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.     

Sexual behavior mutants revisited: molecular and cellular basis of Drosophila mating

The study of Drosophila melanogaster by a combination of forward genetics with specific mutants, and reverse genetics, in which a given gene is expressed in an appropriate brain area to test its effect on behavior, provides a unique opportunity to explore the causal relationship between a particular gene, its function in the cell and the behavioral outcome at the organismic level. Enhanced male-to-male courtship has been shown to occur as a result of mutations in several different genes. For example, the Voila mutant exhibits intense GAL4 reporter expression in the tarsal gustatory sensilla, suggesting the importance of tapping by a male on the female abdomen with his forelegs. Feminization of parts of the antennal lobe and mushroom body by targeted expression of a female-determining gene transformer ⁺ (tra ⁺) drives the male to court other males. Mutations in the tra target gene fruitless (fru), which is expressed in the antennal lobe as well as the suboesophageal ganglion (the gustatory inputs are processed here), also induce homosexual courtship in males. These results suggest that sensory inputs mediated and/or processed by the tarsal receptors, suboesophageal ganglion, antennal lobe and mushroom body contribute to the regulation of male–female courtship. Mosaic analysis localized the neural center for male courtship behavior to the posterior dorsal brain, in which the sensory information processed by the aforementioned neural structures may be integrated. Another mosaic study mapped the neural center for female sexual behavior, as measured by her receptiveness to copulation, to the anterior dorsal brain. The issue as to how the mutations that reduce female sexual receptiveness, e.g. dissatisfaction (dsf), spinster (spin) and chaste (cht), affect the structure and/or function of this neural center deserves to be addressed urgently. Received 27 April 1999; received after revision 21 June 1999; accepted 8 July 1999     

A potent actomyosin ATPase activator from the Okinawan marine spongeAgelas cf.nemoechinata

Summary A bromine-containing alkaloid, oxysceptrin, has been isolated as a potent actomyosin ATPase activator from the Okinawan marine spongeAgelas cf.nemoechinata, and the structure elucidated to be1 on the basis of the 2D NMR spectral data.Acknowledgments. We thank Mr Z. Nagahama for his help in sponge collecting and Ms M. Takamatsu for her technical assistance.     

Phylogeny and rapid Northern and Southern Hemisphere speciation of goldfinches during the Miocene and Pliocene Epochs

Mitochondrial cytochrome b (cyt b) from 25 out of 31 extant goldfinches, siskins, greenfinches and redpolls (genus Carduelis) has been sequenced from living samples taken around the world, specimens have also been photographed. Phylogenetic analysis consistently gave the same groups of birds, and this grouping was generally related to geographical proximity. It has been supposed that Pleistocene glaciations played a crucial role in the origin of extant diversity and distribution of Northern Hemisphere vertebrates. Molecular comparison of most extant songbird species belonging to the genus Carduelis does not support this assertion. The fossil record of chicken and pheasant divergence time has been used to calibrate the molecular clock; cyt b DNA dendrograms suggest that speciation in Carduelinae birds occurred during the Miocene and Pliocene Epochs (9 – 2 million years ago) in both the Northern and Southern Hemispheres. Only about 4% average amount of nucleotide substitution per lineage is found between the most distant Carduelis species; this suggests a remarkably rapid radiation when compared with the radiation of other passerine songbird genera. In addition, a continuum of small songbird speciation may be found during the Miocene Epoch in parallel with speciation of other orders (i.e. Galliformes, chicken/pheasant). Pleistocene glaciations may have been important in subspeciation (i.e. Eastern European grey-headed goldfinches/Western European black-headed goldfinches) and also in ice-induced vicariance (isolation) (i.e. siskin in Western Europe vs. siskin in Far East Asia) around the world. European isolated Serinus citrinella (citril finch) is not a canary, but a true goldfinch. South American siskins have quickly radiated in the last 4 million years coinciding with the emergence of the Isthmus of Panama; probably, a North American siskin related to C. notata invaded a suitable and varied biotope (the South American island) for Carduelis birds. North American goldfinches may be renamed as siskins, because they have a distant genetic relationship with European goldfinches. Genus Acanthis could be dropped, and thus redpolls should be separated from twite and linnet, the latter (Europeans) probably being related to American goldfinches. Also, reproductive barriers are observed between closely related species and not between other more distant ones. Finally, a tentative classification for genus Carduelis species is suggested. Received 6 March 1998; received after revision 3 July 1998; accepted 7 July 1998     

Assembly and genetics of spore protective structures

The sporulation program in Bacillus subtilis ends in the formation of a highly resistant endospore that can withstand extremes of heat, mechanical disruption, ultraviolet irradiation, lytic enzymes and chemical attack. These properties are attributed mainly to the unique structure of spore coat and cortex, as well as to the physical state of the spore cytoplasm. The outermost layer of the spore, called the coat, has two morphologically distinct sublayers: an electron-dense outer coat and an electron-translucent inner coat. The coat is composed of more than 2 dozen proteins of varying size. Many coat genes and coat proteins have been isolated and characterized in detail, and studies of these have identified proteins with important roles in coat assembly, resistance and spore germination. We describe here characteristics of the coat proteins and propose a model for coat assembly based on recent work.     

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">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.     

Differential display analysis of gene expression in invertebrates

Screening for differentially expressed genes is a straightforward approach to study the molecular basis for changes in gene expression. Differential display analysis has been used by investigators in diverse fields of research since it was developed. Differential display has also been the approach of choice to investigate changes in gene expression in response to various biological challenges in invertebrates. We review the application of differential display analysis of gene expression in invertebrates, and provide a specific example using this technique for novel gene discovery in the nematode Caenorhabditis elegans.     

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     

A role for the hinge/ear domain of the β chains in the incorporation of AP complexes into clathrin-coated pits and coated vesicles

The clathrin-associated adaptor protein (AP) complexes drive the polymerization of clathrin in coated pits to form coated vesicles. It has previously been shown that the carboxyl-terminal hinge/ear domain of the β2 chain contains a binding site for clathrin and that removal of this domain from APs or from isolated β2 chains abrogates their ability to form clathrin coats in vitro. We show here that the hinge/ear domain is necessary for efficient incorporation of AP complexes into coated pits and coated vesicles in cells, a result that is consistent with the view that the β chains indeed provide an important interaction between the AP complexes and clathrin. Received 7 April 1997; received after revision 22 May 1997; accepted 28 May 1997     

Leukocyte integrins and inflammation

Leukocyte adhesion is of pivotal functional importance. Without adequate adhesion, T lymphocytes and natural killer cells are not cytotoxic, B cells cannot develop into antibody secreting plasma cells, leukocytes do not home into inflamed tissues and myeloid cells are not able to phagocytize or exhibit chemotactic responses. During evolution several leukocyte adhesion molecules have developed belonging to a few molecular families. Among these, the leukocyte-specific integrins (β ₂ integrins, CD11/CD18 molecules) are among the most important. Much progress has taken place during the past few years, and at present we have a considerable knowledge of their structure and function. Inflammation is critically dependent on integrin activity, and its regulation forms the topic of this short review.     

Research Articles¶Naturally occurring 2′-O-methylpurine nucleosides with hypotensive properties

2′-O-Methylinosine (1) has been isolated for the first time and shown to be an intrinsic hypotensive principle. Its probable in vivo precursor, 2′-O-methyladenosine (3), showed stronger and even orally potent hypotensive activity. Resistance of the methyladenosine (3) against adenosine deaminase is thought to contribute to its long-lasting activity. The effect of both nucleosides (1 and 3) was not accompanied with any significant change in heart rate, which is often observed with adenosine. Received 2 October 1997; accepted 28 October 1997     

Human Genome and Diseases:¶Apaf1 in developmental apoptosis and cancer: how many ways to die?

Apaf1 has been described as the core of the apoptosome. Deficiency in murine Apaf1 leads to embryonic lethality with a phenotype affecting many aspects of developmental apoptosis. In the developing brain, Apaf1 is a death regulator of the neuronal founder cells. Combined intercrosses of mouse lines mutant for members of the mitochondrial death pathway are providing us with some clues about the relative regulation existing among neuronal cell populations. Apaf1-deficient embryos display an interesting phenotype in the inner ear and in limb development, which involves different caspase-dependent and -independent pathways. Moreover, APAF1 is mutated in human melanomas, and its depletion contributes to malignant transformation in a mouse model of cancer. This review has a double aim: the analysis of the alternatives taken by the embryo to bring into the suicidal program different cells at different stages, and the relevance of APAF1 in the onset and progression of cancer. Received 5 March 2001; received after revision 19 April 2001; accepted 4 May 2001     

Clostridial neurotoxins: structure-function led design of new therapeutics

The neurotoxins produced by various species of Clostridia are the causative agents of botulism and tetanus. The ability of the toxins, specifically those of the botulinum neurotoxin family, to disrupt neurotransmission has been exploited for use in several medical indications and now represents the therapeutic option of choice in a number of cases. Clostridial neurotoxins have been discovered to have a multi-domain structure that is shared between the various proteins of the family, and it has also been determined that each domain contributes a specific role to the holotoxin. The extensive use of recombinant expression approaches, along with solution of multiple crystallographic structures of individual domains, has enabled researchers to explore structurefunction relationships of the toxin domains more closely. These advances have facilitated a greater understanding of the potential use of individual domains for a wide variety of purposes, including the development of new therapeutics. Received 21 October 2005; received after revision 10 November 2005; accepted 16 November 2005     

Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide

Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172. Received 7 December 1998; received after revision 15 March 1999; accepted 22 March 1999     

Heat shock protein gene expression during Xenopus development

Stress-induced heat shock protein gene expression is developmentally regulated during early embryogen esis of the frog, Xenopus laevis. For example, a number of heat shock protein genes, such as hsp70, hsp90, and ubiquitin are not heat-inducible until after the midblastula stage of embryogenesis. Furthermore, the family of small heat shock protein genes, hsp30, are differentially expressed after the midblastula stage as well as being regulated at the level of mRNA stability. Many of these stress proteins are also synthesized constitutively during oogenesis and embryogenesis during which they may act as molecular chaperones as well as being involved in sequestering proteins in an inactive state until required by the developing embryo. Furthermore the induction of these stress protein genes has been correlated with enhanced thermoresistance. During stressful conditions heat shock proteins probably prevent aggregation or misfolding of damaged protei ns within the embryo.     

SecB,one small chaperone in the complex milieu of the cell

SecB is only one of a plethora of cytosolic chaperones in E. coli whose common property is that they bind nonnative proteins. It plays a crucial role during protein export via the general secretory pathway by modulating the partitioning of precursors between folding or aggregation and delivery to the membrane-bound translocation apparatus. In this latter role SecB demonstrates specific binding to a unique partner, SecA. SecB has the potential to participate in functions outside of export acting as a general nonspecific chaperone to provide buffering capacity of the nonnative state of proteins in the cytosolic pool. We discuss the interactions of SecB with its many binding partners in light of its recently determined structure, emphasizing both kinetic and thermodynamic parameters. RID="*" ID="*"Corresponding author.     

Structure of effusol: A new phenolic constituent fromJuncus effusus

Summary Effusol, a relatively rare alkylated phenolic 9, 10-dihydrophenanthrene has been isolated fromJuncus effusus. Its structure has been established mainly on the basis of¹H and¹³C-NMR spectra.     

MDR1, cholesterol certification and cell growth: a comparative study in normal and multidrug-resistant KB cell lines

The product of the MDR1 gene (P-gp) has been implicated in the transport of cholesterol from plasma membrane to endoplasmic reticulum for esterification. In previous studies on leukemia cell lines, we suggested that cholesterol esterification may regulate the rate of cell growth and that the MDR1 gene might be involved in this process by modulating intracellular cholesterol esters levels. To further investigate this matter, the rate of cell growth, cholesterol metabolism, expression of the MDR1 gene, and P-gp activity were compared in KB cell lines displaying differences in expression and function of P-gp (drug-sensitive phenotype versus MDR phenotype). The rate of cell growth correlated with cholesterol esterification in all KB cell lines, whereas the over-expression of MDR1 observed in the MDR cell lines was not always associated with an increased capacity of cells to esterify cholesterol. Two known inhibitors of P-gp activity, progesterone and verapamil, strongly inhibited both cholesterol esterification and cell proliferation in all KB cell lines, but they affected intracellular accumulation of labeled vinblastine only in MDR cell lines. These results further support a role for cholesterol esters in the regulation of cell growth and suggest that the P-gp expressed in MDR KB cells is not involved in the general process leading to cholesterol esterification. Received 14 February 2000; received after revision 10 April 2000; accepted 8 May 2000