Sequence Diversity of Genes Encoding Botulinum Neurotoxin Type F

Botulism due to type F botulinum neurotoxin (BoNT/F) is rare (<1% of cases), and only a limited number of clostridial strains producing this toxin type have been isolated. As a result, analysis of the diversity of genes encoding BoNT/F has been challenging. In this study, the entire bont/F nucleotide sequences were determined from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak investigations. We examined proteolytic and nonproteolytic Clostridium botulinum type F strains, bivalent strains, including Bf and Af, and Clostridium baratii type F strains. Phylogenetic analysis revealed that the bont/F genes examined formed 7 subtypes (F1 to F7) and that the nucleotide sequence identities of these subtypes differed by up to 25%. The genes from proteolytic (group I) C. botulinum strains formed subtypes F1 through F5, while the genes from nonproteolytic (group II) C. botulinum strains formed subtype F6. Subtype F7 was composed exclusively of bont/F genes from C. baratii strains. The region of the bont/F5 gene encoding the neurotoxin light chain was found to be highly divergent compared to the other subtypes. Although the bont/F5 nucleotide sequences were found to be identical in strains harboring this gene, the gene located directly upstream (ntnh/F) demonstrated sequence variation among representative strains of this subtype. These results demonstrate that extensive nucleotide diversity exists among genes encoding type F neurotoxins from strains with different phylogenetic backgrounds and from various geographical sources.Botulism is a potentially fatal disease caused solely by the action of serologically distinct neurotoxins (BoNT/A, -B, -C, -D, -E, -F, or -G) which prevent acetylcholine release at neuromuscular junctions, resulting in paralysis. Food-borne botulism may result from the ingestion of a preformed toxin that is produced in inadequately preserved food. Under certain conditions, botulinum neurotoxin-producing Clostridium sp. may colonize and produce toxin in wounds (wound botulism) or in the intestine (infant botulism or adult colonization). Globally, human botulism cases are associated with botulinum neurotoxin serotypes A, B, E, and rarely F. The Centers for Disease Control and Prevention (CDC) maintains active surveillance for botulism cases in the United States. Of 1,269 U.S. cases of botulism reported to the CDC between 1981 and 2002, approximately 1% were due to type F toxin (13). An additional 10 cases of type F botulism were reported to the CDC from 2003 to 2007 (http://www.cdc.gov/nationalsurveillance/botulism_surveillance.html).Type F botulism was first described in 1960 following an outbreak occurring in Denmark involving liver paste (30). The organism isolated in this outbreak metabolically resembled proteolytic Clostridium botulinum strains of types A and B. In a subsequent outbreak, type F toxin was found to be produced by a nonproteolytic C. botulinum strain isolated from venison jerky (29). Bivalent toxin-producing strains have been described, including Bf strains isolated from infants in the United States and England (1, 16, 17, 35) and an Af strain isolated from individuals in Argentina with food-borne botulism (11). Bivalent strains may produce higher titers of one toxin type, which are denoted with a capital letter. The only reported organism isolated from infants with botulism due to type F toxin alone (i.e., not associated with additional serotypes as in bivalent strains) is Clostridium baratii (2, 14, 24). In addition, C. baratii type F has been isolated from adults with botulism (28) as well as suspect foods associated with botulism cases (15; CDC, unpublished data).Botulinum neurotoxin genes (bont) are typically found within toxin gene clusters that include other genes encoding components of the toxin complex (ha70, ha17, ha33, ntnh), regulatory proteins (botR), or proteins with unknown functions (p47, orfX1, orfX2, orfX3). Two general toxin gene cluster arrangements have been described, including the orfX cluster (orfX3-orfX2-orfX1-botR-p47-ntnh-bont) and the ha cluster (ha70-ha17-ha33-botR-ntnh-bont) (21, 22). The bont/F genes of type F and type Bf strains examined by Hill et al. (21) were found in an orfX cluster.The amino acid sequence identities of the BoNT serotypes A to G range from approximately 35 to 70% (36). In addition, within nearly all toxin serotypes, various levels of amino acid sequence variation have been observed, resulting in the identification of toxin subtypes (20, 36, 37).Although a limited number of genes encoding type F botulinum neurotoxin have been sequenced, a comparison of sequences available in public databases indicates that significant diversity exists within this serotype. The nucleotide sequence identity of the type F neurotoxin gene from the proteolytic strain Langeland differs from that of the gene in the nonproteolytic strain 202F by 7%. The type F gene from C. baratii strain ATCC 43756 differs from those of Langeland and 202F by 18% and 20%, respectively. Although the bivalent (Bf) strain CDC3281 is phenotypically proteolytic, the toxin gene shows greater similarity to those from nonproteolytic strains (34). In addition to metabolic differences observed between proteolytic and nonproteolytic C. botulinum strains as well as C. baratii, these organisms are phylogenetically distinct based on differences among their 16S rRNA sequences (5, 20).In order to define the degree of genetic diversity among strains encoding botulinum neurotoxin type F, we sequenced the bont/F gene and partially characterized the toxin gene cluster by using a panel of 33 strains with diverse origins. These strains were selected from those available in the CDC culture collection as well as several isolated in Argentina. The only reported Af strains have been isolated in Argentina. Among 68 outbreaks of serotype-confirmed food-borne botulism in Argentina between 1922 and 2007, type F was isolated in two outbreaks, and type Af was isolated in one outbreak. In addition, Lúquez et al. (26) reported isolation of type F and Af strains from Argentine soils.Here, we report that analysis of the bont/F genes from the strains examined in this study revealed a high degree of nucleotide sequence heterogeneity and the identification of seven type F subtypes (F1 to F7). In addition, the nucleotide sequence of one subtype (F5) has not been previously reported and contains evidence of recombination compared to the other subtypes.     

Almond (Prunus dulcis (Mill.) D.A. Webb) polyphenols: From chemical characterization to targeted analysis of phenolic metabolites in humans

In this paper, a survey of our studies on almond polyphenols including their chemical characterization and further bioavailability in humans is reported. Combination of analytical techniques (LC-DAD/fluorescence, LC/ESI-MS and MALDI-TOF-MS) allowed us, for the first time, the identification of A- and B-type procyanidin, propelargonidin and prodelphinidin polymers in almond skins. Glucuronide, O-methyl glucuronide, sulfate and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and isorhamnetin, and sulfate conjugates of isorhamnetin, together with conjugates of hydroxyphenylvalerolactones were detected in plasma and urine samples after the intake of almond skin polyphenols. In addition, numerous microbial-derived metabolites, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic and hydroxyhippuric acids were also identified. Depending of the type of metabolite, maximum urinary excretion was attained at different time in comparison to the control group in the course of the 24-h period of urine excretion, allowing us to establish the onset of microbial metabolism.     

Impaired muscarinic type 3 (M3) receptor/PKC and PKA pathways in islets from MSG-obese rats

Monosodium glutamate-obese rats are glucose intolerant and insulin resistant. Their pancreatic islets secrete more insulin at increasing glucose concentrations, despite the possible imbalance in the autonomic nervous system of these rats. Here, we investigate the involvement of the cholinergic/protein kinase (PK)-C and PKA pathways in MSG β-cell function. Male newborn Wistar rats received a subcutaneous injection of MSG (4 g/kg body weight (BW)) or hyperosmotic saline solution during the first 5 days of life. At 90 days of life, plasma parameters, islet static insulin secretion and protein expression were analyzed. Monosodium glutamate rats presented lower body weight and decreased nasoanal length, but had higher body fat depots, glucose intolerance, hyperinsulinemia and hypertrigliceridemia. Their pancreatic islets secreted more insulin in the presence of increasing glucose concentrations with no modifications in the islet-protein content of the glucose-sensing proteins: the glucose transporter (GLUT)-2 and glycokinase. However, MSG islets presented a lower secretory capacity at 40 mM K⁺ (P < 0.05). The MSG group also released less insulin in response to 100 μM carbachol, 10 μM forskolin and 1 mM 3-isobutyl-1-methyl-xantine (P < 0.05, P < 0.0001 and P < 0.01). These effects may be associated with a the decrease of 46 % in the acetylcholine muscarinic type 3 (M3) receptor, and a reduction of 64 % in PKCα and 36 % in PKAα protein expressions in MSG islets. Our data suggest that MSG islets, whilst showing a compensatory increase in glucose-induced insulin release, demonstrate decreased islet M3/PKC and adenylate cyclase/PKA activation, possibly predisposing these prediabetic rodents to the early development of β-cell dysfunction.     

The structure of shallow water fish assemblages in sandy beaches of a tropical bay in the southwestern Atlantic

Ichthyological Research - The spatial and seasonal dynamics of shallow water fish assemblages and their relationship with physical-chemical variables were investigated on Guanabara Bay, one of the...     

Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis

As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 μg against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins. Arch. Insect Biochem. Physiol. 38:53–65, 1998. © 1998 Wiley-Liss, Inc.     

A putative vesicular transporter expressed in Drosophila mushroom bodies that mediates sexual behavior may define a neurotransmitter system

Vesicular transporters are required for the storage of?all classical and amino acid neurotransmitters in synaptic vesicles. Some neurons lack known vesicular transporters, suggesting additional neurotransmitter systems remain unidentified. Insect mushroom bodies (MBs) are critical for several behaviors, including learning, but the neurotransmitters released by the intrinsic Kenyon cells (KCs) remain unknown. Likewise, KCs do not express a known vesicular transporter. We report the identification of a novel Drosophila gene portabella (prt) that is structurally similar to known vesicular transporters. Both larval and adult brains express PRT in the KCs of the MBs. Additional PRT cells project to the central complex and optic ganglia. prt mutation causes an olfactory learning deficit and an unusual defect in the male's position during copulation that is rescued by expression in KCs. Because prt is expressed in neurons that lack other known vesicular transporters or neurotransmitters, it may define a previously unknown neurotransmitter system responsible for sexual behavior and a component of olfactory learning.     

The hexokinase gene family in the zebrafish: Structure,expression, functional and phylogenetic analysis

Hexokinase-catalyzed glucose phosphorylation is the first and crucial step for glucose utilization. Although there are reported studies on glucose metabolism in commercial species, knowledge on it is almost nil in zebrafish (Danio rerio), an important model organism for biological research. We have searched these fish hexokinase genes by BLAST analysis; determined their expression in liver, muscle, brain and heart; measured their response to fasting and glucose administration; and performed homology sequences studies to glimpse their evolutionary history. We have confirmed by RT-qPCR studies that the six DNA sequences annotated as possible hexokinases in the NCBI GenBank are transcribed. The organ distribution of the HXK genes is similar in zebrafish as in mammals, to which they are distantly related. Of these, DrGLK and DrSHXK1 are expressed in the fish liver, DrHXK1 in brain and heart, and DrHXK2 in muscle. The only gene responsive to glucose was liver DrGLK. Its expression is induced approximately 1 h after glucose intraperitoneal injection, but not after saline solution injection. The comparison of the fish sequences and the corresponding mammalian ones imply that in both taxa the main muscle and brain isoforms are fusion products of the ancestral gene, their amino halves having separated before than their carboxy ones, followed by the fusion event, whereas fish and mammalian glucokinase genes remained unduplicated.     

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors.