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Recent advances in the characterization of ambient pH regulation of gene expression in filamentous fungi and yeasts.

All microorganisms must adapt to the pH of their environment. One aspect of this adaptation, particularly important for organisms that grow over a wide pH range, is the ability to express appropriately genes whose roles ultimately involve functions at the cell surface or in the environment. Genes encoding permeases, secreted enzymes, enzymes involved in synthesis of exported metabolites such as toxins and antibiotics, and probably enzymes modifying secreted proteins posttranslationally all fall into this category. Here we discuss the most recent findings on the transcriptional regulatory system in fungi that enables such genes to be expressed only when the ambient pH is conducive to their ultimate functions. The intriguing issue of how pH is sensed and how the resulting signal is transmitted to the transcription factor involves at least one late endosome component. Proper functioning of the regulatory system responding to ambient pH is essential for fungal pathogenicity of both animals and plants.

Aspergillus nidulans↗

Bacteria and gastrointestinal secretion and motility.

The composition of the flora of the large bowel is extremely complex, as are the combination of factors involved in its control. As would be expected, although the host affects the gut flora, equally the gut flora affects the host. One of the results of this interplay is that subtle changes can lead to a marked effect on the host resulting in disease. It is now well established that bacteria can alter both secretion and motility of the gastrointestinal tract. However the effect on both of these functions has been best studied in the small bowel, this being especially true for studies on motility. In order to appreciate fully the complexity of the situation and to understand the activities of the flora that can modify gut function a brief overview of the flora of the gastrointestinal tract in health and disease and the factors involved in its regulation will be given. This will be followed by a general description of the effects of this flora on the gut, and a specific account of how large bowel motility and secretion can be altered.

Animals↗

The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host.

Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.

Amino Acid Sequence↗

Thyroliberin action in pituitary cells is not inhibited by pertussis toxin.

The effects of pertussis toxin on the responses of rat pituitary-tumour (GH) cells to thyrotropin-releasing hormone (thyroliberin, TRH) were examined. Treatment of cells with pertussis toxin did not alter the affinity or concentration of TRH receptors, or the sensitivity of the TRH receptor to inhibition by guanine nucleotides. TRH caused an increase in low-Km GTPase activity in membrane-containing fractions from both control and pertussis-toxin-treated cells. TRH stimulation of inositol phosphate formation was insensitive to pertussis toxin. TRH caused a biphasic increase in the concentrations of cytosolic free Ca2+ as monitored by intracellularly trapped Quin 2, and this increase was the same in control and toxin-treated cultures. The toxin did not alter the increase in prolactin and growth-hormone (somatotropin) release stimulated by TRH or shift the TRH dose-response curve, and it did not affect the TRH-induced rise in prolactin synthesis measured over 24 h. However, pertussis toxin did block the ability of somatostatin and muscarinic agonists to inhibit prolactin and growth-hormone secretion stimulated by vasoactive intestinal peptide when analysed under the same conditions as those in which the TRH system was unaffected. These data indicate that the guanine nucleotide effects on TRH binding and activity are not mediated by Ni, but possibly by another member of the family of guanine-nucleotide-dependent regulatory proteins.

Animals↗

The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant.

Systemic and mucosal immune responses were effectively induced following the subcutaneous administration of Haemophilus influenzae type b oligosaccharide conjugated to diphtheria toxoid vaccine in a formulation containing the active form of vitamin D3. IgA and IgG antibodies with specificity for both the protein and oligosaccharide components of the vaccine were detectable in mucosal secretions following immunization. The IgA and IgG mucosal antibodies were produced locally, and were functional as demonstrated by their diphtheria toxin neutralizing activity. Our data suggests that subcutaneous tissues can effectively serve as effective antigen presenting sites for both mucosal and systemic immune responses to antigens administered in combination with vitamin D3.

Adjuvants, Immunologic↗

Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved.

There are common themes among bacteriophage-encoded virulence factors, which include the well-characterized bacterial toxins and proteins that alter antigenicity as well as several new classes of bacteriophage-encoded proteins such as superantigens, effectors translocated by a type III secretion system, and proteins required for intracellular survival and host cell attachment. These virulence factors are encoded by a diversity of bacteriophages, members of the viral families Siphoviridae, Podoviridae, Myoviridae and Inoviridae, with some bacteriophages having characteristics of more than one virus family. The location of virulence genes within the bacteriophage genomes is non-random and consistent with an origin via imprecise prophage excision or as either transferable cassettes or integral components of the bacteriophage genome.

Bacterial Adhesion↗

Toxic effects of tetanus toxin on GG2EE macrophages: prevention of gamma interferon-mediated upregulation of lysozyme-specific mRNA levels.

By using a nonneuronal cell system, evidence has previously been provided that tetanus toxin (TT) intoxication occurs in macrophages, impairing their secretory activity as well as their antitumoral activity. In particular, both secreted and total lysozyme (LZM) activities are reduced by TT treatment, provided that GG2EE macrophages have been preexposed to gamma interferon (IFN-gamma). In an attempt to provide insight into the molecular mechanisms underlying this phenomenon, we focused our attention on the levels of LZM-specific transcripts. GG2EE macrophages preexposed to IFN-gamma exhibited augmented levels of LZM-specific mRNA. Such an effect was detected 1 h after removal of IFN-gamma, peaked at 3 h, and gradually decreased with time in culture. Exposure of IFN-gamma-pretreated GG2EE macrophages to TT resulted in the prevention of the IFN-gamma-mediated upregulation of LZM mRNA levels. The phenomenon was mediated by the holotoxin (> or = 1 micrograms/ml) and abrogated by preexposure of the macrophages to the C fragment of TT. Protein kinase C (PKC) and Ca(2+)-calmodulin-dependent PK were likely involved in the IFN-gamma-mediated upregulation of LZM mRNA levels and biological activity, as assessed by PK inhibitors. Furthermore, PK inhibitors mimicked TT in impairing LZM activity of GG2EE macrophages, thus suggesting that impairment of PKC and/or the Ca(2+)-calmodulin-dependent PK pathway(s) may be one of the events involved in TT intoxication of macrophages.

Animals↗

Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine.

Lactococcus lactis is a nonpathogenic and noncolonizing bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes. To determine whether lactococci can also deliver cytokines to the immune system, we have constructed novel constitutive expression strains of L. lactis which accumulate a test antigen, tetanus toxin fragment C (TTFC), within the cytoplasmic compartment and also secrete either murine interleukin-2 (IL-2) or IL-6. When mice were immunized intranasally with various different expression strains of L. lactis, the anti-TTFC antibody titers increased more rapidly and were substantially higher in mice immunized with the bacterial strains which secreted IL-2 or IL-6 in addition to their production of TTFC. This adjuvant effect was lost when the recombinant strains of L. lactis were killed by pretreatment with mitomycin C and could therefore be attributed to the secretion of IL-2 or IL-6 by the recombinant lactococci. These results provide the first example of the use of a cytokine-secreting, noninvasive experimental bacterial vaccine vector to enhance immune responses to a coexpressed heterologous antigen and point the way to experiments which will test the possible therapeutic efficacy of this mode of cytokine delivery.

Administration, Intranasal↗

Pseudomonas aeruginosa type III-secreted toxin ExoT inhibits host-cell division by targeting cytokinesis at multiple steps.

Pseudomonas aeruginosa is an opportunistic pathogen that requires preexisiting epithelial injury to cause acute infections. We report that P. aeruginosa inhibits mammalian cytokinesis in a type III secretion system and exotoxin T (ExoT)-dependent manner. ExoT is a bifunctional type III secretion system effector protein that contains an N-terminal GTPase-activating protein domain and a C-terminal ADP-ribosyl transferase domain. Each of its domains inhibits cytokinesis in a kinetically, morphologically, and mechanistically distinct manner. The GTPase-activating protein-mediated inhibition of cytokinesis occurs early, likely as a consequence of its inhibitory effect on RhoA. The ADP-ribosyl transferase domain inhibits late steps of cytokinesis by blocking syntaxin-2 localization to the midbody, an event essential for completion of cytokinesis. These findings provide an example of a bacterial pathogen targeting cytokinesis.

ADP Ribose Transferases↗

Effect of systemic pH on models of altered ileal transport in the rat.

Decreases in arterial pH markedly increase sodium, chloride, and water absorption in the normal ileum and can reverse ongoing cholera toxin-induced secretion. In the current study we examined whether these effects of pH are evident in other models of ileal secretion, and in a model of increased absorption. Rats were anesthetized and transport was measured in ileal loops during respiratory acidosis and alkalosis. Decreases in arterial pH increased absorption equally in control loops and in adjacent loops perfused with a Ringer's solution containing ST toxin (cyclic guanosine monophosphate-mediated secretion), hypertonic mannitol (passive, osmotically mediated secretion), or glucose. Decreases in arterial pH increased absorption in a similar way in loops exposed to cholera toxin (cyclic adenosine monophosphate-mediated secretion) that were then perfused with glucose-Ringer's solution. Alterations in arterial and luminal pH did not affect glucose absorption. These results suggest that the effect of arterial pH on ileal absorption occurs by a mechanism that is independent of these various means of altering transport.

Absorption↗

Botulinum toxins--cause of botulism and systemic diseases?

Toxins of Clostridium botulinum (types A-G) are known as 'neurotoxins', causing the clinically well-known picture of flaccid muscular paralysis. The molecular biological background is the blocking of acetylcholine secretion in neuromuscular junctions by enzymatic cleavage of molecules forming the machinery of exocytosis. Two 'non-neurotoxins' (types C2, C3) are produced by some strains of C. botulinum types C and D. These affect the cytoskeleton by ribosylating actin filaments. All these toxins are used as cell biological tools for the study of specific actions and effects in different eukaryotic cells. Pharmaceutical and molecular biological research has shown their influence on several crucial organs (or cell cultures thereof) of humans and animals (brain and spinal cord, cerebellum, hippocampus, hypophysis, pancreas, adrenal glands, salivary glands and others). Under natural conditions, botulinum toxins may pass the intestinal barrier and circulate in the bloodstream for a certain time. Carriers occurring naturally in food, such as wheat germ agglutinin, digitonin or saponin, and bacterial toxins such as streptolysin O, perfringolysins, C2 toxin or botulinolysin may also form pores in cell walls. They facilitate the entry of botulinum toxins into cells that may not have natural binding receptors. It is concluded that in vivo actions of different botulinum toxins after their entry into the organism may contribute to the onset of different diseases of hitherto cryptogenic origin. Some examples are given and future problems are discussed.

Animals↗

Strong mucosal and systemic immunities induced by nasal immunization with anthrax protective antigen protein incorporated in liposome-protamine-DNA particles.

PURPOSE: The very lengthy and complicated dosing schedule of the current anthrax vaccine adsorbed, which was licensed in the USA for the prevention of cutaneous anthrax infection, calls for the development of an efficacious and easily administrable vaccine to prevent against the most lethal form of anthrax infection, the inhalation anthrax. We propose to develop a nasal anthrax vaccine using anthrax protective antigen (PA) protein carried by liposome-protamine-DNA (LPD) particles. METHODS: PA was incorporated in LPD particles and nasally dosed to mice. The resulting PA-specific immune response and lethal toxin neutralization activity were measured. RESULTS: Mice nasally immunized with PA incorporated into LPD particles developed both systemic and mucosal anti-PA responses. The anti-PA immunities induced included the production of anti-PA antibodies (IgG and IgM in the serum and IgA in nasal and lung mucosal secretions) and the proliferation of splenocytes after in vitro stimulation. The anti-PA IgG subtype induced was mainly IgG1. Finally, anthrax lethal toxin neutralization activity was detected both in the serum and in the mucosal secretions. CONCLUSIONS: The anti-PA immune response induced by nasal PA incorporated in LPD was comparable to that induced by nasal PA adjuvanted with cholera toxin or subcutaneously injected PA adjuvanted with aluminum hydroxide.

Administration, Intranasal↗

The role of ADP-ribosylation and G(M1)-binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibrio cholerae cholera toxin.

The mucosal route of vaccination has attracted a great deal of attention recently. Not only is mucosal application of vaccines, for example, orally or intranasally, particularly convenient, it also offers the possibility to induce locally produced and secreted S-IgA antibodies in addition to systemic IgG antibodies. These IgA antibodies are known to play a key role in protection against pathogens that invade the host through mucosal surfaces. Induction of such responses is not readily achieved by currently used vaccination strategies, which generally involve intramuscular or subcutaneous injection with inactivated pathogens or antigens thereof. For the induction of a mucosal immune response, the vaccine needs to be applied locally. However, local vaccination with non-replicating antigens is usually ineffective and may result in tolerance unless a mucosal immunoadjuvant is included. The most potent mucosal immunoadjuvants known to date are probably cholera toxin (CT) and the closely related Escherichia coli heat-labile enterotoxin (LT). Although CT and LT have become standard adjuvants for experimental mucosal vaccines, the intrinsic toxicity has thus far precluded their use as adjuvants for human vaccine formulations. In the present review, the mucosal immunogenic and adjuvant properties of LT and CT are described, with special emphasis on the functional role of the individual subunits on their immune-stimulatory properties.

Adenosine Diphosphate Ribose↗

Cellular and molecular aspects of drug transport in the kidney.

The kidney plays an important role in the elimination of numerous hydrophilic xenobiotics, including drugs, toxins, and endogenous compounds. It has developed high-capacity transport systems to prevent urinary loss of filtered nutrients, as well as electrolytes, and simultaneously to facilitate tubular secretion of a wide range of organic ions. Transport systems for organic anions and cations are primarily involved in the secretion of drugs in renal tubules. The identification and characterization of organic anion and cation transporters have been progressing at the molecular level. To date, many members of the organic anion transporter (OAT), organic cation transporter (OCT), and organic anion-transporting polypeptide (oatp) gene families have been found to mediate the transport of diverse organic anions and cations. It has also been suggested that ATP-dependent primary active transporters such as MDR1/P-glycoprotein and the multidrug resistance-associated protein (MRP) gene family function as efflux pumps of renal tubular cells for more hydrophobic molecules and anionic conjugates. Tubular reabsorption of peptide-like drugs such as beta-lactam antibiotics across the brush-border membranes appears to be mediated by two distinct H+/peptide cotransporters: PEPT1 and PEPT2. Renal disposition of drugs is the consequence of interaction and/or transport via these diverse secretory and absorptive transporters in renal tubules. Studies of the functional characteristics, such as substrate specificity and transport mechanisms, and of the localization of cloned drug transporters could provide information regarding the cellular network involved in renal handling of drugs. Detailed information concerning molecular and cellular aspects of drug transporters expressed in the kidney has facilitated studies of the mechanisms underlying renal disposition as well as transporter-mediated drug interactions.

Animals↗

Mercury intoxication: it still exists.

A3-year-old boy presented to the Hospital for Sick Children with systemic symptoms and oropharyngeal and peripheral extremity changes suggestive of Kawasaki disease. He was found to have severe hypertension. Investigation for a catecholamine-secreting tumor was negative. Toxins were considered when the patient's 20-month-old brother presented with similar symptoms, and the boys were subsequently diagnosed with elemental mercury poisoning. We review the literature on mercury intoxication and discuss the historical context, clinical syndrome (acrodynia), treatment, and radiologic findings of this unusual diagnosis.

Acrodynia↗

Yeast dsRNA viruses: replication and killer phenotypes.

The cytoplasmic L-A dsRNA virus of Saccharomyces cerevisiae consists of a 4.5 kb dsRNA and the two gene products it encodes; the capsid (cap) and at least one copy of the capsid-polymerase (cap-pol) fusion protein. Virion cap-pol catalyses transcription of the plus (sense)-strand; this is extruded from the virus and serves as messenger for synthesis of cap and cap-pol. Nascent cap-pol binds to a specific domain in the plus strand to initiate encapsidation and then catalyses minus-strand synthesis to complete the replication cycle. Products of at least three host genes are required for replication, and virus copy number is kept at tolerable levels by the SKI antivirus system. S. cerevisiae killer viruses are satellite dsRNAs that use a similar encapsidation domain to parasitize the L-A replication machinery. They encode precursors of secreted polypeptide toxins and immunity (specific resistance) determinants and are self-selecting. Three unique killer types, K1, K2 and K28, are currently recognized. They are distinguished by an absence of cross-immunity and by toxin properties and lethal mechanisms; while K1 and K2 toxins bind to cell-wall glucan and disrupt membrane functions, K28 toxin binds to mannoprotein and causes inhibition of DNA synthesis.

DNA Topoisomerases, Type I↗

Renal tubular drug transporters.

The kidney plays an important role in the elimination of numerous hydrophilic xenobiotics, including drugs, toxins, and endogenous compounds. It has developed high-capacity transport systems to prevent urinary loss of filtered nutrients, as well as electrolytes, and simultaneously to facilitate tubular secretion of a wide range of organic ions. Transport systems for organic anions and cations are primarily involved in the secretion of drugs in renal tubules. The identification and characterization of organic anion and cation transporters have been progressing at the molecular level. To date, many members of the organic anion transporter, organic cation transporter, and organic anion-transporting polypeptide families have been found to mediate the transport of diverse organic ions. It has also been suggested that ATP-dependent primary active transporters such as MDR1/P-glycoprotein and the multidrug resistance-associated protein family function as efflux pumps of renal tubular cells for more hydrophobic molecules and anionic conjugates. Tubular reabsorption of peptide-like drugs such as beta-lactam antibiotics across the brush-border membranes appears to be mediated by two distinct H+/peptide cotransporters: PEPT1 and PEPT2. Renal disposition of drugs occurs through interaction with these diverse secretory and absorptive transporters in renal tubules. Studies of the functional characteristics, such as substrate specificity and transport mechanisms, and of the localization of drug transporters could provide information regarding the cellular network involved in renal handling of drugs. Detailed information concerning molecular and cellular aspects of drug transporters expressed in the kidney has facilitated studies of the mechanisms underlying renal disposition as well as transporter-mediated drug interactions.

Animals↗