New endo-beta-1,4-glucanases from the parabasalian symbionts,Pseudotrichonympha grassii and Holomastigotoides mirabile of Coptotermes termites

An endo-β-1,4-glucanase (EG) was purified from the hindgut of an Australian mound-building termite, Coptotermes lacteus. The hindgut extract had a peak separate from those for extracts obtained from the salivary glands and the midgut based on sephacryl S-200 gel chromatography, and also demonstrated an origin different from the endogenous EGs of the termite itself. The recovery was further purified by SDS-PAGE, and its N-terminal amino acid sequence analyzed. This showed high homology to EGs from glycoside hydrolase family (GHF) 7. PCR-based cloning methods were applied to the hindgut contents of C. lacteus and individual protozoan symbionts from C. formosanus. cDNAs encoding putative EGs homologous to GHF7 members were then identified. The functionality of one of the putative proteins was confirmed by its expression in Escherichia coli. Received 18 September 2002; accepted 20 September 2002 RID="*" ID="*"Corresponding author.     

Small,novel proteins from the mistletoe <Emphasis Type="Italic">Phoradendron tomentosum</Emphasis> exhibit highly selective cytotoxicity to human breast cancer cells

Four novel proteins (phoratoxins C–F) have been isolated from the North American mistletoe Phoradendron tomentosum. The amino acid sequences of these phoratoxins were determined unambiguously using a combination of Edman degradation and trypsin enzymatic digestion, and by electrospray ionization tandem mass spectrometry sequencing. Phoratoxins C, E and F consist of 46 amino acid residues; and phoratoxin D of 41. All proteins had six cysteines, similar to the earlier described phoratoxins A and B, which are thionins. The cytotoxicity of each protein was evaluated in a human cell line panel that represented several cytotoxic drug-resistance mechanisms. For the half-maximal inhibitory concentrations (IC₅₀ values) of the different cell lines in the panel, correlation with those of standard drugs was low. The most potent cytotoxic phoratoxin C was further tested on primary cultures of human tumor cells from patients. The solid tumor samples from breast cancer cells were 18 times more sensitive to phoratoxin C than the tested hematological tumor samples. Received 30 September 2002; received after revision 28 October 2002; accepted 7 November 2002 RID="*" ID="*"Corresponding author.     

The DnaK/ClpB chaperone system from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Proteins of thermophilic organisms are adapted to remain well structured and functional at elevated temperatures. Nevertheless like their 'cousins' that reside at medium temperatures, they require the assistance of molecular chaperones to fold properly and prevent aggregation. This review compares structural and functional properties of the DnaK/ClpB systems of Thermus thermophilus and, mainly, Escherichia coli (Dnak_Tth and Dnak_Eco). Many elemental properties of these systems remain conserved. However, in addition to a general increase of the thermal stability of its components, the Dnak_Tth system shows profound differences in its regulation, and genetic as well as oligomeric organization. Whether these differences are unique or represent general strategies of adaptation to life at elevated temperatures remains to be clarified. RID="*" ID="*"Corresponding author.     

A role for <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine as a nutrient sensor and mediator of insulin resistance

The ability to regulate energy balance at both the cellular and whole body level is an essential process of life. As western society has shifted to a higher caloric diet and more sedentary lifestyle, the incidence of type 2 diabetes (non-insulin-dependent diabetes mellitus) has increased to epidemic proportions. Thus, type 2 diabetes has been described as a disease of 'chronic overnutrition'. There are abundant data to support the relationship between nutrient availability and insulin action. However, there have been multiple hypotheses and debates as to the mechanism by which nutrient availability modulates insulin signaling and how excess nutrients lead to insulin resistance. One well-established pathway for nutrient sensing is the hexosamine biosynthetic pathway (HSP), which produces the acetylated aminosugar nucleotide uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) as its end product. Since UDP-GlcNAc is the donor substrate for modification of nucleocytoplasmic proteins at serine and threonine residues with N-acetylglucosamine (O-GlcNAc), the possibility of this posttranslational modification serving as the nutrient sensor has been proposed. We have recently directly tested this model in adipocytes by examining the effect of elevated levels of O-GlcNAc on insulin-stimulated glucose uptake. In this review, we summarize the existing work that implicates the HSP and O-GlcNAc modification as nutrient sensors and regulators of insulin signaling. RID="*" ID="*"Corresponding author.     

<Emphasis Type="Italic">LAPTM4A</Emphasis> interacts with <Emphasis Type="Italic">hOCT2</Emphasis> and regulates its endocytotic recruitment

Human organic cation transporter 2 (hOCT2) is involved in the transport of endogenous and exogenous organic cations mainly in cells of the kidney and the brain. Here, we focus on the regulation of hOCT2 by direct protein–protein interaction. Screening within a mating-based split-ubiquitin-yeast-two-hybrid system (mBSUS) revealed the lysosomal-associated protein transmembrane 4 alpha (LAPTM4A) as a potential interacting protein. Interaction of LAPTM4A and hOCT2 was confirmed by pulldown assays, FRET microscopy analysis and immunofluorescence microscopy. Functionally, overexpression of LAPTM4A significantly decreased ASP⁺ uptake in HEK293 cells stably transfected with hOCT2, suggesting a negative regulation of hOCT2-mediated transport. Furthermore, overexpression of LAPTM4A leads to a significantly decreased hOCT2 plasma membrane expression in surface biotinylation experiments. In addition, significant expression of LAPTM4A in human kidney was demonstrated by immunoblotting and immunofluorescence.     

The role of conjugative transposons in the <Emphasis Type="Italic">Enterobacteriaceae</Emphasis>

Although widely studied in Gram-positive Streptococci and in the Gram-negative Bacteroides, there is a scarcity of information on the occurrence and nature of conjugative transposon-like elements in the well-studied Enterobacteriaceae. In fact, some of the major reviews on conjugative transposons prior to 1996 failed to mention their occurrence in this group. Recently, their presence has been reported in Salmonella, Vibrio and Proteus species, and in some cases such as the SXT element in Vibrio and the IncJ group element CTnR391, there has been some molecular characterization. The elements thus far examined appear to be larger than the common Gram-positive conjugative transposons and to be mosaic in structure, with genes derived from several sources. Recent evidence suggests that in the Enterobacteriaceae the elements may be related to enteric pathogenicity islands. The evolution, distribution and role of these elements in the Enterobacteriaceae is discussed.     

A new lysozyme from the eastern oyster (<Emphasis Type="Italic">Crassostrea virginica</Emphasis>) indicates adaptive evolution of <Emphasis Type="Italic">i</Emphasis>-type lysozymes

A new lysozyme (cv-lysozyme 2) with a MALDI molecular mass of 12 984.6 Da was purified from crystalline styles and digestive glands of eastern oysters (Crassostrea virginica) and its cDNA sequenced. Quantitative real time RT-PCR detected cv-lysozyme 2 gene expression primarily in digestive gland tissues, and in situ hybridization located cv-lysozyme 2 gene expression in basophil cells of digestive tubules. Cv-lysozyme 2 showed high amino acid sequence similarity to other bivalve mollusk lysozymes, including cv-lysozyme 1, a lysozyme recently purified from C. virginica plasma. Differences between cv-lysozyme 2 and cv-lysozyme 1 molecular characteristics, enzymatic properties, antibacterial activities, distribution in the oyster body and site of gene expression indicate that the main role of cv-lysozyme 2 is in digestion. While showing that a bivalve mollusk employs different lysozymes for different functions, findings in this study suggest adaptive evolution of i type lysozymes for nutrition. Received 30 August 2006; received after revision 14 October 2006; accepted 6 November 2006     

<Emphasis Type="Italic">O</Emphasis>-fucose modifications of epidermal growth factor-like repeats and thrombospondin type 1 repeats: unusual modifications in unusual places

Recent discoveries revealing that carbohydrate modifications play critical roles in a wide variety of biological processes have brought wide recognition to the field of glycobiology. Growing attention has focused on the function of unusual O-linked carbohydrate modifications such as O-fucose. O-fucose modifications have been described in several different protein contexts, including epidermal growth factor-like repeats and thrombospondin type 1 repeats. The O-fucose modifications on thrombospondin type 1 repeats have only recently been described, but the site of modification occurs in a region proposed to play a role in cell adhesion. O-fucose modifications on epidermal growth factor-like repeats have been described as important players in several signal transduction systems. For instance, Notch, a cell-surface signaling receptor required for many developmental events, bears multiple O-fucose saccharides on the epidermal growth factor-like repeat of its extracellular domain. The O-fucose moieties serve as a substrate for the β1,3 N-acetylglucosaminyltransferase activity of Fringe, a known modifier of Notch function. The alteration of O-fucose structures by Fringe influences the ability of Notch ligands to activate the receptor and provides a means to regulate Notch signaling. Thus, O-fucose and Fringe provide a clear example of how carbohydrate modifications can have direct functional consequences on the proteins they modify. RID="*" ID="*"Corresponding author.     

Cellular microbiology of intracellular <Emphasis Type="Italic">Salmonella enterica</Emphasis>: functions of the type III secretion system encoded by <Emphasis Type="Italic">Salmonella</Emphasis> pathogenicity island 2

The facultative intracellular pathogen Salmonella enterica resides in a special membrane compartment of the host cell and modifies its host to achieve intracellular survival and proliferation. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has a central role in the interference of intracellular Salmonella with host cell functions. SPI2 function affects antimicrobial defense mechanisms of the host, intracellular transport processes, integrity and function of the cytoskeleton and host cell death. These modifications are mediated by translocation of a large number of effector proteins by the SPI2 system. In this review, we summarize recent work on the cellular phenotypes related to SPI2 function and contribution of SPI2 effector proteins to these manipulations. These studies reveal a complex set of pathogenic interferences between intracellular Salmonella and its host cells.Received 11 June 2004; received after revision 8 July 2004; accepted 12 July 2004     

Identification of chicken lysozyme <Emphasis Type="Italic">g</Emphasis>2 and its expression in the intestine

Lysozyme is an important component of the innate immune system, protecting the gastrointestinal tract from infection. The aim of the present study was to determine if lysozyme is expressed in the chicken (Gallus gallus) intestine and to characterise the molecular forms expressed. Immunohistochemical staining localised lysozyme to epithelial cells of the villous epithelium along the length of the small intestine. There was no evidence for lysozyme expression in crypt epithelium and no evidence for Paneth cells. Immunoblots of chicken intestinal protein revealed three proteins: a 14-kDa band consistent with lysozyme c, and two additional bands of approximately 21 and 23 kDa, the latter consistent with lysozyme g. RT-PCR analyses confirmed that lysozyme c mRNA is expressed in 4-day, but not older chicken intestine and lysozyme g in 4- to 35-day chicken intestine. A novel chicken lysozyme g2 gene was identified by in silico analyses and mRNA for this lysozyme g2 was identified in the intestine from chickens of all ages. Chicken lysozyme g2 shows similarity with fish lysozyme g, including the absence of a signal peptide and cysteines involved in disulphide bond formation of the mammalian and bird lysozyme g proteins. Analyses using SecretomeP predict that chicken lysozyme g2 may be secreted by the non-classical secretory pathway. We conclude that lysozyme is expressed in the chicken small intestine by villous enterocytes. Lysozyme c, lysozyme g and g2 may fulfil complimentary roles in protecting the intestine.Received 4 August 2004; received after revision 1 September 2004; accepted 7 September 2004     

Neuroprotective effects of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> extract

Ginkgo biloba extract has been therapeutically used for several decades to increase peripheral and cerebral blood flow as well as for the treatment of dementia. The extract contains multiple compounds such as flavonoids and terpenoids that are thought to contribute to its neuroprotective and vasotropic effects. In this review, we summarize the experimental results on the mechanism of neuroprotection induced by standardized extract of Ginkgo biloba leaves (EGb 761) and its constituents. The effects described mostly in animals include those on cerebral blood flow, neurotransmitter systems, cellular redox state and nitric oxide level. Furthermore, we discuss the current status of clinical trials as well as undesired side effects of EGb 761.Received 21 November 2002; received after revision 8 March 2003; accepted 17 March 2003     

Fatty acid metabolism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid -oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the -oxidation products out of the peroxisome to mitochondria for full oxidation to CO₂ and H₂O.Received 19 February 2003; received after revision 27 March 2003; accepted 27 March 2003     

Notable diversity in hemoglobin expression patterns among species of the deep-sea clam, <Emphasis Type="Italic">Calyptogena</Emphasis>

The deep-sea clams Calyptogena nautilei and C. tsubasa, which live in the cold-seep area at a depth of 3570 m in the Nankai Trough, Japan, have abundant hemoglobins (Hbs) in erythrocytes, similar to other Calyptogena species. We determined the cDNA-derived amino acid sequences of Hbs from two Calyptogena species. C. tsubasa was found to contain two dimeric Hbs, Hb I consisting of 145 amino acid residues and Hb II with 137 residues, similar to known Hbs from C. soyoae and C. kaikoi. Sequence identity was over 90% among the orthologous chains of Calyptogena Hbs. On the other hand, surprisingly, C. nautilei contained two monomeric Hbs, Hb III containing 141 residues and Hb IV with 134 residues. In addition, Hbs III and IV showed only 33–42% sequence identity with Hbs I and II from other Calyptogena species. The distal (E7) histidine, one of the functionally important residues of the heme protein, is replaced by glutamine in all Hb chains of Calyptogena species. A phylogenetic analysis indicated that C. nautilei Hb III is closer to Hb I from other Calyptogena species. We suppose that a Hb gene was duplicated at least three times in an immediate ancestor of Calyptogena and, presumably depending on physiological conditions different Hb sets are being expressed: dimeric Hbs I and II in C. soyoae, C. kaikoi and C. tsubasa, and monomeric Hbs III and IV in C. nautilei. Received 13 May 2003; received after revision 5 June 2003; accepted 12 June 2003     

Soluble proteins of chemical communication in the social wasp <Emphasis Type="Italic">Polistes dominulus</Emphasis>

Members of the odorant-binding protein (OBP) and chemosensory protein (CSP) families were identified and characterised in the sensory tissues of the social wasp Polistes dominulus (Hymenoptera: Vespidae). Unlike most insects so far investigated, OBPs were detected in antennae, legs and wings, while CSPs appeared to be preferentially expressed in the antennae. The OBP is very different from the homologous proteins of other Hymenopteran species, with around 20% of identical residues, while the CSP appears to be much better conserved. Both OBP and CSP, not showing other post-translational modifications apart from disulphide bridges, were expressed with high yields in a bacterial system. Cysteine pairing in the recombinant and native proteins follows the classical arrangements described for other members of these classes of proteins. OBPs isolated from the wings were found to be associated with a number of long-chain aliphatic amides and other small organic molecules. Binding of these ligands and other related compounds was measured for both recombinant OBP and CSP.Received 14 May 2003; received after revision 8 June 2003; accepted 12 June 2003     

High affinity recognition of a <Emphasis Type="Italic">Phytophthora</Emphasis> protein by <Emphasis Type="Italic">Arabidopsis</Emphasis> via an RGD motif

The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [¹²⁵I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53–56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp⁵⁶ into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.Received 23 October 2003; received after revision 5 December 2003; accepted 12 December 2003     

Molecular characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> PHO80-like proteins,a novel class of CDKA;1-interacting cyclins

Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle. In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T). A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs). They all share a conserved central region of 100 amino acids (cyclin box) displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T. brucei. The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast. The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments. P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues. The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed.Received 9 February 2004; received after revision 18 March; accepted 19 April 2004     

Hepatic effects of <Emphasis Type="Italic">Cimicifuga racemosa</Emphasis> extract <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Extracts of Cimicifuga racemosa are used frequently for menopausal complaints. Cimicifuga is well tolerated but can occasionally cause liver injury. To assess hepatotoxicity of cimicifuga in more detail, ethanolic C. racemosa extract was administered orally to rats, and liver sections were analyzed by electron microscopy. Tests for cytotoxicity, mitochondrial toxicity and apoptosis/necrosis were performed using HepG2 cells. Mitochondrial toxicity was studied using isolated rat liver mitochondria. Microvesicular steatosis was found in rats treated with > 1,000 mg/kg [DOSAGE ERROR CORRECTED] body weight cimicifuga extract. In vitro, cytotoxicity was apparent at concentrations > or =75 microg/mL, and mitochondrial beta-oxidation was impaired at concentrations > or =10 microg/mL. The mitochondrial membrane potential was decreased at concentrations > or =100 microg/mL, and oxidative phosphorylation was impaired at concenq trations > or =300 microg/mL. The mechanism of cell death was predominantly apoptosis. C. racemosa exerts toxicity in vivo and in vitro, eventually resulting in apoptotic cell death. The results are compatible with idiosyncratic hepatotoxicity as observed in patients treated with cimicifuga extracts.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> innate immunity: an emerging role for peptidoglycan recognition proteins in bacteria detection

Over the past years, parallel studies conducted in mammals and flies have emphasized the existence of common mechanisms regulating the vertebrate and invertebrate innate immune systems. This culminated in the discovery of the central role of the Toll pathway in Drosophila immunity and in the implication of Toll-like receptors (TLRs)/interleukin-1(IL-1) in the mammalian innate immune response. In spite of clear similarities, such as shared intracellular pathway components, important divergences are expected between the two groups, whose last common ancestor lived more than half a billion years ago. The most obvious discrepancies lie in the mode of activation of the signalling receptors by microorganisms. In mammals, TLRs are part of protein complexes which directly recognize microbe-associated patterns, whereas Drosophila Toll functions like a classical cytokine receptor rather than a pattern recognition receptor. Recent studies demonstrate that members of the evolutionarily conserved peptidoglycan recognition protein family play an essential role in microbial sensing during immune response of Drosophila.Received 26 June 2003; received after revision 29 July 2003; accepted 25 August 2003     

<Emphasis Type="Italic">O</Emphasis>-Mannosylation and human disease

Glycosylation of proteins is arguably the most prevalent co- and post-translational modification. It is responsible for increased heterogeneity and functional diversity of proteins. Here we discuss the importance of one type of glycosylation, specifically O-mannosylation and its relationship to a number of human diseases. The most widely studied O-mannose modified protein is alpha-dystroglycan (α-DG). Recent studies have focused intensely on α-DG due to the severity of diseases associated with its improper glycosylation. O-mannosylation of α-DG is involved in cancer metastasis, arenavirus entry, and multiple forms of congenital muscular dystrophy [1, 2]. In this review, we discuss the structural and functional characteristics of O-mannose-initiated glycan structures on α-DG, enzymes involved in the O-mannosylation pathway, and the diseases that are a direct result of disruptions within this pathway.