Isolation and identification of paralytic peptides from hemolymph of the lepidopteran insects Manduca sexta, Spodoptera exigua, and Heliothis virescens

Seven paralytic peptides were isolated and identified from lepidopteran hemolymph. All of these peptides cause rapid, rigid paralysis when injected into Manduca sexta and some other lepidopteran larvae. Each peptide contains 23 amino acid residues including 2 cysteines and the carboxyl termini are acidic. Synthetic peptides in the disulfide or reduced forms, and as carboxyl-terminal acids or amides were equally paralytic. The most potent paralytic peptide, Mas PP I, has the following sequence: H-Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu- Arg-Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-OH. The two peptides from M. sexta hemolymph are remarkable in that they are autoparalytic (i.e. factors in collected hemolymph that are paralytic when injected into the same larvae).     

Quantitative proteomics for identifying biomarkers for Rabies

Introduction

Rabies is a fatal acute viral disease of the central nervous system, which is a serious public health problem in Asian and African countries. Based on the clinical presentation, rabies can be classified into encephalitic (furious) or paralytic (numb) rabies. Early diagnosis of this disease is particularly important as rabies is invariably fatal if adequate post exposure prophylaxis is not administered immediately following the bite.

Methods

In this study, we carried out a quantitative proteomic analysis of the human brain tissue from cases of encephalitic and paralytic rabies along with normal human brain tissues using an 8-plex isobaric tags for relative and absolute quantification (iTRAQ) strategy.

Results and conclusion

We identified 402 proteins, of which a number of proteins were differentially expressed between encephalitic and paralytic rabies, including several novel proteins. The differentially expressed molecules included karyopherin alpha 4 (KPNA4), which was overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A), which was upregulated in paralytic rabies group and glutamate ammonia ligase (GLUL), which was overexpressed in paralytic as well as encephalitic rabies. We validated two of the upregulated molecules, GLUL and CAMK2A, by dot blot assays and further validated CAMK2A by immunohistochemistry. These molecules need to be further investigated in body fluids such as cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies. This is the first study to systematically profile clinical subtypes of human rabies using an iTRAQ quantitative proteomics approach.     

家蚕瘫痪肽结合蛋白基因克隆与分析

ENF肽家族具有保守的N末端结构（Glu—Asn—Phe-）。该家族成员肽大多具有重叠功能活性,在鳞翅目昆虫的免疫反应,生长调控和自体调节等方面都发挥着重要的作用。在昆虫的免疫反应中,血细胞尤其是淋巴液的黏附性是针对外来侵入物的免疫应答过程中的重要因素。家蚕瘫痪肽（paralytic peptide）是ENF肽家族的一种,其具有多种的生物学活性,包括致瘫痪性及在家蚕血细胞免疫反应中的促吞噬细胞扩散活性。ENF肽家族的另一成员,粘虫（Pseudaletia separata）的生长阻抑肽（Growth-blocking peptide）,同家蚕瘫痪肽一样能够在粘虫的血细胞免疫反应中起到调节吞噬细胞的功能。目前,关于昆虫细胞免疫应答的终端调控分子机制的研究还比较少,有文献报道粘虫的生长阻抑肽结合蛋白（GBP—BP）能够起到沉默生长阻抑肽活性的功能,从而可能参与调节细胞免疫应答的终端调控。在本研究中,利用荧光差异显示技术（FDD）分析了家蚕感染BmNPV病毒后基因表达差异情况,在血淋巴中获得了一条差异条带G12782＊通过5＇-RACE技术,首次在家蚕中克隆得到了该基因的全长cDNA序列。通过同源性分析得知,该基因所编码的蛋白质与粘虫的生长阻抑肽结合蛋白具有很大的同源性,并被命名为家蚕瘫痪肽结合蛋白（Bmori paralytic peptide binding protein,PP-BP）。通过RT-PCR研究发现,该蛋白基因在血淋巴中大量表达。同时,利用实时荧光定量PCR（Real-time quantitative PCR）技术分析了该基因在正常饲养家蚕与添食BmNPV病毒的家蚕中的表达差异,结果显示该基因在家蚕添食BmNPV病毒后的表达量大大增强,这就暗示该基因可能与BmNPV病毒刺激后所引起的家蚕血液细胞免疫反应相关。利用生物信息学方法对该基因的结构进行了分析,发现该基因具有两个外显子和一个内含子。这个基因已经登入GenBank数据库,收入号为DQ306881。     

Insect larvae contain substances toxic to adults: the discovery of paralysins

Acidic methanolic extracts of larvae of nine different insect species were found to contain substances that cause a lethal effect in the adult stage of the same species and of other species. These endogenous toxic substances, apparently being widely spread over the class of insects, were designated as paralysins, because of their immediate and observable paralytic effect upon injection. The developmental concentration curves of five different species of insects (Galleria mellonella (Lepidoptera), Neobellieria bullata (Diptera), Spodoptera frugiperda (Lepidoptera), Tenebrio molitor (Coleoptera) and Schistocerca gregaria (Orthoptera) indicate that the toxins are not present throughout all the developmental stages in the same concentration. The strongest paralytic activity was found in late instar larvae or in the early pupal stage. The temporal distribution of paralysins during development suggests that they might be involved in metamorphosis.     

Human intoxication with paralytic shellfish toxins: clinical parameters and toxin analysis in plasma and urine

This study reports the data recorded from four patients intoxicated with shellfish during the summer 2002, after consuming ribbed mussels (Aulacomya ater) with paralytic shellfish toxin contents of 8,066 +/- 61.37 microg/100 gr of tissue. Data associated with clinical variables and paralytic shellfish toxins analysis in plasma and urine of the intoxicated patients are shown. For this purpose, the evolution of respiratory frequency, arterial blood pressure and heart rate of the poisoned patients were followed and recorded. The clinical treatment to reach a clinically stable condition and return to normal physiological parameters was a combination of hydration with saline solution supplemented with Dobutamine (vasoactive drug), Furosemide (diuretic) and Ranitidine (inhibitor of acid secretion). The physiological condition of patients began to improve after four hours of clinical treatment, and a stable condition was reached between 12 to 24 hours. The HPLC-FLD analysis showed only the GTX3/GTX2 epimers in the blood and urine samples. Also, these epimers were the only paralytic shellfish toxins found in the shellfish extract sample.     

Cross training effects of non-paralytic dorsiflexion muscle strengthening exercise on paralytic dorsiflexor muscle activity,gait ability,and balancing ability in patients with chronic stroke: A randomized,controlled, pilot trial

Objective:To investigate the effects of non-paralytic dorsiflexion muscle strengthening exercise on functional abilities in chronic hemiplegic patients after stroke.Methods:A total of 21 patients with chronic stroke underwent dorsiflexion muscle strengthening exercise (MST) 5 times a week for 6 weeks (the experimental group, MST to non-paralytic dorsiflexion muscles, n=11; the control group, MST to paralytic dorsiflexion muscles; n=10). Paralytic dorsiflexor muscle activities (DFA) and 10 m walking tests (10MWT) and timed up and go tests (TUG) were measured before and after intervention.Results:A significant increase in DFA was observed after intervention in the experimental and control groups (p<0.05) (experimental 886.6% for reference voluntary contraction (RVC), control 931.6% for RVC). TUG and 10MWT results showed significant reductions post-intervention in the experimental and control groups (experimental group -5.6 sec, control -4.8 sec; experimental group -3.1 sec, control, -3.9 sec; respectively). No significant intergroup difference was observed between changes in DFA or between changes in TUG and 10MWT results after intervention (p>.05).Conclusion:Strengthening exercise performed on non-paralytic dorsiflexion muscles had positive cross-training effects on paralytic dorsiflexor muscle activities, balance abilities, and walking abilities in patients with chronic stroke.     

Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov.

Lyngbya wollei (Farlow ex Gomont) comb. nov., a perennial mat-forming filamentous cyanobacterium prevalent in lakes and reservoirs of the southeastern United States, was found to produce a potent, acutely lethal neurotoxin when tested in the mouse bioassay. Signs of poisoning were similar to those of paralytic shellfish poisoning. As part of the Tennessee Valley Authority master plan for Guntersville Reservoir, the mat-forming filamentous cyanobacterium L. wollei, a species that had recently invaded from other areas of the southern United States, was studied to determine if it could produce any of the known cyanotoxins. Of the 91 field samples collected at 10 locations at Guntersville Reservoir, Ala., on the Tennessee River, over a 3-year period, 72.5% were toxic. The minimum 100% lethal doses of the toxic samples ranged from 150 to 1,500 mg kg of lyophilized L. wollei cells-1, with the majority of samples being toxic at 500 mg kg-1. Samples bioassayed for paralytic shellfish toxins by the Association of Official Analytical Chemists method exhibited saxitoxin equivalents ranging from 0 to 58 micrograms g (dry weight)-1. Characteristics of the neurotoxic compound(s), such as the lack of adsorption by C18 solid-phase extraction columns, the short retention times on C18 high-performance liquid chromatography (HPLC) columns, the interaction of the neurotoxins with saxiphilin (a soluble saxitoxin-binding protein), and external blockage of voltage-sensitive sodium channels, led to our discovery that this neurotoxin(s) is related to the saxitoxins, the compounds responsible for paralytic shellfish poisonings. The major saxitoxin compounds thus far identified by comparison of HPLC fluorescence retention times are decarbamoyl gonyautoxins 2 and 3. There was no evidence of paralytic shellfish poison C toxins being produced by L. wollei. Fifty field samples were placed in unialgal culture and grown under defined culture conditions. Toxicity and signs of poisoning for these laboratory-grown strains of L. wollei were similar to those of the field collection samples.     

Banking of embryos of mutated, paralytic tremor rabbit by means of vitrification

Cryopreservation enables banking of embryos for future use in medicine and in animal breeding. It also enables protection of germ plasm of endangered species and unique strains or lanes of laboratory animals. This paper describes an example of employing a vitrification method for banking of embryos of a unique lane of rabbit. The paralytic tremor (pt) rabbit is an X-linked recessive mutant lane of the Chinchilla breed characterized by hypomyelination of the central nervous system. In order to obtain a sufficient number of embryos, pt females were subjected to superovulation and surgical embryo collection. All suitable embryos were vitrified in 0.25 mL insemination straws in a modified EFS vitrification solution comprised of ethylene glycol (40%), Ficoll 70 (18%) and sucrose (0.3 M) in Hepes buffered TCM medium containing 20% fetal calf serum. In order to assess the efficiency of the vitrification procedure, a representative portion of vitrified embryos was warmed after a period of storage. Warmed embryos were subjected to in vitro culture for 72 h or were transferred to the uterus of synchronized recipients. The majority of the 141 warmed embryos survived vitrification and 100/141 (71%) developed to the blastocyst stage. Moreover, out of an additional 34 warmed embryos transferred to four recipients, eight (23.5%) developed to term and seven live pups were born. Six of the rabbit pups exhibited paralytic tremor symptoms typical for the pt lane. Although the overall efficiency of the vitrification method was lower compared with the effects usually achieved for 'healthy' embryos, results presented confirm the real possibility of the future restoration of the colony of pt rabbit, if sufficient number of embryos are cryopreserved.     

A temperature-sensitive allele of Drosophila sesB reveals acute functions for the mitochondrial adenine nucleotide translocase in synaptic transmission and dynamin regulation

Rapidly reversible, temperature-sensitive (ts) paralytic mutants of Drosophila have been useful in delineating immediate in vivo functions of molecules involved in synaptic transmission. Here we report isolation and characterization of orangi (org), an enhancer of shibire (shi), a ts paralytic mutant in Drosophila dynamin. org is an allele of the stress sensitive B (sesB) locus that encodes a mitochondrial adenine nucleotide translocase (ANT) and results in a unique ts paralytic behavior that is accompanied by a complete loss of synaptic transmission in the visual system. sesB(org) reduces the restrictive temperature for all shi(ts) alleles tested except for shi(ts1). This characteristic allele-specific interaction of sesB(org) with shi is shared by abnormal wing discs (awd), a gene encoding nucleoside diphosphate kinase (NDK). sesB(org) shows independent synergistic interactions, an observation that is consistent with a shared pathway by which org and awd influence shi function. Genetic and electrophysiological analyses presented here, together with the observation that the sesB(org) mutation reduces biochemically assayed ANT activity, suggest a model in which a continuous mitochondrial ANT-dependent supply of ATP is required to sustain NDK-dependent activation of presynaptic dynamin during a normal range of synaptic activity.     

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as gamma-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4-100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.     

Plasma procalcitonin in patients with ileus. Relations to other inflammatory parameters

Plasma procalcitonin (PCT) is a highly specific marker for the diagnosis of bacterial infections and sepsis. PCT levels are usually low in viral infections, chronic inflammation or postsurgical states. The purpose of this study was to characterize PCT plasma levels in patients with various types of ileus at preoperative stage, where the other inducing factors suchas a surgical stress are excluded. The prospective study was performed on 54 patients admitted to in-patient surgical department with a proven diagnosis of ileus. Patients were divided to three groups--obstructive, vascular and paralytic ileus. Plasma levels of PCT (Kryptor analysis), TNFalpha, IL-1beta, IL-6, cortisol (ELISA) and CRP (Kryptor ultrasensitive analysis) were estimated before any invasive procedure was realized. We demonstrated significant elevation of PCT in both obstructive ileus in adhesions and vascular ileus compared with healthy subjects (p 0.01). PCT levels were not elevated in paralytic ileus. The regression coefficient was the highest for PCT and CRP (r=0.78, p 0.01), for TNFalpha and IL-8 (r=0.76, p 0.01) in vascular ileus. There was no significant correlation between PCT and other inflammatory parameters. The different types of ileus induce an elevation of plasma PCT levels and PCT shows itself as an acute phase reactant. The highest PCT concentrations were presented in patients with vascular ileus, whereas paralytic ileus revealed similar cytokine and PCT pattern as in healthy subjects. Plasma PCT estimation extended to a measurement of CRP and IL-6 may become a useful complementary examination for diagnostics of acute abdomen in patients.     

Paralytic activity of (des-Glu1)conotoxin GI analogs in the mouse diaphragm

A series of 20 peptide analogs of (des-Glu1)conotoxin GI were prepared by solid phase synthesis. The peptides were tested for their abilities to inhibit contractions in the mouse-diaphragm-with-phrenic-nerve assay. (Des-Glu1)conotoxin has an IC50 of 2.7 x 10(-7) M in this assay. Results from this assay show that total loss of paralytic activity occurs when Pro is replaced by Gly, Tyr by D-Tyr, or Gly by D-Phe. In most cases loss or change in length of one of the disulfide rings eliminates paralytic activity except with compound 17, which is weakly active, IC50 = 7.0 x 10(-5) M. Replacement of the Cys1-Cys6 disulfide bond with an amide bond (compound 9) greatly lowers paralytic activity, IC50 = 3.7 x 10(-5) M.     

The effect of mussel size, temperature, seston volume, food quality and volume-specific toxin concentration on the uptake rate of PSP toxins by mussels (Mytilus galloprovincialis Lmk)

The accumulation of paralytic shellfish poisoning (PSP) toxins by bivalves is a serious threat to public health all over the world. However, very little is known about the uptake kinetics of these toxins and the environmental factors that may modify this process. We have studied the effect of mussel size, temperature, seston volume, food quality, and volume-specific toxin concentration (VOSTOC), on the uptake rate of paralytic shellfish poisoning (PSP) toxins by mussels (Mytilus galloprovincialis), by means of a second order factorial experiment. Over a 3-day period, the mussels were fed artificial diets containing Alexandrium minutum AL1V (a PSP toxin producer), Tetraselmis suecica, Ensiculifera sp1 and silt, to the levels required by each treatment. Mussel size, seston volume and VOSTOC were found to be statistically significant when the total toxin accumulated per weight of wet tissue was considered. Mussel size affected the uptake negatively and latter two positively. The interactions, mussel size-VOSTOC and mussel size-food quality were also significant. The response was not linear as shown by the significance of the quadratic term of mussel size. Notwithstanding, when the PSP toxins accumulation per mussel was analysed, only one factor, the VOSTOC and the interactions, food quality-mussel size and food quality-seston volume, were found to be significant. VOSTOC was the most important factor in the accumulation of toxins, in our opinion, probably due to toxin assimilation being mainly regulated by the probability of contact between the toxins and the cellular walls of the digestive system. The size of the bivalve is also especially important because toxin concentration is usually calculated per weight of bivalve tissue and because the weight-specific ingestion increases with mussel size. The food quality, which was directly related to the assimilation of organic matter, had an inverse effect on toxin assimilation. In our opinion, this is probably due to the effect of inorganic particles in enhancing the disruption of Alexandrium cells. Temperature had no effect on the uptake rate except for the accumulation of the gonyautoxin GTX1.     

Effects of the toxic dinoflagellate, Gymnodinium catenatum on hydrolytic and antioxidant enzymes, in tissues of the giant lions-paw scallop Nodipecten subnodosus

This study documents effects of the toxic dinoflagellate Gymnodinium catenatum, a producer of paralytic shellfish poison, on juvenile farmed (5.9+/-0.39 cm) giant lions-paw scallop Nodipecten subnodosus. Scallops were fed bloom concentrations of toxic dinoflagellate G. catenatum for 7 h. The effect of the toxic dinoflagellate in different tissues was determined by analysis of antioxidant enzymes (catalase, superoxide dismutase, gluthathione peroxidase), thiobarbituric acid reactive substances (lipid peroxidation), and hydrolytic enzymes (proteases, glycosidases, phosphatases, lipases, and esterases). Histopathological photos record the effects of the toxic dinoflagellate in various tissues. The results show that juvenile lions-paw scallops produce pseudo-feces, partially close their shell, increase melanization, and aggregate hemocytes. Several enzymes were affected and could serve as biological markers. In general, the adductor muscle was not affected. In the digestive gland, some enzymes could be the result of defensive and digestive processes. Gills and mantle tissue were markedly affected because these sites respond first to toxic dinoflagellates, leading to the idea that proteolytic cascades could be involved.     

Hydralysin,a novel animal group-selective paralytic and cytolytic protein from a noncnidocystic origin in hydra

In Cnidaria, the production of neurotoxic polypeptides is attributed to the ectodermal stinging cells (cnidocytes), which are discharged for offensive (prey capture) and/or defensive purposes. In this study, a new paralysis-inducing (neurotoxic) protein from the green hydra Chlorohydra viridissima was purified, cloned, and expressed. This paralytic protein is unique in that it (1) is derived from a noncnidocystic origin, (2) reveals a clear animal group-selective toxicity, (3) possesses an uncommon primary structure, remindful of pore-forming toxins, and (4) has a fast cytotoxic effect on insect cells but not on the tested mammalian cells. The possible biological role of such a noncnidocystic toxin is discussed.     

Genetic analysis of a synaptic calcium channel in Drosophila: intragenic modifiers of a temperature-sensitive paralytic mutant of cacophony

Our previous genetic analysis of synaptic mechanisms in Drosophila identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel alpha1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cac(TS2), indicated cac encodes a primary calcium channel alpha1 subunit functioning in neurotransmitter release. To further examine the functions and interactions of cac-encoded calcium channels, a genetic screen was performed to isolate new mutations that modify the cac(TS2) paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cac(TS2), including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Here we report molecular characterization of three intragenic modifiers and examine the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs. These mutations may further define the structural basis of calcium channel alpha1 subunit function in neurotransmitter release.     

Structures of Two Paralytic Shellfish Toxins,Gonyautoxins V and VI,Isolated from a Tropical Dinoflagellate,Pyrodinium bahamense var. compressa

Two paralytic shellfish toxins, gonyautoxin V and gonyautoxin VI, isolated from a tropical dinoflagellate, Pyrodinium bahamense var. compressa, were identified respectively to be derivatives of saxitoxin and neosaxitoxin with a sulfonatocarbamoyl moiety.     

Genetic modifiers of the Drosophila NSF mutant, comatose, include a temperature-sensitive paralytic allele of the calcium channel alpha1-subunit gene, cacophony

The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Our previous work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1 function and to broaden our analysis of synaptic transmission to other gene products, we have performed a genetic screen for mutations that interact with comt. Here we report the isolation and analysis of four mutations that modify TS paralysis in comt, including two intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel alpha1-subunit gene, cacophony (cac). Analysis of synaptic function in these mutants alone and in combination will further define the in vivo functions and interactions of specific gene products in synaptic transmission.     

In vivo and in vitro models of demyelinating disease: JHM virus in the rat central nervous system localized by in situ cDNA hybridization and immunofluorescent microscopy.

In situ probing of central nervous system (CNS) tissues has made it possible to associate the presence of JHM virus (JHMV) RNA with individual cells in the rat CNS. The presence of viral RNA was not always associated with antigen expression. The in situ hybridization revealed that cerebellar Purkinje cells and hippocampal neurons were highly susceptible to JHMV infection during either acute or paralytic disease. In the paralytic disease, Purkinje cell neurons frequently contained viral RNA. This observation suggests that these neurons, and perhaps others, may be repositories for JHMV in rats that undergo prolonged infections.