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Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (Kd = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 µm) with the neuronal α7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve–muscle preparations revealed no blocking activity of sWntx-5 at µm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.

The possible involvement of specific regions/loops of cardiotoxin from Naja sputatrix venom in mediating its cytolytic activity is evaluated using a new cytolytic assay. In this assay, the amount of chloramphenicol acetyltransferase (CAT) that is released upon lysis of the cellular membranes by the cytotoxin has been measured as an index of cytolysis. This newly developed CAT system is more sensitive than the traditional haemolysis method utilizing red blood cells or the lactate dehydrogenase assay for cytolysis. Series of chimaeric toxin molecules have been constructed by swapping the loops between highly hydrophilic neurotoxin and highly hydrophobic cardiotoxin molecules from Naja sputatrix, which are known to exhibit structural similarity (three-finger conformation) but to have different functional properties. Comparison of the cytolytic activities of the recombinant chimaeric toxins demonstrated the possible involvement of all three loops of cardiotoxin in its cytolytic potency. However, the first two loops of the protein appear to make the major contribution to its lytic activity. cDNAs encoding cardiotoxin and the chimaeric toxins, when expressed in transfected cultured Chinese hamster ovary cells, resulted in cell lysis, indicating that these cDNAs can be developed as useful cytolytic agents.

Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.

MicroRNAs (miRNA), small sequences of non-coding RNA which interact with complementary sequences on the 3’untranslated region of target messenger RNAs to modulate translation, have a pivotal role in the development of the heart and its response to injury. Myocardial infarction (MI) triggers a dynamic miRNA response with the potential of yielding therapeutic targets. Following miRNA array profiling in rat hearts 2, 7 and 14 days after MI induced by coronary ligation, we identified a progressive time-dependent up-regulation of miR-31 compared to sham rats. Increase of miR-31 in heart tissue in the acute and subacute phases after MI (up to 90-fold) was also detected by Real-Time PCR (P=0.02 at day 2; P

Natriuretic peptide receptor 3 (NPR3) is the clearance receptor for the cardiac natriuretic peptides (NPs). By modulating the level of NPs, NPR3 plays an important role in cardiovascular homeostasis. Although the physiological functions of NPR3 have been explored, little is known about its regulation in health or disease. MicroRNAs play an essential role in the post-transcriptional expression of many genes. Our aim was to investigate potential microRNA-based regulation of NPR3 in multiple models. Hypoxic challenge elevated levels of NPPB and ADM mRNA, as well as NT-proBNP and MR-proADM in human left ventricle derived cardiac cells (HCMa), and in the corresponding conditioned medium, as revealed by qRT-PCR and ELISA. NPR3 was decreased while NPR1 was increased by hypoxia at mRNA and protein levels in HCMa. Down-regulation of NPR3 mRNA was also observed in infarct and peri-infarct cardiac tissue from rats undergoing myocardial infarction. From microRNA microarray analyses and microRNA target predictive databases, miR-100 was selected as a candidate regulator of NPR3 expression. Further analyses confirmed up-regulation of miR-100 in hypoxic cells and associated conditioned media. Antagomir-based silencing of miR-100 enhanced NPR3 expression in HCMa. Furthermore, miR-100 levels were markedly up-regulated in rat hearts and in peripheral blood after myocardial infarction and in the blood from heart failure patients. Results from this study point to a role for miR-100 in the regulation of NPR3 expression, and suggest a possible therapeutic target for modulation of NP bioactivity in heart disease.

An Australian common brown snake, Pseudonaja textilis, is known to contain highly lethal neurotoxins. Among them, a long-chain alpha-neurotoxin, pseudonajatoxin b, has been identified. In this report, while presenting evidence for the presence of at least four such long-chain alpha-neurotoxins in the venom of P. textilis, we describe the characteristics of both the mRNA and the gene responsible for the synthesis of these neurotoxins. A precursor toxin synthesized from the gene has been identified as being capable of producing the isoforms possibly by post-translational modifications at its C-terminal end. Recombinant toxins corresponding to the precursor and its product have been found to possess similar binding affinities for muscular acetylcholine receptors (IC(50)=3x10(-8) M) and a lethality, LD(50), of 0.15 microg/g in mice.

This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the cardiotoxin (CTX)-2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500-bp (spliced) CTX-2 mRNA as the final transcript. However, the liver produces two types of CTX-2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP-1, C/EBP, CACCC-binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.

From the serum of the nonvenomous snake Python reticulatus, a new phospholipase A(2) (PLA(2)) inhibitor termed phospholipase inhibitor from python (PIP) was purified by sequential chromatography and cloned to elucidate its primary structure and fundamental biochemical characteristics. A cDNA clone encoding PIP was isolated from the liver total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). It contained a 603 bp open reading frame that encoded a 19-residue signal sequence and a 182-residue protein. PIP showed about 60% sequence homology with those PLA(2) inhibitors having a urokinase-type plasminogen activator receptor-like domain structure. PIP was also functionally expressed as a fusion protein in Escherichia coli to explore its potential therapeutic significance. The recombinant PIP was shown to be identical to the native form in chromatographic behavior and biochemical characteristics. Both the native and recombinant PIP appear to exist as a hexamer of 23-kDa subunits having an apparent molecular mass of approximately 140 kDa. PIP showed ability to bind to the major PLA(2) toxin (daboiatoxin, DbTx) of Daboia russelli siamensis at 1-2-fold molar excess of inhibitor to toxin. It exhibited broad spectra in neutralizing the toxicity of various snake venoms and toxins and inhibited the formation of edema in mice. Our data demonstrate the venom neutralizing potential of the recombinant PIP and suggest that the proline-rich hydrophobic core region may play a role in binding to PLA(2).

The structure and organization of five genes responsible for the synthesis of six isoforms of short-chain alpha-neurotoxins in Pseudonaja textilis venom are reported in this paper. This also forms the first report which describes the synthesis of two neurotoxin mRNA variants from one of these genes (Pt-sntx1) as a result of alternative splicing. Each gene consists of three exons which are separated by two introns and each has a functional promoter. The promoter activity was confirmed by both CAT assay and Real-Time PCR. A transcription initiation site, two putative TATA boxes, one CCAAT box and the transcription factor binding consensus sites for AP-1, GATA-2, c/EBPb were identified in the 5' non-coding region of each gene. Phylogenetic analysis showed that these five genes from P. textilis constituted a distinct group which has evolved by gene duplication followed by accelerated evolution from an ancestral gene.

Four phospholipase A 2 (PLA 2 ) enzymes (Superbins a, b, c, and d) with varying platelet aggregation inhibitor activities have been purified from Austrelaps superbus by a combination of gel filtration, ion-exchange, and reversed-phase high-pressure liquid chromatography. Purity and homogeneity of the superbins have been confirmed by high-performance capillary zone electrophoresis and mass spectrometry. The electron spray ionization mass spectrometry data showed that their molecular masses range from 13,140 to 13,236 Da. Each of the proteins has been found to be basic and exhibit varying degrees of PLA 2 activity. They also displayed different platelet aggregation inhibitory activities. Superbin a was found to possess the most potent inhibitory activity with an IC 50 of 9.0 nM, whereas Superbin d was found to be least effective with an IC 50 of 3.0 μM. Superbins b and c were moderately effective with IC 50 values of 0.05 and 0.5 μM, respectively. The amino-terminal sequencing confirmed the identity of these superbins. cDNA cloning resulted in the identification of 17 more PLA 2 isoforms in A. superbus venom. It has also provided complete information on the precursor PLA 2 . The precursor PLA 2 contained a 27-amino-acid signal peptide and 117- to 125-amino-acid PLA 2 (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA 2 enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA 2 enzymes containing an extra pancreatic loop also have been identified among the isoforms.

A sensitive and highly reproducible method has been used to show that Aquaporin 3 (AQP 3 ) mRNA is present in the ovine placenta and chorion from at least 60 days of gestation (term=145–150d) with levels increasing substantially (>16 fold) at 100 days, and remaining constant thereafter. By immuno- and hybridization histochemistry, the epithelial cells expressing AQP 3 were found to be the trophoblast cells. Some AQP 3 was expressed in fibroblasts of the amnion and allantois but none was expressed in the epithelia of these membranes. AQP 1 was expressed in endothelial cells of fetal and maternal blood vessels but not in any epithelial cell of the ovine placenta and fetal membranes. The level of AQP 3 expression is consistent with known ovine placental permeabilities to water, glycerol and urea.

We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding cardiotoxins (CTX) of Naja naja sputatrix . The cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP-2, NFIL-6/C/EBP, NF-κB and PuF have been identified in the 5′-region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three-finger loop structure of the cardiotoxin molecule. The diverse functions of cardiotoxins together with our findings suggest that the cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.

In obese individuals, a high efflux of glycerol from accumulated fat in adipose tissue into the liver is known to be associated with the development of type 2 diabetes. Aquaporin 9 (AQP9) is an aquaglyceroporin which serves as the primary route of hepatic glycerol uptake for gluconeogenesis. Hence, development of AQP9 blockers/regulators may be of potential benefit in controlling hyperglycaemia especially in obese and diabetic individuals. HTS13286 was recently identified as a specific AQP9 inhibitor, even so its limited solubility renders the molecule unsuitable for in vivo application. microRNAs are naturally occurring gene regulators that are often associated with various diseases. The feasibility in selective modulation of microRNAs has introduced a new paradigm for therapeutic applications. In this study, we explore the possibility of using microRNAs to regulate AQP9 expression and eventually glycerolbased gluconeogenesis. In silico prediction of microRNAs targeting the 3’ untranslated region of AQP9 was conducted using miRWalk database. Among the list of potential microRNAs, miR-22 and miR-23a were shortlisted for their high expression in liver and further confirmed to interact with AQP9 via luciferase assay. Over-expression of miR-22 or miR- 23a was able to reduce AQP9 expression (mRNA and protein) and ultimately inhibit glycerol-based gluconeogenesis in HepG2 cells. Livers of diabetic rats were also observed to exhibit an inverse correlation between miR-22, miR-23a and AQP9 expression. As negative modulators of AQP9 expression, miR-22 and miR-23a suggest a potential role in regulating glycerol entry into hepatocytes which could be beneficial in managing glycerol-dependent hyperglycaemic conditions.

Neuronal development is a pro-survival process that involves neurite growth, synaptogenesis, synaptic and neuronal pruning. During development, these processes can be controlled by temporal gene expression that is orchestrated by both long non-coding RNAs and microRNAs. To examine the interplay between these different components of the transcriptome during neuronal differentiation, we carried out mRNA, long non-coding RNA and microRNA expression profiling on maturing primary neurons. Subsequent gene ontology analysis revealed regulation of axonogenesis and dendritogenesis processes by these differentially expressed mRNAs and non-coding RNAs. Temporally regulated mRNAs and their associated long non-coding RNAs were significantly over-represented in proliferation and differentiation associated signalling, cell adhesion and neurotrophin signalling pathways. Verification of expression of the Axin2, Prkcb, Cntn1, Ncam1, Negr1, Nrxn1 and Sh2b3 mRNAs and their respective long non-coding RNAs in an in vitro model of ischemic-reperfusion injury showed an inverse expression profile to the maturation process, thus suggesting their role(s) in maintaining neuronal structure and function. Furthermore, we propose that expression of the cell adhesion molecules, Ncam1 and Negr1 might be tightly regulated by both long non-coding RNAs and microRNAs.

cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.

Wound healing is a major problem in diabetic patients and current treatments have met with limited success. We evaluated the treatment of excisional and diabetic wounds using a stem cell isolated from the human umbilical cord Wharton's jelly (hWJSC) that shares unique properties with embryonic and adult mesenchymal stem cells. hWJSCs are non-controversial, available in abundance, hypo-immunogenic, non-tumorigenic, differentiate into keratinocytes, and secrete important molecules for tissue repair. When human skin fibroblasts (CCD) in conventional scratch-wound assays were exposed to hWJSC-conditioned medium (hWJSC-CM) the fibroblasts at the wound edges migrated and completely covered the spaces by day 2 compared to controls. The number of invaded cells, cell viability, total collagen, elastin, and fibronectin levels were significantly greater in the hWJSC-CM treatment arm compared to controls (P 0.05). When a single application of green fluorescent protein (GFP)-labeled hWJSCs (GFP-hWJSCs) or hWJSC-CM was administered to full-thickness murine excisional and diabetic wounds, healing rates were significantly greater compared to controls (P 0.05). Wound biopsies collected at various time points showed the presence of green GFP-labeled hWJSCs, positive human keratinocyte markers (cytokeratin, involucrin, filaggrin) and expression of ICAM-1, TIMP-1, and VEGF-A. On histology, the GFP-hWJSCs and hWJSC-CM treated wounds showed reepithelialization, increased vascularity and cellular density and increased sebaceous gland and hair follicle numbers compared to controls. hWJSCs showed increased expression of several miRNAs associated with wound healing compared to CCDs. Our studies demonstrated that hWJSCs enhance healing of excisional and diabetic wounds via differentiation into keratinocytes and release of important molecules.

The 3' UTR of insulin has been identified as a critical region that confers mRNA stability, which is crucial for promoting transcription in response to glucose challenge. miRNAs are endogenously encoded non-coding RNAs that function as regulators of gene expression. This regulatory function is generally mediated by complementary binding to the 3'UTR of its mRNA targets that affects subsequent translational process. Genes involved in the regulation of glucose homeostasis, particularly in insulin production, have been found as targets of several miRNAs. Yet, no direct miRNA-based regulators of insulin biosynthesis have been identified. In this study, identification of possible miRNA-based regulators of insulin production is explored. Members of a miRNA family, miR-25 and miR-92a, are found as direct modulators of insulin expression. Overexpression of miR-25 or miR-92a reduced insulin expression while inhibition of miR-25 and miR-92a expression using corresponding antagomiRs promoted insulin expression and ultimately enhanced glucose-induced insulin secretion. Furthermore, suppression of insulin secretion by pre miR-9 could be attenuated by treatment with anti-miR-25 or miR-92a. Interestingly, we found the binding site of miR-25 and miR-92a to overlap with that of PTBP1, an important RNA binding molecule that stabilizes insulin mRNA for translation. Despite the increase in PTBP1 protein in the pancreas of diabetic rats, we observed insulin expression to be reduced alongside upregulation of miR-25 and miR-92a, suggesting an intricate regulation of insulin (bio)synthesis at its mRNA level.

Over the past decade, scientific discoveries have highlighted new roles for a unique class of non-coding RNAs. Transcribed from the genome, these non-coding RNAs have been implicated in determining the biological complexity seen in mammals by acting as transcriptional and translational regulators. Non-coding RNAs, which can be sub-classified into long non-coding RNAs, microRNAs, PIWI-interacting RNAs and several others, are widely expressed in the nervous system with roles in neurogenesis, development and maintenance of the neuronal phenotype. Perturbations of these non-coding transcripts have been observed in ischemic preconditioning as well as ischemic brain injury with characterization of the mechanisms by which they confer toxicity. Their dysregulation may also confer pathogenic conditions in neurovascular diseases. A better understanding of their expression patterns and functions has uncovered the potential use of these riboregulators as neuroprotectants to antagonize the detrimental molecular events taking place upon ischemic-reperfusion injury. In this review, we discuss the various roles of non-coding RNAs in brain development and their mechanisms of gene regulation in relation to ischemic brain injury. We will also address the future directions and open questions for identifying promising non-coding RNAs that could eventually serve as potential neuroprotectants against ischemic brain injury.

Venoms and toxins are of significant interest due to their ability to cause a wide range of pathophysiological conditions that can potentially result in death. Despite their wide distribution among plants and animals, the biochemical pathways associated with these pathogenic agents remain largely unexplored. Impoverished and underdeveloped regions appear especially susceptible to increased incidence and severity due to poor socioeconomic conditions and lack of appropriate medical treatment infrastructure. To facilitate better management and treatment of envenomation victims, it is essential that the biochemical mechanisms of their action be elucidated. This review aims to characterize downstream envenomation mechanisms by addressing the major neuro-, cardio-, and hemotoxins as well as ion-channel toxins. Because of their use in folk and traditional medicine, the biochemistry behind venom therapy and possible implications on conventional medicine will also be addressed.

Scorpion venoms are rich sources of bioactive peptides and are widely known for their ion channel inhibiting properties. We have isolated, cloned and characterized a venom protein (Bumarsin) from the Chinese scorpion, Mesobuthus martensii Karsch. Bumarsin cDNA encodes a 8132 Da, 72 amino acid mature protein that most probably exists in its native form as a Cys-bridged homodimer. We have identified this novel protein to be an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. 0.6 μM of Bumarsin inhibits 32% of the HMG-CoA reductase activity, in comparison to 10 μM simvastatin which only inhibits 35% of the activity. RT-PCR and SELDI-TOF mass spectrometric studies demonstrate that bumarsin regulates the expression of both genes and proteins involved in cholesterol homeostasis. Our results suggest that bumarsin may provide a model for the design of novel drugs that can be used to modulate cholesterol homeostasis.

Aquaporins (AQPs) are transmembrane water channels ubiquitously expressed in mammalian tissues. They play prominent roles in maintaining cellular fluid balance. Although expression of AQP1, -3, -4, -5, -8, -9, and -11 has been reported in the central nervous system, it is AQP4 that is predominately expressed. Its importance in fluid regulation in cerebral edema conditions has been highlighted in several studies, and we have also shown that translational regulation of AQP4 by miR-320a could prove to be useful in infarct volume reduction in middle cerebral artery occluded rat brain. There is evidence for the existence of two AQP4 transcripts (M1 and M23) in the brain arising from two alternative promoters. Because the AQP4 M1 isoform exhibits greater water permeability, in this study, we explored the possibility of microRNA-based transcriptional regulation of the AQP4 M1 promoter. Using RegRNA software, we identified 34 microRNAs predicted to target the AQP4 M1 promoter region. MicroRNA profiling, quantitative stem-loop PCR, and luciferase reporter assays revealed that miR-130a, -152, -668, -939, and -1280, which were highly expressed in astrocytes, could regulate the promoter activity. Of these, miR-130a was identified as a strong transcriptional repressor of the AQP4 M1 isoform. In vivo studies revealed that LNA(TM) anti-miR-130a could up-regulate the AQP4 M1 transcript and its protein to bring about a reduction in cerebral infarct and promote recovery.

MicroRNAs (miRNAs) are endogenous short (20–22 nucleotides) non-coding RNA molecules that mediate gene expression. This is an important regulatory mechanism to modulate fundamental cellular processes such as differentiation, proliferation, death, metabolism, and pathophysiology of many diseases. The miRNA expression profile of the kidney differs greatly from that of other organs, as well as between the different regions in the kidney. In kidneys, miRNAs are indispensable for development and homeostasis. In this review, we explore the involvement of miRNAs in the regulation of blood pressure, hormone, water, and ion balance pertaining to kidney homeostasis. We also highlight their importance in renal pathophysiology, such as in polycystic disease, diabetic nephropathy, nephrogenic diabetes insipidus, hypertension, renal cancer, and kidney fibrosis (epithelial–mesenchymal transition). In addition, we highlight the need for further investigations on miRNA-based studies in the development of diagnostic, prognostic, and therapeutic tools for renal diseases.

Venoms and toxins are of significant interest due to their ability to cause a wide range of pathophysiological conditions that can potentially result in death. Despite their wide distribution among plants and animals, the biochemical pathways associated with these pathogenic agents remain largely unexplored. Impoverished and underdeveloped regions appear especially susceptible to increased incidence and severity due to poor socioeconomic conditions and lack of appropriate medical treatment infrastructure. To facilitate better management and treatment of envenomation victims, it is essential that the biochemical mechanisms of their action be elucidated. This review aims to characterize downstream envenomation mechanisms by addressing the major neuro-, cardio-, and hemotoxins as well as ion-channel toxins. Because of their use in folk and traditional medicine, the biochemistry behind venom therapy and possible implications on conventional medicine will also be addressed.

MicroRNAs are gaining importance as regulators of gene expression with the capability to fine-tune and modulate cellular events. The complex network with their selective targets (mRNAs/genes) pave way for regulation of many physiological processes. Dysregulation of normal neuronal activities could result in accumulation of substances that are detrimental to neuronal functions and subsequently result in neurotoxicity. Neurotoxicity-mediated pathophysiological conditions could then manifest as diseases or disabilities like Parkinson’s and Alzheimer’s which have debilitating implications. Such toxicity can be a result of individuals predisposed due to genetic inheritance or from other sources such as brain tumours. Neurotoxicity can also be brought about by external agents like drugs and alcohol as well as brain injury with miRNAs playing a pivotal role in diseases. It is therefore vital to understand the expression of these microRNAs and their impact on neuronal activities. In this paper, we discuss some of the neuronal pathophysiological conditions that could be caused by dysregulated microRNAs.

To elucidate the role of chloride currents in erectile function through characterizing the behavior of corpus cavernosum (CC) in chloride-free (Cf) medium, which has not been evaluated before.Isolated rabbit CC strips were suspended in thermo-regulated organ baths containing oxygenated Tyrode for isometric tension recording. Cf Tyrode was prepared by substituting sodium chloride, calcium chloride, and potassium chloride (KCl) with equivalent molar concentrations of sodium acetate, calcium acetate, and potassium acetate salts. Resting cavernosal tone and contractions by noradrenaline, histamine, and KCl were assessed in Cf Tyrode with or without chloride channel blockers, niflumic acid (NFA), and anthracene-9-carboxylic acid (A9C).Withdrawal of extracellular chloride caused myogenic contractions in the unstimulated CC strips (n = 18). In addition, peak contractions by noradrenaline (n = 14) and histamine (n = 13) were augmented in Cf buffer by 47.2 ± 5.9% and 85.4 ± 13.2%, respectively (P.05), whereas KCl contractions were not significantly altered (17.6 ± 4.6%; n = 7). Interestingly, Cf buffer exerted opposing effects, potentiation and reduction, respectively, on the plateau phase of contractions mediated by noradrenaline and histamine. The stimulatory effect of Cf buffer on the intrinsic myogenic tone was diminished by NFA (30 μM), and A9C (300 μM-1 mM). NFA (30-100 μM), however, specifically reduced the plateau phase without significantly modifying the peak contraction of noradrenaline in Cf buffer.These results reiterate the importance of chloride currents as a mechanism underlying the maintenance of penile cavernosal tone. Thus, chloride channel could be an effective alternative target to regulate penile erection.

Stroke is one of the leading causes of death and disability worldwide. There are two major types of stroke: cerebral ischemia caused by obstruction of blood vessels in the brain and haemorrhagic stroke that is triggered by the disruption of blood vessels. Thrombolytic therapy involving recombinant tissue plasminogen activator (rtPA) has been shown to be beneficial only when used within 4.5 hours of onset of acute ischemic stroke. rtPA treatment beyond this time window has been found to be unsuitable and usually resulting in haemorrhagic transformation. Stroke is a multifactorial disease that forms a possible end state for majority of patients suffering from diabetes, atherosclerosis and hypertension which are known risk factors. Although the biochemistry of stroke and related diseases is quite well understood, the knowledge on the molecular mechanisms underlying these diseases is still at its infancy. microRNAs that form a unique class of endogenous riboregulators of gene function, offer tremendous potential in unraveling the mechanisms underlying stroke pathogenesis. microRNA expression also reflects the response of individuals to drugs and therapy. Several microRNAs and their target genes, known to be involved in endothelial dysfunction, dysregulation of neurovascular integrity, edema formation, pro-apoptosis, inflammation and extra-cellular matrix remodeling contribute to the critical processes in the pathogenesis of stroke. In this review, we will also be discussing the role of microRNAs as possible diagnostic and prognostic biomarkers as well as potential therapeutic targets in stroke pathogenesis.

Background Phospholipase A2 liberates free fatty acids and lysophospholipids upon hydrolysis of phospholipids and these products are often associated with detrimental effects such as inflammation and cerebral ischemia. The neuroprotective effect of neutral phospholipase from snake venom has been investigated. Results A neutral anticoagulant secretory phospholipase A2 (nPLA) from the venom of Naja sputatrix (Malayan spitting cobra) has been found to reduce infarct volume in rats subjected to focal transient cerebral ischemia and to alleviate the neuronal damage in organotypic hippocampal slices subjected to oxygen-glucose deprivation (OGD). Real-time PCR based gene expression analysis showed that anti-apoptotic and pro-survival genes have been up-regulated in both in vivo and in vitro models. Staurosporine or OGD mediated apoptotic cell death in astrocytoma cells has also been found to be reduced by nPLA with a corresponding reduction in caspase 3 activity. Conclusion We have found that a secretory phospholipase (nPLA) purified from snake venom could reduce infarct volume in rodent stroke model. nPLA, has also been found to reduce neuronal cell death, apoptosis and promote cell survival in vitro ischemic conditions. In all conditions, the protective effects could be seen at sub-lethal concentrations of the protein.

Background The methods currently available for diagnosis and prognosis of cerebral ischaemia still require further improvements. Micro-RNAs (small non-coding RNAs) have been recently reported as useful biomarkers in diseases such as cancer and diabetes. We therefore carried out microRNA (miRNA) profiling from peripheral blood to detect and identify characteristic patterns in ischaemic stroke. Methods/Principal Findings The ischaemic stroke patients aged between 18–49 years, characterized based on World Health Organization clinical criteria were further classified according to TOAST classification, a) Large-vessel atherosclerosis [n = 8] b) Small-vessel disease [n = 3] c) Cardioembolism [n = 5] d) Undetermined cause [n = 3]. The patients' functional status at the time of blood sampling (at the outpatient clinics) was evaluated with the modified Rankin Scale (mRS). Blood samples from normal (n = 5) individuals were used as controls. Total RNA extracted from whole blood was subjected to miroRNA profiling and real-time PCR analysis. miRNAs that are implicated in the endothelial/vascular function, erythropoiesis, angiogenesis and neural function showed differential expression profile as compared to the normal control. Interestingly, miRNAs that are involved in hypoxic conditions have also been found in our miRNA profiles. Conclusion We demonstrate that the peripheral blood miRNAs and their profiles can be developed as biomarkers in diagnosis and prognosis of cerebral ischaemic stroke. The dysregulated miRNAs have been detectable even after several months from the onset of stroke in what is usually regarded as neurologically stable patients.

MicroRNAs (miRNAs) belong to the group of non-coding, small RNAs (19 to 21 nucleotide-long oligonucleotides) that are endogenously produced from their own genes. miRNAs interfere with or affect the transcription or translation of other genes, resulting in gene silencing or activation by a process known as RNAi or RNA activation, respectively. The discovery of miRNAs and related small RNAs within the last 10 years has opened up a new field in biology, called RNomics, which investigates differences in RNA populations and offers great potential in correcting the regulation of abnormally expressed and disease-causing genes, as well as in the development of novel therapeutic agents. This review focuses on the developments in miRNAs and their potential therapeutic applications.

The in vivo interactions between alpha-neurotoxin, cardiotoxin and two phospholipases A2 (sputa-phospholipase A2-1 and 3) isolated from Malayan cobra venom were assessed by examining the effects of simultaneous injection of sub-LD50 dose of one toxin on (i) i.v. LD50 S of the other toxins in mice; and (ii) mean survival times of mice injected with lethal doses of the other toxins. While LD50 measurements did not reveal any interaction between the toxins in vivo, survival time measurements suggest a synergy between the neurotoxin and sputa-phospholipase A2-1 and between sputa-phospholipase A2-1 and sputa-phospholipase A2-3. Our results also suggest that both sputa-phospholipases A2 interfere with the lethal action of the cardiotoxin, resulting in prolongation of the mean survival time of mice injected with a lethal dose of cardiotoxin. The patterns of in vivo interactions between phospholipase A2 and other venom toxins appear to depend on the nature and mode of pharmacological action of the phospholipase A2.

Cardiotoxin-4b (CTX-4b), isolated from Naja naja sputatrix venom, shows lethality in several cell types. Employing murine primary cortical neurons, this study was undertaken to investigate the molecular mechanisms of CTX-4b in the induction of neuronal death. CTX-4b induced a dose- and time-dependent neuronal death. Strong induction of calpains as early as 4 h post-CTX-4b 75 nM treatment was detected in neurons with negligible caspase 3 activation. For the first time in cultured murine primary cortical neurons, it was noted that CTX-4b-mediated cell death triggered oxidative stress with an increase in reactive oxygen species (ROS) levels, and that application of antioxidants showed effective attenuation of cell death. Taken together, these results indicate that CTX-4b-mediated neuronal death is associated with (i) early calpain activation and (ii) oxidative stress. Most importantly, antioxidants have proved to be a promising therapeutic avenue against CTX-4b-induced neuronal death.

Aquaporins (AQPs) are a family of small hydrophobic, integral membrane proteins that are expressed in all living organisms and play critical roles in controlling the water flow into and out of cells. So far, 13 different AQPs have been identified in mammals (AQP 0-12). AQPs have recently been implicated in various diseases such as cancer, cataract, brain oedema, gallstone disease and nephrogenic diabetes insipidus, as well as in the development of obesity and polycystic kidney disease. Interfering with the expression of AQPs will undoubtedly have therapeutic applications. Hence, in this review, the authors look at each AQP and its association with various pathological conditions in humans and demonstrate that they form potential targets for the treatment of such diseases.

The structure and organization of the genes encoding the long-chain neurotoxins and four other isoforms of weak neurotoxins in the venom of Naja sputatrix are reported. The genes contained three exons interrupted by two introns, a structure similar to other members of the three-finger toxin family. The proteins encoded by these genes, however, show varied affinity towards nicotinic acetylcholine receptors. Phylogenetic analysis of these genes showed that the weak neurotoxin gene is confined to a distinct group. We also observe that specific mutations of the gene provide the diversity in function in these toxins while maintaining a common structural scaffold. This forms the first report where the molecular basis of evolution of postsynaptic neurotoxins from an ancestral gene can be demonstrated using the same species of snake.

Victims of snakebite quickly succumb to severe respiratory failure, which can be fatal if left untreated. One of the most toxic components of snake venom is phospholipase A2 (PLA2; EC 3.1.1.4). PLA2 isolated from the elapid, Naja sputatrix, induced pulmonary inflammation and edema when administered intravenously and intratracheally to rats. Analysis of pulmonary gene expression profiles using oligonucleotide microarrays revealed 60 genes whose expression was altered by at least 3-fold in response to intratracheal instillation of PLA2 for 3 h as compared with controls. In addition to genes encoding cytokines and chemokines responsible for inflammatory processes, the Na+/K+-ATPase gene has been found to be involved in edema formation. Real-time PCR, Western blot, and immunohistochemical analyses confirmed that the expression of AQP1 and AQP5 mRNAs and proteins was decreased. Besides providing an experimental model for studies on the pathophysiology of the lung, this investigation yields a clue to the mechanisms by which endogenous PLA2s could mediate inflammation in conditions such as allergy and rheumatoid arthritis.

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (K(d) = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 microm) with the neuronal alpha7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve-muscle preparations revealed no blocking activity of sWntx-5 at microm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.

We have characterized tamulustoxin, a novel 35-amino-acid peptide found in the venom of the Indian red scorpion (Mesobuthus tamulus). Tamulustoxin was identified through a [125I]toxin I screen, designed to identify toxins that block voltage-activated potassium channels. Tamulustoxin has also been cloned by RT-PCR, using RNA extracted from scorpion venom glands. Tamulustoxin shares no homology with other scorpion venom toxins, although the positions of its six cysteine residues would suggest that it shares the same structural scaffold. Tamulustoxin rapidly inhibited both peak and steady-state currents (18.9 +/- 1.0 and 37 +/- 1.1%, respectively) produced by injecting CHO cells with mRNA encoding the hKv1.6 channel.

This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the cardiotoxin (CTX)-2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500-bp (spliced) CTX-2 mRNA as the final transcript. However, the liver produces two types of CTX-2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP-1, C/EBP, CACCC-binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.

The elongation factor-1 alpha (EF-1alpha) is a highly conserved ubiquitous protein that is involved in translation and is desirable for use in phylogenetic studies on Blastocystis, an enigmatic intestinal parasite with a contentious taxonomic position. In the present study, a PCR product (BEalpha) that codes for a major part of the coding region of the EF-lalpha protein was amplified. Genome walking experiments together with cloning were implemented to elucidate the 5' and 3' ends of the EF-1alpha gene of the human isolate, Blastocystis hominis C. The genomic organization and the potential transcription factor binding sites of the 5' end of B. hominis C EF-1alpha were identified. A comparative study on the deduced amino acid sequences of BEalpha of 13 Blastocystis isolates from various hosts was done to evaluate the phylogenetic relationship among the species. A phylogenetic reconstruction analysis with other eukaryotic EF-1alpha sequences was carried out to trace the phylogenetic position of Blastocystis among eukaryotic organisms.

Two lethal proteins, which specifically bind to the nAChR from Torpedo californica, were isolated from the venom of Pseudonaja textilis, the common brown snake from Australia. The isolated proteins have masses of 6236 and 6345 Da and are structurally related to short-chain neurotoxins from other elapids. Six cDNAs encoding isoforms of related neurotoxins were cloned using the RT-PCR of the venom gland mRNAs. The sequences of the corresponding proteins consist of 57-58 amino acid residues and display several unique features when compared with all known short-chain neurotoxins. Accordingly, they grouped separately in phylogenetic analysis. The six cDNAs were expressed in Escherichia coli and the recombinant proteins were characterized. They have similar masses and display similar toxicities and binding constants to the nAChR as the native toxins isolated from the venom. Thus, a new group of short-chain postsynaptic neurotoxins from the venom of an Australian elapid has been characterized.

cDNAs encoding 4 short chain α-neurotoxins from Malayan spitting cobra (Naja naja sputatrix) venom were cloned and expressed in Escherichia coli. The recombinant toxins possessed identical amino acid sequences to α-neurotoxins. This is the first report on cloning and expression of isoforms of neurotoxins from a species of spitting cobra. Two of these isoforms were also identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), capillary electrophoresis followed by mass spectrometry and characterized by protein sequencing. Based on the variable amino acid residues, the neurotoxins in N. n. sputatrix could be assigned to 2 major groups. 10 E 11 T and 10 Q 11 A, which could be further subdivided into 10 E 11 T 28 S; 10 E 11 T 28 G and 10 Q 11 A 28 S: 10 Q 11 A 28 G respectively. These substitutions were also found to be unique to N. n. sputatrix neurotoxins. Phylogenetic analysis based on molecular properties of the toxins provided further support for the classification of N. n. sputatrix neurotoxin into 2 fundamental groups.

We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding cardiotoxins (CTX) of Naja naja sputatrix. The cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP-2, NFIL-6/C/EBP, NF-kappaB and PuF have been identified in the 5'-region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three-finger loop structure of the cardiotoxin molecule. The diverse functions of cardiotoxins together with our findings suggest that the cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.

Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.

Aquaporins (AQPs) are transmembrane water channel proteins that serve as nano-filters to selectively transport water molecules through plasma membranes in living cells at high speeds while blocking all other solutes from going through. It is envisioned that biomimetic membranes composed of aquaporin and amphiphilic phospholipids or copolymers may have great potential for use in high flux and low energy consumption water purification processes. However, the major barrier for using such a biomimetic membrane is that the ultrathin biomimetic bilayers are often too fragile to withstand high hydraulic or osmotic pressures in industrial water purification processes. Here, we show an innovative method to overcome membrane fragility by using these water channel proteins incorporated into vesicles and surface imprinting onto a substrate membrane. The water channel protein Aquaporin Z (AQPz) was reconstituted into self-assembled polymer vesicles and then immobilized onto a porous support membrane. In situ “surface imprinting” polymerization was then conducted to generate a dense hydrophobic polymer layer. This novel membrane was shown to have a well-controlled nano-structured selective layer that exhibits high mechanical strength and stability during the water filtration process. The water purification performance is significantly improved by using the AQPz-vesicle-imprinted membrane in either nanofiltration or forward osmosis mode.

A highly permeable yet highly selective pore-spanning biomimetic membrane embedded with aquaporin Z is molecularly designed and constructed via a combination of pressure-assisted vesicle adsorption and covalent-conjugation-driven vesicle fusion on a porous support. This approach represents a significant breakthrough in the architecture of biomimetic membranes embedded with aquaporin in a planar form.

M. C. M. Chung, N.-H. Tan and A. Armugam. The amino acid sequences of two postsynaptic neurotoxins isolated from Malayan cobra (Naja naja sputatrix) venom. Toxicon32, 1471–1474, 1994.—The complete amino acid sequences of two postsynaptic neurotoxins (toxin-3 and toxin-5) isolated from Malayan cobra (Naja naja sputatrix) venom were determined by direct automated Edman degradation of peptides obtained from digests with various proteases. Toxin-3 and toxin-5 are both short-chain neurotoxins and their amino acid sequences are highly homologous to Naja naja atra and Naja naja philippinensis neurotoxin, respectively. Toxin-3 is unique in possessing aspartic acid (D) as the fifth residue, while all other homologous short-chain neurotoxins have asparagine (N) at the corresponding position.

Aquaporin, a transmembrane protein that functions as a highly selective water channel, has intrigued membrane researchers around the world who have attempted to synthesize membranes embedded with aquaporin for water purification. We have molecularly designed pore-suspending biomimetic membranes embedded with Aquaporin Z (AQPz) based on different lipid protein ratios and investigated their local mechanical stabilities by force indentation using atomic force microscopy (AFM). The compatibility between 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and AQPz was proved by stop-flow experiments, in which the DMPC-AQPz (a lipid to protein molar ratio of 2000 : 1) exhibited 3000-fold higher permeability than the pristine DMPC vesicles. A series of vesicles were then ruptured on pristine porous alumina and carboxylated polyethylene glycol (PEG) coated porous alumina. The carboxylated-PEG polymer cushion significantly enhances the flexibility of the DMPC bilayer on the substrate and also gives rise to higher breakthrough forces from AFM when AQPz is reconstituted into the pore-suspending membrane on the carboxylated-PEG coated substrate. The local membrane mechanical stability is further improved by increasing the AQPz content, suggesting that AQPz increases the energy barrier required for a normal force to punch through the resultant biomimetic membrane. Therefore, the carboxylated-PEG grafted porous alumina exhibits a promising platform for the fabrication of pore-suspending biomimetic membranes. Nevertheless, to achieve the highest performance of DMPC-AQPz biomimetic membranes, a compromise may be needed to optimize the DMPC-AQPz ratio.

We have developed a new method for quantification of promoter activity in cell lines transfected with recombinant plasmids containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) by real-time PCR. As the efficiency of transfection has a direct influence on the total mRNA produced, we have used the neomycin-resistance gene present within the same vector DNA to normalize the measurement of mRNA levels. Three promoters from genes encoding toxins (pre-synaptic neurotoxin phospholipase A2, post-synaptic α neurotoxin and cardiotoxin), believed to have evolved from the same ancestor but exhibiting different promoter activities, have been employed in this study to demonstrate the feasibility and accuracy of the method in CAT gene reporter analysis.