STIMULATION OF PHOSPHOLIPASE C-BETA(2) BY RECOMBINANT GUANINE-NUCLEOTIDE-BINDING PROTEIN BETA-GAMMA DIMERS PRODUCED IN A BACULOVIRUS/INSECT CELL EXPRESSION SYSTEM - REQUIREMENT OF GAMMA-SUBUNIT ISOPRENYLATION FOR STIMULATION OF PHOSPHOLIPASE-C

Recombinant wild-type beta(1) gamma(1) dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and beta(1) gamma 1 dimers carrying a mutation known to block gamma-subunit isoprenylation (beta(1) gamma(1)C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant beta(1) gamma(1) dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble beta(1) gamma(1) preparation contained approximately equal amounts of non-isoprenylated and isoprenylated beta(1) gamma(1) dimers. Soluble wild-type and mutant beta(1) gamma(1) dimers and native beta(1) gamma(1) dimers purified from bovine retina were reconstituted with recombinant phospholipase C-beta(2). Only isoprenylated beta(1) gamma(1) dimers were capable of stimulating phospholipase C-beta(2). The results show that gamma-subunit isoprenylation and/or additional post-translational processing of the protein are required for beta gamma subunit stimulation of phospholipase C.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF A SALMFAMIDE-LIKE NEUROPEPTIDE IN THE NERVOUS-SYSTEM OF ADULT AND LARVAL STAGES OF THE HUMAN BLOOD FLUKE, SCHISTOSOMA-MANSONI

The localization and distribution of SALMFamide immunoreactivity (IR), SI(GFNSALMFamide), in the nervous system of both the adult and larval stages of the trematode Schistosoma mansoni has been determined by an indirect immunofluorescent technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the nervous system of adult male and female S. mansoni. In the central nervous system (CNS), IR was evident in nerve cells and fibres in the anterior ganglia, cerebral commissure and dorsal and ventral nerve cords. In the peripheral nervous system (PNS), IR was apparent in nerve plexuses associated with the subtegmental musculature, oral and ventral suckers, the lining of the gynaecophoric canal, and in fine nerve fibres innervating the dorsal tubercles of the male worm. In the reproductive system of male and female worms, S1-IR was only observed around the ootype/Mehlis' gland complex in the female. Immunostaining was also evident in the nervous system of both miracidium and cercarial larval stages. A post-embedding, IgG-conjugated colloidal gold immunostaining technique was employed to examine the subcellular distribution of SALMFamide-IR in the CNS of S. mansoni. Gold labelling of peptide was localized over dense-cored vesicles within nerve cell bodies and fibres constituting the neuropile of the anterior ganglia, cerebral commissure and nerve cords of the CNS. Antigen pre-absorption studies indicated that the results obtained do suggest S1-like immunostaining and not cross-reactivity with other peptides, in particular FMRFamide.

ULTRASTRUCTURAL-LOCALIZATION OF PANCREATIC POLYPEPTIDE AND FMRFAMIDE IMMUNOREACTIVITIES WITHIN THE CENTRAL-NERVOUS-SYSTEM OF THE NEMATODE, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

A post-embedding immunogold technique has been used to examine the subcellular distribution of immunoreactivities to vertebrate pancreatic polypeptide (PP) and to the invertebrate peptide, FMRFamide within the central nervous system (CNS) of the nematode, Ascaris suum. Gold labelling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the main ganglia and nerve cords in the CNS. Double-labelling of peptides demonstrated an apparent co-localization of PP and FMRFamide immunoreactivities in the same dense-cored vesicles, although populations of dense-cored vesicles that labelled solely for FMRFamide were also evident. Antigen preabsorption studies indicated little or no cross-reactivity between the two antisera.

ULTRASTRUCTURAL-LOCALIZATION OF FMRFAMIDE-IMMUNOREACTIVITY AND PANCREATIC POLYPEPTIDE-IMMUNOREACTIVITY WITHIN THE CENTRAL-NERVOUS-SYSTEM OF THE LIVER FLUKE, FASCIOLA-HEPATICA (TREMATODA, DIGENEA)

A post-embedding immunogold technique was used to examine the subcellular distribution of immunoreactivities to the invertebrate peptide, FMRFamide, and to vertebrate pancreatic polypeptide (PP) within the central nervous system of the trematode, Fasciola hepatica. Gold labeling of peptide was localised exclusively over both dense-cored and ellipsoidal electron-dense vesicles (with a homogeneous matrix) present within nerve cell bodies, small and 'giant' nerve processes of the neuropile in the cerebral ganglia and transverse commissure, as well as in the main longitudinal nerve cords. Double labeling demonstrated an apparent co-localisation of FMRFamide and PP immunoreactivities in the same dense-cored vesicles, although populations of ellipsoidal electron-dense vesicles that labeled solely for FMRFamide were also evident. Antigen pre-absorption studies indicated little, if any, cross-reactivity of the two antisera.

IMMUNOELECTRON MICROSCOPIC STUDIES OF REGULATORY PEPTIDES IN THE NERVOUS-SYSTEM OF THE MONOGENEAN PARASITE, DICLIDOPHORA-MERLANGI

Specific antisera, directed against the highly conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) and the invertebrate peptide FMRFamide, have been used in conjunction with post-embedding, IgG-conjugated colloidal gold immunostaining to demonstrate peptide immunoreactivity at subcellular level in the nervous system of adult Diclidophora merlangi. Gold labelling revealed that immunoreactivity for PP and FMRFamide was localized exclusively in dense-cored vesicles occupying the majority of axons in the central nervous system. Double-labelling demonstrated an apparent co-localization of PP and FMRFamide in the same dense-cored vesicles. Antigen preabsorption experiments indicated cross-reactivity of the two antisera as unlikely, and that some if not all of the PP/FMRFamide immunostaining in the parasite was due to a neuropeptide F-like peptide.

Low-dose hypersensitivity in Chinese hamster V79 cells targeted with counted protons using a charged-particle microbeam

The Gray Laboratory charged-particle microbeam has been used to assess the clonogenic ability of Chinese hamster V79 cells after irradiation of their nuclei with a precisely defined number of protons with energies of 1.0 and 3.2 MeV. The microbeam uses a 1-mum. silica capillary collimator to deliver protons to subcellular targets with high accuracy. The detection system is based on a miniature photomultiplier tube positioned above the cell dish, which detects the photons generated by the passage of the charged particles through an 18-mum-thick scintillator placed below the cells. With this system, a detection efficiency of greater than 99% is achieved. The cells are plated on specially designed dishes (3-mum-thick Mylar base), and the nuclei are identified by fluorescence microscopy. After an incubation period of 3 days, the cells are revisited individually to assess the formation of colonies from the surviving cells. For each energy investigated, the survival curve obtained for the microbeam shows a significant deviation below I Gy from a response extrapolated using the LQ model for the survival data above 1 Gy. The data are well fitted by a model that supports the hypothesis that radioresistance is induced by low-dose hypersensitivity. These studies demonstrate the potential of the microbeam for performing studies of the effects of single charged particles on cells in vitro. The hypersensitive responses observed are comparable with those reported by others using different radiations and techniques. (C) 2001 by Radiation Research Society.

A focused ultrasoft X-ray microbeam for targeting cells individually with submicrometer accuracy

The application of microbeams is providing new insights into the actions of radiation at the cell and tissue levels. So far, this has been achieved exclusively through the use of collimated charged particles. One alternative is to use ultrasoft X rays, focused by X-ray diffractive optics. We have developed a unique facility that uses 0.2-0.8-mm-diameter zone plates to focus ultrasoft X rays to a beam of less than 1 mum diameter. The zone plate images characteristic K-shell X rays of carbon or aluminum, generated by focusing a beam of 5-10 keV electrons onto the appropriate target. By reflecting the X rays off a grazing-incidence mirror, the contaminating bremsstrahlung radiation is reduced to 2%. The focused X rays are then aimed at selected subcellular targets using rapid automated cell-finding and alignment procedures; up to 3000 cells per hour can be irradiated individually using this arrangement. (C) 2001 by Radiation Research Society.

Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using GO-G, confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser(15) (p53(Ser15)), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologicafly distinct subpool of p53(Ser15) is ATM dependent and resistant to 26S-proteasomal degradation. p53(Ser15) colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear micro-beam irradiation studies confirm p53 S,,15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser(15) or Ser(18) phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21(WAF) decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis. (Cancer Res 2005; 65(23): 10810-21).

NEUROPEPTIDE F-IMMUNOREACTIVITY IN THE TETRATHYRIDIUM OF MESOCESTOIDES-CORTI (CESTODA, CYCLOPHYLLIDEA)

The distribution pattern and subcellular localisation of neuropeptide F (NPF) immunoreactivity (IR) in the tetrathyridium stage of Mesocestoides corti were investigated by whole-mount immunocytochemistry in conjunction with confocal scanning laser microscopy (CSLM) and by immunoelectron microscopy using immunogold labeling. Using an antiserum directed to the C-terminal decapeptide amide (residues 30-39) of synthetic NPF (Moniezia expansa), CSLM revealed NPF-IR throughout the central and peripheral nervous systems of parental and dividing tetrathyridia. Ultrastructurally, gold labeling of NPF-IR was confined to the contents of the smaller of the two sizes of electron-dense neuronal vesicle identified.

NEUROPEPTIDE F-IMMUNOREACTIVITY IN THE MONOGENEAN PARASITE DICLIDOPHORA-MERLANGI

The localisation and distribution of neuropeptide F (NPF)-immunoreactivity (IR) in the monogenean fish-gill parasite, Diclidophora merlangi, have been investigated by whole-mount immunocytochemistry interfaced with confocal scanning laser microscopy and, at the ultrastructural level, by indirect immunogold labeling. Using antisera directed to intact synthetic NPF (Moniezia expansa, residues 1-39) or to the C-terminal decapeptide (residues 30-39) of synthetic NPF (M. expansa), immunostaining was found throughout the central (CNS) and peripheral nervous systems (PNS), including the innervation of the reproductive system. Immunoreactivity was found to be more intense using the antiserum to the C-terminal decapeptide fragment of NPF. At the subcellular level, gold labeling of NPF-IR was found exclusively over the contents of dense-cored vesicles that occupied nerve axons of both the CNS and the PNS. The distribution pattern of immunostaining for NPF mirrored exactly that previously documented for the vertebrate pancreatic polypeptide (PP) family of peptides and for FMRFamide. This finding and the results of preabsorption experiments strongly suggest that NPF is the predominant native neuropeptide in D. merlangi and that it accounts for most of the immunostaining previously obtained with PP and FMRFamide antisera.

Protein kinases C: potential targets for intervention in diabetic nephropathy

Protein kinases C are a family of serine threonine protein kinases that play key roles in extracellular signal transduction. Inappropriate activation of protein kinase C has been implicated in the pathophysiology of many diseases, including diabetes mellitus. Indeed, protein kinase C activation may contribute not only to the pathogenesis of diabetic complications such as nephropathy and retinopathy, but also to insulin resistance. Growing awareness that protein kinase C isoforms subserve specific subcellular functions has led to the development of isoform-specific inhibitors, which may be useful investigational tools and therapeutic agents for attenuating the effects of inappropriate protein kinase C activity. Here we review the role played by protein kinases C in diabetic nephropathy and the recent progress that has been made to modulate its activity using specific inhibitors. Curr Opin Nephrol Hypertens 7:563-570. (C) 1998 Lippincott Wiiliams & Wilkins.

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx(EcO157)) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx(EcO157) consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.

FUS expression alters the differentiation response to all-trans retinoic acid in NB4 and NB4R2 cells

The FUS gene is overexpressed in acute myeloid leukaemia (AML) patients and has roles in transcription and mRNA processing. We used ectopic expression of FUS and FUS antisense sequences to assess the effect of modulation of FUS expression in all-trans retinoic acid (ATRA)-sensitive (NB4) and insensitive (NB4R2) human acute promyelocytic (APL) cell lines which express the t(15:17) translocation. Growth, viability and differentiation patterns were maintained, but the expression of the FUS antisense construct in both the cell lines altered the response to ATRA: the previously ATRA-sensitive NB4 cells exhibited resistance; whilst the previously resistant NB4R2 cells showed a differentiation response to treatment.

Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides

Translocation of lipid-linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni-encoded ABC-type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate-linked heptasaccharide intermediate involved in the recently identified bacterial N-linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N-linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O-antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.

Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit

One of the most common pathways for the export of O-specific lipopolysaccharide (LPS) across the plasma membrane requires the participation of the Wzx protein. Wzx belongs to a family of integral membrane proteins that share little conservation in their primary amino acid sequence, making it difficult to delineate functional domains. This paper reports the cloning and expression in Escherichia coli K-12 of various Wzx homologues from different bacteria as FLAG epitope-tagged protein fusions. A reconstitution system for O16 LPS synthesis was used to assess the ability of each Wzx protein to complement an E. coli K-12 Deltawzx mutant. The results demonstrate that Wzx proteins from O-antigen systems that use N-acetylglucosamine or N-acetylgalactosamine for the initiation of the biosynthesis of the O repeat can fully complement the formation of O16 LPS. Partial complementation was seen with Wzx from Pseudomonas aeruginosa, a system that uses N-acetylfucosamine in the initiation reaction. In contrast, there was negligible complementation with the Wzx protein from Salmonella enterica, a system in which galactose is the initiating sugar. These results support a model whereby the first sugar of the O repeat can be recognized by the O-antigen translocation machinery.