Christensen M et al. (1999) Worm Breeder's Gazette "Identification of a Novel Mechanosensitive Anion Current in Embryonic Cells of C. elegans*"

Strange K, Christensen M

[Worm Breeder's Gazette, 1999]

Mechanotransduction plays a central role in fundamental physiologic processes such as detection of touch and sound, regulation of cell volume, and control of motility. Mechanosensitive channels have been studied extensively using electrophysiology. Little is known, however, about the molecular structure of mechanosensitive channels or about how mechanical stress is transduced into altered channel gating. We developed techniques to patch-clamp and study ion channels in C. elegans embryonic cells. In cell-attached and inside-out patches, application of gentle suction activated a mechanosensitive current. Suction caused an immediate increase in current amplitude of 6.4 2.8 fold (n = 20) at +100 mV in inside-out patches. The current rapidly inactivated when suction was discontinued and could be repeatedly reactivated by additional suction. When membrane voltage was ramped from +100 to -100 mV at 100 mV/second, the current showed moderate outward rectification (outward:inward current = 2.0 0.05 at 100 mV). Current amplitude was largely unaffected when bath Na+ was replaced with NMDG+ (n = 10). However, replacement of bath Cl- with either gluconate or glutamate reduced inward and outward currents by 46 10% and 39 7%, respectively (n = 9). Replacement of 120 mM bath Cl- with a mixture of 60 mM Cl- and 60 mM SCN- increased the inward current at -100 mV by 3.0 0.4 fold and shifted Erev by 16 2 mV (n=7). We conclude that membrane stretch activates a novel mechanosensitive anion current in inside-out patches from C. elegans embryonic cells.

Pokharel DR et al. (2006) Vaccine "Vaccination with Setaria cervi 175 kDa collagenase induces high level of protection against Brugia malayi infection in jirds."

Nandakumar Kodumudi K, Rai R, Pokharel DR, Rathaur S, Reddy MV

[Vaccine, 2006]

A zinc containing metalloprotease, 175 kDa collagenase, purified from adult female Setaria cervi showed strong cross-reactivity with sera from putatively immune (PI) individuals (unpublished observation) and induced cytotoxicity to B. malayi L3 larvae and microfilariae by ADCC mechanism [Srivastava Y, Bhandari YP, Reddy MVR, Harinath BC, Rathaur S. An adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi. J Helminth 2004;78:347-52]. These preliminary observations suggested the immunoprotective nature of collagenase. To confirm the vaccine potential of this protease, a vaccine trial was conducted in jirds (Meriones unguiculatus) against human filarial parasite B. malayi. The vaccination resulted into a mean protection level of 75.86% and produced high level of protease neutralizing antibodies. Cytokine analysis in immune jirds sera suggested a mixed Th1/Th2 type cellular immune response whereas ELISA, immunoblotting and enzyme antibody inhibition assay revealed the presence of specific anti-collagenase antibodies. Taken together, all these results suggest that S. cervi 175 kDa collagenase could form the basis of an effective molecular vaccine against human lymphatic filariasis.

Goodman MB et al. (1996) West Coast Worm Meeting "TOWARD LINKING MOLECULES TO NEURONS TO BEHAVIOR: A PROGRESS REPORT ON C. ELEGANS ELECTROPHYSIOLOGY"

Lockery SR, Goodman MB

[West Coast Worm Meeting, 1996]

Genetic analyses of behavior have identified gene products important for neuronal function in C. elegans, including putative sensory and neurotransmitter receptors. As a first step in forging a link between these proteins and cellular physiology, we have recorded from neurons in the heads of semi-intact, L1 worms. The technique was modified from that reported previously [WBG 13(5): 32] and regularly yields tight-seal, whole-cell recordings. In current clamp, neurons responded to current steps with graded potentials whose time course depended on the injected current (n=24). Action potentials could not be elicited. Some cells (n=3) exhibited spontaneous, sustained shifts in membrane potential from approximately -60 to -20 mV. At -60 mV, smaller amplitude, transient depolarizations were frequently observed. Voltage pulses to greater than -10 mV elicited an outward current with transient and sustained components. The rate of decay of the transient component varied between cells. The range (tau = 10-100 ms) is similar to that reported for the superfamily of cloned, voltage-gated potassium currents. Thus, the variation in decay rate could reflect heterogeneity in potassium channel expression among different classes of neurons. Hyperpolarizing pulses to less than -80 mV elicited a voltage-dependent inward current with no obvious time dependence. Between -80 and -10 mV, most cells had a region of high resistance. We are currently characterizing the main outward current and investigating how it might contribute to temporal processing in individual C. elegans neurons.

Cheirmaraj K et al. (1990) J Immunoassay "Diagnostic use of polyclonal antibodies raised in mouse ascitic fluid in bancroftian filariasis."

Reddy MV, Cheirmaraj K, Harinath BC

[J Immunoassay, 1990]

Polyclonal antibodies were produced against Brugia malayi adult antigens (BmA (PBS) SAg and BmA (SDS) SAg) in mouse ascitic fluid by immunising Balb/c mice intraperitoneally with high ratio of adjuvant to immunogen. The diagnostic use of these antibodies in detecting circulating filarial antigen in bancroftian filariasis was studied by sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using stick assay system. Both antibodies raised against PBS and SDS soluble antigens were found to be equally sensitive and relatively specific in detection of circulating filarial antigen. When anti BmA (PBS) SAg antibody was used in sandwich ELISA, 90% of microfilaraemic sera, 30-40% of acute and sub acute filarial sera, 20% of chronic filarial sera, 7% of endemic normal sera and none of 15 non-endemic normal sera were positive for filarial antigen. Using anti BmA (SDS) Sag antibody, 93% of microfilarial sera, 40% of acute and sub acute filarial sera, 20% of chronic filarial sera and none of 15 endemic and non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detection using anti BmA S Ag antibodies produced in mouse ascitic fluid in sandwich ELISA may be useful in detection of active stage (microfilaraemia) of infection.

Vasu C et al. (2000) Parasitol Int "A 43-kDa circulating filarial antigen fraction of Wuchereria bancrofti in immunoprophylaxis against Brugia malayi in jirds."

Harinath BC, Vasu C, Reddy MV

[Parasitol Int, 2000]

A 43 kiloDaltan (kDa) antigen fraction (CFA2-6) isolated from microfilaraemic plasma of bancroftian filarial patients showed selective reactivity with sera samples collected from endemic normals. Antibodies raised against this antigen showed a strong reactivity with the surface of Brugia malayi infective larvae as well as microfilariae. Similar antigenic determinants were detected in the parasite extracts, but not in the excretory-secretory products. Further analysis was done on the immunoprophylactic potential of CFA2-6 in inducing immunity against Brugia malayi in Meriones unguiculatus (jird) in vivo. A strong protective response of approximately 84% was observed against the development of the filarial parasite in the jirds immunized with CFA2-6. The immunized jirds also showed a significant clearance (87%) of microfilariae inoculated intravenously. Approximately 65% of infective larvae failed to survive in jirds transferred with anti-CFA2-6 serum compared to the jirds transferred with sera from the control jirds. Passive transfer of anti-CFA2-6 antibody to the jirds followed by intravenous inoculation of microfilariae resulted in the reduction of 77% of circulating microfilariae. This study suggests that the 43-kDa CFA2-6 could stimulate a strong protective immune response against infective larvae and microfilariae in experimental animals.

Tom Nickell et al. (2002) Mid-west Worm Meeting "Calcium-activated Potassium Channels in C. elegans chemosensory neurons"

Ray Pun, Steve Kleene, Tom Nickell

[Mid-west Worm Meeting, 2002]

Calcium-activated potassium channels are present in the C. elegans nervous system, including many neurons of the nerve ring. Properties of these channels have been reported in a heterologous expression system; however, it was of interest to determine whether channels of similar properties could be recorded from C. elegans neurons. We recorded single channels in excised patches from the chemosensory neuron AWA, which were labeled with GFP. Neurons were exposed by cutting the worm with a scalpel blade just behind the terminal bulb. Excised patches were formed in conventional manner. Channels corresponding to BK potassium channels were present in AWA neurons. In patches containing the channels, channels were absent or only activated at high positive potentials (+100 mV). Addition of 100 mM calcium markedly shifted the activation of the channels toward more negative potentials, with channel openings visible at 0 mV and more positive potentials. Mean conductance of these channels was about 65 pS, with a reversal potential of -50 mV.

Anand SB et al. (2011) J Helminthol "A combination of two Brugia malayi filarial vaccine candidate antigens (BmALT-2 and BmVAH) enhances immune responses and protection in jirds."

Anand SB, Reddy MV, Kaliraj P, Kodumudi KN

[J Helminthol, 2011]

In this study filarial recombinant protein or DNA vaccine constructs encoding BmALT-2 and BmVAH as single or as cocktail antigens were evaluated. Male jirds were immunized intramuscularly with DNA vaccine constructs or were immunized intraperitoneally with protein vaccine. The single and bicistronic DNA constructs induced substantial interferon- responses in spleen cells; antigen-specific responses were higher following immunization with the bicistronic cocktail construct and evoked a significant protective response of 57% in jirds challenged with Brugia malayi that was similar in the antibody-dependent cellular cytotoxicity (ADCC) assay and micropore chamber experiment. The cocktail protein vaccines induced a mixture of IgG2a (Th1) and IgG1 (Th2) responses with 80% protective response when challenged with B. malayi infective larvae. However, the single protein vaccine rALT-2 induced Th2 (IgG1/IgG3) with a 70% protective response and rVAH induced Th1 (IgG2a) with a lower proliferative response with 60% protection following challenge with B. malayi infective larvae. These results suggest that filarial cocktail protein vaccines are able to elicit substantial immune and protective responses when compared with single antigen vaccination in suitably vaccinated jirds.

I Vinogradova et al. (2002) European Worm Meeting "Patch-clamp study of the cationic currents in the pharyngeal muscle cells of the adult nematode C.elegans"

R Walker, I Vinogradova, L Holden-Dye

[European Worm Meeting, 2002]

Continuing contractions and extensive intercellular coupling prevent reliable whole-cell current recording from the enzymatically cleaned muscle cells of the C.elegans pharynx. However, confirming data previously obtained with intracellular recording with sharp microelectrodes (Pemberton et al., 2002), it has been shown that sodium omission from the Dent's physiological saline blocks inward currents in response to depolarisations from the holding potential of 120 mV to 40 mV both in wild type worms and egl 19 (n582). In outside-out patches tested with Cs-internal solution and Dent's saline and held at 80 mV channels with very low opening probability were observed. Amplitude of the unitary currents in these patches was reduced in 0Na+ Dent's saline but not in 0Ca2+ solution. Currents were further reduced and then abolished in 0Na+ 0Ca2+ solution. Exposure of the patches to the Dent's saline with 10-times reduced concentration of Na+ caused a shift in reversal potential smaller that expected in accordance with the Nernst equation. Channels can be blocked by gadolinium ions and their mean opening time increased by veratridine. In inside out patches tested in symmetrical 150 mM Na+ solution containing no Ca2+ and K+ ions opening probability of the recorded channels at 80 mV was 3 times higher than in outside-out patches but was reduced to the level observed in outside-out patches when 3 mM Ca2+ were added to the solution contacting the external surface of the membrane patches. Taken together these data suggest the presence of some sort of sodium permeable channels in pharyngeal muscle cells of the adult worms.

Byerly L et al. (1978) Worm Breeder's Gazette "voltage-clamp studies of the electrical currents of the Ascaris pharyngeal muscle."

Masuda M, Byerly L

[Worm Breeder's Gazette, 1978]

It has proven difficult to study the currents that generate the electrical activity in the somatic muscle cells of Ascaris due to the inability to control the voltage across the excitable membrane. Therefore, we have directed our attention to the pharyngeal muscle, where it is possible to directly measure the voltage and pass large currents across the excitable membrane. We have developed a system which allows us to do current-clamp and voltage-clamp experiments on an isolated segment of the pharyngeal membrane. We find that this membrane has no pacemaker activity. In the absence of nervous input the membrane potential is flat at a level near -40 mV. Two types of spontaneous postsynaptic potentials are frequently seen; one type has a reversal potential near -40 mV and the other has a reversal potential near -10 mV. When the membrane is at the resting level, this second type PSP triggers a positive-going action potential, which reaches a level between +30 and +50 mV. The membrane potential then falls to a plateau near O mV, where it remains until a negative-going PSP triggers a negative-going action potential that reaches about -50 mV (the potassium reversal potential). The membrane potential remains at the plateau level for periods ranging from 100 msec to several minutes. The positive-going action potential is produced by an inward current that appears to be carried by both Na+ and Ca++. This current is prolonged, showing little inactivation by 200 msec after a positive voltage step. Clamping the membrane to positive potentials elicits essentially no delayed-rectification K current, the current that normally repolarizes active membranes. However, stepping the membrane potential back to the resting level after a large positive pulse elicits a strong, transient outward K+ current; this is the current that produces the negative-going action potential. We have done a detailed analysis of this current and have shown that it is a voltage- inverted analogue of the Hodgkin-Huxley Na+ current. It is activated by negative steps in potential. It shows inactivation, being completely inactivated at the resting potential. Conditioning pulses to levels more positive than -10 mV are necessary to remove the inactivation from the channel. This is a new K+ conductance that has not been found in any other animal. This demonstration of unique mechanisms in nematode physiology should serve as a caution in trying to interpret the function of the nematode nervous system. The pharyngeal nervous system must not only generate the signal that triggers the pharynx to contract (open the lumen), but also the signal to relax (close the lumen). Since the relaxation of the pharynx is the 'power stroke' of this muscle, it is not surprising that the membrane has developed a special K+ current to produce a fast, separately-triggerable repolarization.

Dabir S et al. (2008) Vaccine "Prophylactic evaluation of recombinant extracellular superoxide dismutase of Brugia malayi in jird model."

Dabir P, Dabir S, Reddy MV, Goswami K

[Vaccine, 2008]

The immunoscreening of Brugia malayi adult cDNA library with pooled endemic normal sera identified several seroreactive clones including, EC-SOD which contained a 612 bp insert and showed significant nucleotide and deduced amino acid sequence homologies with superoxide dismutase (SOD) of other nematode parasites. The SODs are known to play an important role in the protection of parasite against reactive oxygen species of the host. The coding region of the B. malayi EC-SOD (BmEC-SOD) was cloned and expressed in Escherichia coli followed by affinity purification on nickel agarose resin. Staining of native polyacrylamide gel for SOD activity of the expressed recombinant protein revealed that SOD activity inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide, indicating presence of Cu/Zn-SOD. The rBm EC-SOD protein showed its activity over a broad range of pH.7.0-11.0. Further the immune protective activity of recombinant EC-SOD antigen was evaluated in susceptible host, jirds (gerbils) (Meriones unguiculatus) against B. malayi filarial infection. The immunized jirds showed 33.5% and 36% cytotoxicity against microfilariae and 42.8% and 45.5% cytotoxicity against infective larvae in in vitro antibody dependent cellular cytotoxicity (ADCC) assay and in in situ micropore chamber methods respectively. This study suggests that the rBm EC-SOD antigen could stimulate a partial protective immune response against microfilariae and infective larvae in experimental animals against filarial infection.