs. Thus, we showed that it is possible to structurally modify peptide epitopes to obtain analogues that are still specifically recognized by CTLs. Such analogues may represent interesting leads for antitumor synthetic vaccines.     

In vitro T cell-mediated killing of Pseudomonas aeruginosa. I. Evidence that a lymphokine mediates killing

Previous studies have demonstrated in vivo that T cells can provide protective immunity, in the absence of antibody, against infection with the extracellular Gram-negative bacterium Immunotype 1 (IT-1) Pseudomonas aeruginosa. We established an in vitro system in which immune T cells, after reexposure to bacterial antigens and to macrophages, secrete a product that kills the bacteria. Although macrophages are required for in vitro killing, they function neither as antigen-presenting nor as phagocytic cells in this system. T cells from animals immunized against a different P. aeruginosa immunotype will not kill IT-1 organisms; but the supernatants produced by IT-1 immune T cells after exposure to macrophages and IT-1 P. aeruginosa organisms are nonspecifically effective in killing unrelated bacteria. Because the supernatants from immune T cells lose their bactericidal properties upon minimal dilution, we conclude that if this mechanism is active in vivo, it must play a role in local immunity.     

The human lactoferrin-derived peptide hLF1-11 primes monocytes for an enhanced TLR-mediated immune response

Earlier we reported that the peptide corresponding to the first eleven N-terminal amino acids of human lactoferrin (hLF1-11) is active against multi-drug resistant pathogens in mice. The mechanisms underlying this anti-infective activity remain unclear. Since hLF1-11 is ineffective against pathogens at physiological salt concentrations and hLF1-11 directs differentiation of monocytes toward a macrophage subset with enhanced effector functions, we investigated the effects of hLF1-11 on human and murine monocytes. Results revealed that human and murine monocytes exposed for 1 h to hLF1-11 and then stimulated with the Toll-like receptor (TLR)-ligand LPS for 18 h, displayed enhanced cytokine and chemokine production as compared to control (peptide-treated) monocytes. We also found that expression of mRNA, cell-surface receptor expression, and NF-κB activation by hLF1-11-exposed human monocytes were enhanced as compared to control (peptide-treated) monocytes. Furthermore, the kinetics of the cytokine production was unchanged as mRNA levels and protein levels paralleled the enhanced response of hLF1-11-exposed monocytes to LPS. The cytokine production by human monocytes in response to TLR4, TLR5, and TLR7 stimulation, but not to TLR2 stimulation, was elevated by hLF1-11. In concordance, translocation of NF-κB subunits to the nucleus was enhanced in hLF1-11-exposed monocytes after TLR stimulation, except for TLR2, as compared to control (peptide-exposed) monocytes. In conclusion, monocytes were primed by hLF1-11 for an enhanced inflammatory response upon TLR4, TLR5, and TLR7 stimulation, but not TLR2 stimulation. Such effects of hLF1-11 on monocyte reactivity should be taken into account when considering the clinical development of this peptide for a therapeutic intervention in patients.     

The oxidative modification hypothesis of atherogenesis: an overview

The literature relating lipid and lipoprotein oxidation to atherosclerosis has expanded enormously in recent years. Papers on the “oxidative modification hypothesis” of atherogenesis have ranged from the most basic studies of the chemistry and enzymology of LDL oxidation, through studies of the biological effects of oxidized LDL on cultured cells, and on to in vivo studies of the effects of antioxidants on atherosclerosis in animals and humans. The data in support of this theory are mounting but many key questions remain unanswered. For example, while it is generally agreed that LDL undergoes oxidation and that oxidized LDL is present in arterial lesions, it is still not known how and where LDL gets oxidized in vivo nor which of its many biological effects demonstrable in vitro are relevant to atherogenesis in vivo. This brief review is not intended to be comprehensive but rather to offer a perspective and a context for this Forum. We discuss the strengths and weaknesses of each line of evidence, try to identify areas in which further research is needed, assess the relevance of the hypothesis to the human disease, and point to some of the potential targets for therapy.     

Integrative understanding of immune-metabolic interaction

Recent studies have revealed that the immune system plays a critical role in various physiological processes beyond its classical pathogen control activity. Even under a sterile condition, various cells and tissues can utilize the immune system to meet a specific demand for proper physiological functions. Particularly, a strong link between immunity and metabolism has been identified. Studies have identified the reciprocal regulation between these two systems. For example, immune signals can regulate metabolism, and metabolism (cellular or systemic) can regulate immunity. In this review, we will summarize recent findings on this reciprocal regulation between immunity and metabolism, and discuss potential biological rules behind this interaction with integrative perspectives.     

Towards a functional understanding of "good genes"

The Hamilton & Zuk hypothesis (1982) underpins our understanding of the relationship between secondary sexual characters, parasites, and immunological function. However, despite the wealth of empirical studies that attempt to address issues raised by the Hamilton & Zuk hypothesis, there have been no overt attempts to identify the "good genes" that females select or how those good genes influence the host's immune system. Behavioural ecologists have generally viewed this aspect of immunity as a black box. In this review we propose candidate good genes in vertebrates, discuss their role in immune function and parasite resistance, and discuss several aspects of the assumptions that pervade studies of parasite mediated sexual selection in vertebrates. We also examine invertebrates (specifically insects) where our current knowledge of these systems suggests the patterns apparent in vertebrates are not underpinned by the same genetic mechanisms.     

Extracellular heat shock proteins and cancer: New perspectives

Heat shock proteins (HSPs) are a large family of molecular chaperones aberrantly expressed in cancer. The expression of HSPs in tumor cells has been shown to be implicated in the regulation of apoptosis, immune responses, angiogenesis and metastasis. Given that extracellular vesicles (EVs) can serve as potential source for the discovery of clinically useful biomarkers and therapeutic targets, it is of particular interest to study proteomic profiling of HSPs in EVs derived from various biological fluids of cancer patients. Furthermore, a divergent expression of circulating microRNAs (miRNAs) in patient samples has opened new opportunities in exploiting miRNAs as diagnostic tools. Herein, we address the current literature on the expression of extracellular HSPs with particular interest in HSPs in EVs derived from various biological fluids of cancer patients and different types of immune cells as promising targets for identification of clinical biomarkers of cancer. We also discuss the emerging role of miRNAs in HSP regulation for the discovery of blood-based biomarkers of cancer. We outline the importance of understanding relationships between various HSP networks and co-chaperones and propose the model for identification of HSP signatures in cancer. Elucidating the role of HSPs in EVs from the proteomic and miRNAs perspectives may provide new opportunities for the discovery of novel biomarkers of cancer.     

Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea

Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.