A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics

The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom subpopulations of proteins in future studies.     

Application of phage-displayed single chain antibodies in western blot

A phage display single chain fragment variable library constructed on pⅢ protein of M13 filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules. After four rounds of panning, there was a remarkable enrichment in the titer of bound phages. Twenty phage clones were selected from the last round and screened by means of phage-ELISA. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. The dilutions of antibody phages depend on the affinity between antibody-displayed phage particles and antigens.     

Preparation and preliminary application of monoclonal antibodies against Trichokirin-S1, a small ribosome-inactivating peptide from the seeds of Trichosanthes kirilowii

Trichokirin-S1,a small ribosome-inactivating peptide recently purified from the seeds ofTrichosanthes kirilowii,has potential clinical applications because of its small molecular mass.Two stablestrains of hybridomas (1F11 and 2A5) that can secrete highly specific monoclonal antibodies (mAbs) againstTrichokirin-S1 have been developed using the hybridoma technique.The isotypes of these two mAbs,1F11and 2A5,were determined to be IgG_(2a) and IgG_1,respectively.The affinity constants,which were measuredby non-competitive ELISA,were found to be 2.3×10~8 M~(-1) and 2.8×10~8 M~(-1),respectively.An immunoaffinitymethod using 2A5-coupled Sepharose 4B was successfully developed to purify Trichokirin-S1.These twoantibodies have also been used to detect Trichokirin-S1 in Western blot.     

Identification of a gene encoding a putative phosphotransferase system enzyme IIBC in Listeria welshimeri and its application for diagnostic PCR

AIMS: To identify a Listeria welshimeri-specific gene that can be used for identification of this species by PCR. METHODS AND RESULTS: Through comparative analysis of genomic DNA from Listeria species using dot blot hybridization, an L. welshimeri-specific clone was isolated that contained a gene segment whose translated protein sequence is similar to enzyme IIBC from phosphotransferase systems in other bacteria. Using oligonucleotide primers derived from this L. welshimeri-specific clone, a 608-bp fragment was amplified from L. welshimeri genomic DNA and not from other Listeria species or other Gram-negative and Gram-positive species. CONCLUSION AND SIGNIFICANCE: The PCR employing L. welshimeri-specific primers shows promise as a useful method for differentiating L. welshimeri from other Listeria species and related bacteria.     

ClC-5 Chloride Channel Alters Expression of the Epithelial Sodium Channel (ENaC)

ClC-5 chloride channels and epithelial sodium channels (ENaC) are present in many cell types including airway and retinal epithelia. Since ENaC activity is known to be affected by chloride transport, we co-injected Xenopus oocytes with cRNAs encoding ENaC and ClC-5 to investigate whether channel currents are impacted by heterologous co-expression of these proteins. ClC-5 currents were not detectably affected by co-expression with ENaC, whereas amiloride-sensitive ENaC currents were significantly lower compared to control oocytes expressing ENaC alone. Co-expression of ENaC with cRNA sequences encoding non-conducting fragments of ClC-5 revealed that the amino acid sequence region between positions 347 and 647 was sufficient for inhibition of ENaC currents. Co-expression of ENaC and another transport protein, the sodium dicarboxylate co-transporter (NaDC-1), did not affect ENaC currents. To test whether the inhibitory effects of ClC-5 were specific for ENaC, ClC-5 was also co-expressed with CFTR. CFTR currents were also inhibited by co-expression with ClC-5, whereas ClC-5 currents were unaffected. Western blot analysis of biotinylated oocyte surface membranes revealed that the co-expression of ClC-5 with ENaC, CFTR, or NaDC-1 decreased the abundance of these proteins at the surface membrane. We conclude that overexpression of ClC-5, specifically amino acids 347–647, can alter the normal translation or trafficking of ENaC and other ion transport proteins by a mechanism that is independent of the chloride conductance of ClC-5.     

Detection of Desulfotomaculum in an Italian rice paddy soil by 16S ribosomal nucleic acid analyses

Two specific primers were developed for the amplification of 16S rRNA genes of Desulfotomaculum lineage 1 to detect members of the genus Desulfotomaculum in rice field soil. The combination of both primers in PCR allowed the specific amplification and cloning of ten 16S rDNA sequences of this group from rice paddy soil DNA extracts. The phylogenetic analysis showed that these sequences formed a deeply branching cluster within Desulfotomaculum lineage 1, together with two sequences from the database and two sequences from a hydrocarbon-contaminated aquifer. Dissimilarity values to validly described species, including recently isolated strains of Desulfotomaculum from rice paddy microcosms, were higher than 12%. Within the new cluster the cloned sequences formed three separate groups which were each represented by at least two sequences with identities of >/=99% while one sequence represented an additional group. The sequences should represent sulfate-reducing organisms because they clearly fell into the physiologically coherent group of Gram-positive sulfate reducers. The relative abundance of bacteria of the Desulfotomaculum lineage 1 in rice paddy soil and root samples was estimated with rRNA dot blot hybridizations of extracted RNA. The relative RNA content of Desulfotomaculum lineage 1 was 0.55% in the bulk soil and 1% in the rice root samples, respectively, of the total 16S rRNA content (probe Eub338). Hybridization of rRNA with a probe targeting the new cluster represented by the cloned sequences confirmed the high abundance of 16S rRNA sequences from this cluster in the rice paddy field samples. Another hybridization probe detecting Desulfotomaculum acetoxidans and two closely related Desulfotomaculum isolates from rice paddy soil indicated that these bacteria were less abundant.     

MRNA encoding a putative RNA helicase of the DEAD-box gene family is up-regulated in trypomastigotes of Trypanosoma cruzi

Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.     

Characterization of High Density Lipophorin (HDLP) Receptor from the Fat Body of Galleria mellonella by Ligand Blot Analysis

ABSTRACT To identify and characterize the HDLP (high density lipophorin) receptor from Galleria mellonella (LPR_Gm), we used techniques of ligand blotting. This method was, to our knowledge, firstly used to characterize the lipophorin receptor (LPR) in insect. LPR_gm had the approximate molecular weight of 100 kDa under non-reducing conditions and bound the HDLP very rapidly and specifically. The receptor had an absolute requirement of Ca²⁺ but exceeding concentration of Ca²⁺ has shown some inhibitory effect, and this result was supported by the effect of EDTA, here used for Ca²⁺ chelating reagent. LPR_Gm has the high binding affinity to HDLP and its binding with HDLP was rapid and specific. Suramin (polysulfated polycyclic hydrocarbon) effectively inhibited the LPR_Gm and HDLP binding. LPR_Gm showed the stage specific binding activity especially in day 1-3 last instar larval, prepupal, and adult stages.     

Topiramate reduces AMPA-induced Ca(2+) transients and inhibits GluR1 subunit phosphorylation in astrocytes from primary cultures

Topiramate (TPM) is a structurally novel broad spectrum anticonvulsant known to have a negative modulatory effect on the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate subtypes of glutamate receptors and some types of voltage-gated Na(+) and Ca(2+) channels, and a positive modulatory effect on some types of gamma-aminobutyric acid(A) (GABA(A)) receptors and at least one type of K(+) channels in neurons. In an earlier work, we showed that the negative modulatory effect of TPM (100 mum) on AMPA/kainate receptors in neurons is dependent on TPM modulation of the phosphorylation state of these receptors. In this work, we investigated the effect of TPM on AMPA-induced intracellular calcium ([Ca(2+)](i)) responses in cultured rat cortical astrocytes, with special interest in intracellular mechanisms. Here, we report that the ability of TPM (1-100 mum) to inhibit AMPA-induced accumulation of Ca(2+) in astrocytes is inversely related to the level of protein kinase A (PKA) -mediated phosphorylation of channels activated by AMPA. The level of receptor phosphorylation was further determined with western blot using phosphorylation specific antibodies that recognize the glutamate receptor 1 (GluR1) subunit phosphorylated on Ser845. These results demonstrated that, even in cultured cortical astrocytes, TPM significantly reduced the phophorylation level of GluR1 subunits. Furthermore, it was shown that TPM binds to AMPA receptors in the dephosphorylated state and thereby exerts an allosteric modulatory effect on the ion channel.