Inhibition of matrix metalloproteinase-9 secretion by dimethyl sulfoxide and cyclic adenosine monophosphate in human monocytes

BACKGROUND Matrix metalloproteinases(MMPs),including MMP-9,are an integral part of the immune response and are upregulated in response to a variety of stimuli.New details continue to emerge concerning the mechanistic and regulatory pathways that mediate MMP-9 secretion.There is significant evidence for regulation of inflammation by dimethyl sulfoxide(DMSO)and 3',5'-cyclic adenosine monophosphate(cAMP),thus investigation of how these two molecules may regulate both MMP-9 and tumor necrosis factorα(TNFα)secretion by human monocytes was of high interest.The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFαsecretion by distinct mechanisms.AIM To investigate the regulation of lipopolysaccharide(LPS)-stimulated MMP-9 and tumor necrosis factorαsecretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP.METHODS The paper describes a basic research study using THP-1 human monocyte cells.All experiments were conducted at the University of Missouri-St.Louis in the Department of Chemistry and Biochemistry.Human monocyte cells were grown,cultured,and prepared for experiments in the University of Missouri-St.Louis Cell Culture Facility as per accepted guidelines.Cells were treated with LPS for selected exposure times and the conditioned medium was collected for analysis of MMP-9 and TNFαproduction.Inhibitors including DMSO,cAMP regulators,and anti-TNFαantibody were added to the cells prior to LPS treatment.MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software.TNFαsecretion was analyzed by enzyme-linked immuno sorbent assay.All data is presented as the average and standard error for at least 3 trials.Statistical analysis was done using a two-tailed paired Student t-test.P values less than 0.05 were considered significant and designated as such in the Figures.LPS and cAMP regulators were from Sigma-Aldrich,MMP-9 standard and antibody and TNFαantibodies were from R&D Systems,and amyloid-βpeptide was from rPeptide.RESULTS In our investigation of MMP-9 secretion from THP-1 human monocytes,we made the following findings.Inclusion of DMSO in the cell treatment inhibited LPSinduced MMP-9,but not TNFα,secretion.Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dosedependent fashion.A cell-permeable cAMP analog,dibutyryl cAMP,inhibited both LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFαin a dose-dependent fashion.Pre-treatment of monocytes with an anti-TNFαantibody blocked LPSinduced MMP-9 and TNFαsecretion.Amyloid-βpeptide induced MMP-9 secretion,which occurred much later than TNFαsecretion.The latter two findings strongly suggested an upstream role for TNFαin mediating LPS-stimulate MMP-9 secretion.CONCLUSION The cumulative data indicated that MMP-9 secretion was a distinct process from TNFαsecretion and occurred downstream.First,DMSO inhibited MMP-9,but not TNFα,suggesting that the MMP-9 secretion process was selectively altered.Second,cAMP inhibited both MMP-9 and TNFαwith a similar potency,but at different monocyte cell exposure time points.The pattern of cAMP inhibition for these two molecules suggested that MMP-9 secretion lies downstream of TNFαand that TNFαmay a key component of the pathway leading to MMP-9 secretion.This temporal relationship fit a model whereby early TNFαsecretion directly led to later MMP-9 secretion.Lastly,antibody-blocking of TNFαdiminished MMP-9 secretion,suggesting a direct link between TNFαsecretion and MMP-9 secretion.     

The associated regulators and signal pathway in rIL-16／CD4 mediated growth regulation in Jurkat cells

IL-16 is a ligand and chemotactic factor for CD4 T cells. IL-16 inhibits the CD3 mediated lymphocyte activation and proliferation. The effects of IL-16 on the target cells are dependent on the cell type, the presence of co-activators etc. To understand the regulation function and mechanism of IL-16 on target cells, we used a 130 a.a. recombinant IL-16 to study its effects on the growth of Jurkat T leukemia cells in vitro. We found that the rIL-16 stimulated the proliferation of Jurkat cells at low dose (10^-9M), but inhibited the growth of the cells at higher concentration (10^-5M). Results showed that 10^-5 M of rIL-16 treatment induced an enhanced apoptosis in Jurkat cells. The treatment blocked the expression of FasL, but up-regulated the c-myc and Bid expression in the cells. Pre-treatment of PKC inhibitor or MEK1 inhibitor markedly increased or decreased the rIL-16 induced growth-inhibiting effects on Jurkat cells, respectively.The results suggested that the rIL-16 might be a regulator for the growth or apoptosis of Jurkat cells at a dose-dependent manner. The growth-inhibiting effects of rIL-16 might be Fas/FasL independent, but,associated with the activation of PKC, up-regulated expression of c-Myc and Bid, and the participation of the ERK signal pathway in Jurkat cells.     

LPS-induced activation of phospholipase A_2 phospholipase Cand protein kinase C of murine macrophage-like cell lines （J774 and P388D1）

A murine macrophage-like cell line,J774,acquried,in response to LPS,an ability to kill tumor necrosisfactor(TNF)-insensitive target P815 mastocytoma cells,whereas another cell line,P388D1,did not.LPS-triggered signaling mechanisms between the two celllines were compared with an aim to inquire about thepossible nature of the above-mentioned difference.Theresults showed that two cell lines respond to LPS-treatment by parallel activation of both phospholipasesC and A_2(PLC and PLA_2)to approximately the sameextent.The maximum response of both enzymes of J774cells was noted within 10 min of the treatment,whereas that of P388D1 cells required more than 20min.The other properties of LPS-responsive enzymesstudied were similar between two cell lines,ineludingActivation of PLC and PLA_2 and PKC in macrophages by LPSCa~(2 )augmentation of enzyme activation,participationof guanine nucleotide binding (G) proteins in theinitial activation processes,and inhibition of enzymeactivation by the prior treatment of cells with choleraorpartussis toxins etc.Moreover,LPS-triggered activationof PLC and PLA2 was found to be followed by theincrease of PKC activities in both cell lines.In spite ofthese similarities,J774 cells possessed both basic andacidic forms of PKC activities,while P 388D1 cells ownedonly PKC of basic form.Nevertheless,the question whyJ774 cells,but not P388D1 cells,can acquire thetumoricidal actiyity,aganist P815 cells following LPS-treatment remains to be answered.     

PKCa translocation from mitochondria to nucleus is closely related to induction of apoptosis in gastric cancer cells

PKCs have been implicated in the regulation of cellular differentiation, proliferation, apoptosis and signal transduction. It was demonstrated in this study that PKCα was located both at mitochondria and in cytosol in gastric cancer cell line BGC-823. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the translocation of PKCα from both mitochondria and cytosol to nucleus as clearly shown by laser-scanning-confocal microscopy, while the protein level of PKCα was not changed by TPA treatment as detected by Western blot. The results also revealed that TPA-induced translocation of PKCα was in close association with apoptosis induction, and such association was further affirmed by other experiments where various apoptotic stimuli and specific inhibitors of PKC were used. Taken together, these findings indicate that translocation of PKCα from both mitochondria and cytosol to nucleus in gastric cancer cell is accompanied by induction of apoptosis, and may imply a new mechanism of the potential linking between cell apoptosis and PKCα translocation.     

Caspase-1 activation and mature interleukin-1β release are uncoupled events in monocytes

AIM:To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β(pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.METHODS:All experiments were performed on fresh or overnight cultured human peripheral blood monocytes(PBMCs) that were isolated from healthy donors.PBMCs were activated by lipopolysaccharide(LPS) stimulation before being treated with Adenosine triphosphate(ATP,1 mmol/L),human α-defensin-5(HD-5,50 μg/mL),and/or nigericin(Nig,30 μmol/L).For each experiment,the culture supernatants were collected separately from the cells.Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies.RESULTS:We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation.In the presence of HD-5,this release of IL-1β,but not the processing of pro-IL-1β to IL-1β,was completely inhibited.Similarly,in the presence of HD-5,the release of IL-1β,but not the processing of IL-1β,was significantly inhibited from LPS-activated monocytes stimulated with Nig.Finally,we treated LPS-activated monocytes with ATP and Nig and collected the supernatants.We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes.Interestingly,and contrary to IL-1β processing and release,caspase-1 cleavage and release was not blocked by HD-5.All images are representative of three independent experiments.CONCLUSION:These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.     

The associated regulators and signal pathway in rIL-16/CD4 mediated growth regulation in Jurkat cells

IL-16 is a ligand and chemotactic factor for CD4+ T cells. IL-16 inhibits the CDS mediated lymphocyte activation and proliferation. The effects of IL-16 on the target cells are dependent on the cell type, the presence of co-activators etc. To understand the regulation function and mechanism of IL-16 on target cells, we used a 130 a.a. recombinant IL-16 to study its effects on the growth of Jurkat T leukemia cells in vitro. We found that the rIL-16 stimulated the proliferation of Jurkat cells at low dose (10-9M), but inhibited the growth of the cells at higher concentration (10-5M). Results showed that 10-5 M of rIL-16 treatment induced an enhanced apoptosis in Jurkat cells. The treatment blocked the expression of FasL, but up-regulated the c-myc and Bid expression in the cells. Pre-treatment of PKC inhibitor or MEK1 inhibitor markedly increased or decreased the rIL-16 induced growth-inhibiting effects on Jurkat cells, respectively. The results suggested that the rIL-16 might be a regulator for the growth     

LPS—induced activation of phospholipase A2 phospholipase C and protein kinase C of murine macrophage—like cell lines （J774 and P388D1）

A murine macrophage-like cell line J774,acquired,in response to LPS,an ability to kill tumor necrosis factor(TNF)-insensitive target P815 mastocytoma cells whereas another cell line,P388D1 did not ,LPS triggered signaling mechanisms between the two cell lines were compared with an aim to inquire about the possible nature of the above-mentioned difference,The results whowed that two cell lines respond to LPS-treatment by parallel activation of both phospholipases C and A2 (PLC and PLA2) to approximately the same extent.The maximum response of toth enzymes of J774 cells was noted within 10 min the treatment whereas that of P388D1 cells required more than 20 min,The other properties of LPS-responsive enzymes studied were similar between two cell lines,including Activation of PLC and PLA2 and PKC in macrophages by LPS. Ca2 augmentation of enzyme activation,participation of guanine nucleotide binding(G) proteins in the initial activation preocesses,and inhibition of enzyme activation by the prior treatment of cells with choleraor pertussis toxinsetc.Moreover,LPS-triggered activation of PLC and PLA2 was found to be followed by the increase of PKC activities in both cell lines.Inspite of these similarities.J774 cells possessed both basic and acidicforms of PKC activities,while P 388 D1 cells owned only PKC of basic form,Nevertheless,the question why J774 cells but not P388D1 cells,can acquire the tumoricidal activity,aganist P815,cells following LPStreatment rematins to be answered.     

Transient and sustained oxidative stress differentially activate the JNK1/2 pathway and apoptotic phenotype in H9c2 cells

The aim of this study was to investigate the activation of JNK1/2 signalling pathway and the respective cellular phenotype of H9c2 cardiac myoblasts during two distinct types of oxidative insult. We examined the dose- and time-dependent activation of JNK1/2 pathway by exogenous H₂O₂, both under transient and sustained stimulation. At 2 h of either sustained or transient treatment, maximal phosphorylation of c-Jun was observed, coincidently with the activation of nuclear JNK1/2; under sustained stress, these phosphorylation levels remained elevated above basal for up to 6 h, whereas under transient stress they declined to basal ones within 4 h of withdrawal. Furthermore, the JNK1/2 selective inhibitor SP600125 abolished the c-jun phosphorylation induced by oxidative stress. Our results using cell viability assays and light microscopy revealed that sustained H₂O₂ stimulation significantly and time-dependently decreased H9c2 viability, in contrast to transient stimulation; SP600125 (10 μM) abolished cell death induced by sustained as well as cell survival induced by transient oxidative stress. Hoechst staining showed an increase in DNA condensation during sustained, but not during transient stimulation. Moreover, from the antioxidants tested, catalase and superoxide dismutase prevented oxidative stress-induced cell death. Flow cytometry studies reconfirmed that sustained oxidative stress induced apoptosis, whereas transient resulted in the recovery of cardiac myoblasts within 24 h. We conclude that in H9c2 myoblasts, sustained activation of JNK1/2 signalling pathway during oxidative stimulation is followed by an apoptotic phenotype, while transient JNK1/2 activation correlates well with cell survival, suggesting a dual role of this signalling pathway in cell fate determination.     

Elicitation of anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> and the involvement of methyl jasmonate and salicylic acid

The effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the anthocyanin accumulation, endogenous titres of polyamines and ethylene production in callus cultures of Daucus carota were studied. The interaction of these signaling molecules with elicitors from Aspergillus niger was investigated and the involvement of MeJA was elucidated through the use of the jasmonic acid (JA) biosynthetic inhibitor ibuprofen. MeJA and SA were both found to stimulate the anthocyanin production in the callus cultures. The highest levels of anthocyanin was observed in the cultures treated with 200 μM SA 0.36 % and 0.01 μM MeJA 0.37 %. The MeJA and SA treatments were also found to result in higher activity of Ca²⁺ ATPase suggesting that the enhancement of anthocyanin by SA and MeJA could be mediated through the involvement of the calcium channel. The treatment of the callus cultures with SA was found to result in marginally higher titres of endogenous polyamines (PAs) whereas MeJA resulted in lower levels of PAs as compared to the control. The SA treatment was found to result in lower ethylene production and the treatment with MeJA stimulated the ethylene production. These results suggest that the stimulation of anthocyanin production by MeJA and SA in callus cultures of D. carota is not related to the ethylene production.     

Immunomodulated signaling in macrophages: Studies on activation of Raf-1, MAPK, cPLA_2 and secretion of IL-12

Little is known about the mechanism and signal transduction by LPS-mediated immunomodulation of murine peritoneal macrophages. It is found that the signal molecules of the down-stream of Ras, Raf-1, MAPK p44, and MAPK p42 are phosphorylated, and cPLA2 is activated with a significant increase of the release of [ H3 ] AA by macrophages in response to LPS and PMA. Compared with the very recent finding that LPS and PMA trigger the activation and translocation of PKC-α and PKC-ε, these findings suggest that there is a connection between PKC signaling pathway and the Raf-1/MAPK pathway and that the activation of these main signaling events may be closely related to the secretion of IL-12 during LPS-induced modulation of macrophages.     

Fluorescence resonance energy transfer analysis of bid activation in living cells during ultraviolet-induced apoptosis

Ultraviolet (UV) irradiation is a DNA-damaging agent that triggers apoptosis through both themembrane death receptor and mitochondrial apoptotic signaling pathways.Bid,a pro-apoptotic Bcl-2family member,is important in most cell types to apoptosis in response to DNA damage.In this study,arecombinant plasmid,YFP-Bid-CFP,comprised of yellow and cyan fluorescent protein and a full length Bid,was used as a fluorescence resonance energy transfer analysis (FRET) probe.Using the FRET techniquebased on YFP-Bid-CFP,we found that Bid activation was initiated at 9±1 h after UV irradiation,and theaverage duration of the activation was 75±10 min.Bid activation coincided with a collapse of the mitochondrialmembrane potential with an average duration of 50±10 min. When cells were pretreated with Z-IETD-fmk(caspase-8 specific inhibitor) the process of Bid activation was completely inhibited,but the apoptosis wasonly partially affected.Z-DEVD-fmk (caspase-3 inhibitor) and Z-FA-fmk (non asp specific inhibitor) didnot block Bid activation.Furthermore,the endogenous Bid activation with or without Z-IETD-fmk in responseto UV irradiation was confirmed by Western blotting.In summary, using the FRET technique,we observedthe dynamics of Bid activation during UV-induced apoptosis and found that it was a caspase-8 dependentevent.     

Visualization of Golgia apparatus as an intracellular calcium store by laser scanning confocal microscope

Using laser scanning confocal microscopy,we have found that the in cells loaded with fluo-3/AM,highest intracellular Ca^2 in the perinuclear region is associated with the Golgi apparatus.The spatiotemporal subcellular distribution of Ca^2 in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated.PDGF,which releases Ca^2 from intracellular stores by inositol(1,4,5)-trisphosphate pathway ,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the golgi apparatus.Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin,an inhibitor of the Ca^2 transport ATPase of intracellular calcium store,Permeablizing the plasma membrane by 10μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store.These results suggest that the Golgi apparatus plays a role in Ca^2 regulation in signal transduction.     

PKCα translocation from mitochondria to nucleus is closely related to induction of apoptosis in gastric cancer cells

PKCs have been implicated in the regulation of cellular differentiation, proliferation, apoptosis and signal transduction. It was demonstrated in this study that PKCa was located both at mitochondria and in cytosol in gastric cancer cell line BGC-823. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the translocation of PKCa from both mitochondria and cytosol to nucleus as clearly shown by laser-scanning-confocal microscopy, while the protein level of PKCa was not changed by TPA treatment as detected by Western blot. The results also revealed that TPA-induced translocation of PKCa was in close association with apoptosis induction, and such association was further affirmed by other experiments where various apoptotic stimuli and specific inhibitors of PKC were used. Taken together, these findings indicate that translocation of PKCa from both mitochondria and cytosol to nucleus in gastric cancer cell is accompanied by induction of apoptosis, and may imply a new mechanism of th     

Enhanced NTPDase and 5′-nucleotidase activities in diabetes mellitus and iron-overload model

The activity of the enzymes NTPDase and 5′-nucleotidase was studied in both diabetes mellitus and an associated model of iron-overload. Rats were divided in five groups: citrate (CC), saline (S), diabetic (D), iron-overload (IO), and diabetic iron-overload (DIO). Diabetes was induced with alloxan (150 mg/kg), and iron-overload was induced with iron-dextran (10 intramuscular applications of ±80 mg/kg). The enzymatic activities were evaluated in the platelets. The results demonstrated an increase in the activity of NTPDase with substrates ATP and ADP (60% and 120%, respectively; P < 0.001), and 5′-nucleotidase (60%, P < 0.001). This increase was more intense in the IO and DIO groups. The results obtained in vitro showed an activation in ATP, ADP, and AMP hydrolysis between 1 μM and 1,000 μM ferric nitrate concentrations, being more pronounced at 100 μM and decreasing at 1,000 μM. We concluded that diabetes mellitus in association with iron-overload increased the hydrolysis of adenine nucleotides in platelets, contributing to the abnormalities found in these pathological conditions.     

Response of T cells in vivo induced by repeated superantigen treatments at different time intervals

We have investigated the response of T cells to staphylococcal enterotoxin A （SEA） injections in vivo. We found that a single injection of SEA with an optimal dose of 10μg increased the expression of both CD4 and CD8 significantly. There was expansion of SEA-reactive T cells in vivo after SEA re-injection and the time interval between injections strongly influenced the responsiveness of CD4^＋ and CD8^＋ T cells. Anergy of T cells was observed after three SEA treatments. The time interval between injections mainly affected the unresponsiveness of CD4^＋ T cells, not CD8^＋ T cells. Marked deletion followed by anergy of CD4^＋ T cells was induced at short intervals, and anergy without obvious deletion of CD4^＋ T cells was induced at long intervals. We also found that the anergic state was reversible in vivo. Repeated SEA stimulation led to down-regulation of interleukin （IL）-2, and high levels of IL-10. This study showed that both CD4^＋ and CD8^＋ SEA-primed T cells were responsive to SEA rechallenge in vivo, and a third injection was needed to induce the anergy of T cells.     

Cardiolipin is essential for higher proton translocation activity of reconstituted F_o

The Fo membrane domain of FoF1-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of Fo was about 85% and the sample contained no subunits of F1-ATPase. The purified Fo was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H+ translocation activity of Fo was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of Fo with the order of CL>PA>>PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of Fo vesicles containing CL, which suggested that CL's stimulation of the activity of reconstituted Fo might correlate with its non-bilayer propensity. After Fo was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphospha     

Inhibition of associative long-term depression by activation of β-adrenergic receptors in rat hippocampal CA1 synapses

The aim of this study was to investigate the role of β-adrenergic receptors in modulating associative long-term depression (LTD) at CA1 synapses in rat hippocampal slices. Standard extracellular electrophysiological techniques were employed to record field excitatory post-synaptic potential (fEPSP) activity and to induce associative LTD. Two independent Schaffer collateral pathways were elicited in hippocampal CA1 areas. In one (weak) pathway, the stimulating intensity was adjusted to elicit small fEPSP activity (20–30% of the maximum response). In contrast, 80–90% of the maximum response was evoked in the other (strong) pathway. Associative LTD of weak pathway could be induced by paired stimulation of weak and the strong pathways, repeated 100 times at 0.167 Hz. The associative LTD of weak pathway was NMDA receptor- and phophatase 2B dependent, because bath application of 50 μM D, L-AP5 or 10 μM cypermethrin blocked its induction. Bath application of 1 μM isoproterenol inhibited associative LTD, and this effect was blocked by timolol, suggesting the involvement of β-adrenergic receptors. The inhibitory effect of β-adrenergic receptors on LTD induction was blocked in slices pretreated with inhibitors of protein kinase A and mitogen-activated protein kinase, suggesting that these signal cascades are downstream effectors following activation of β-adrenergic receptors. Nevertheless, bath application of timolol or cypermethrin alone did not have significant effect on associative LTD induction, suggesting neither endogenous function of β-adrenergic receptor nor endogenous PKA activity does have a role in associative LTD induction.     

Evaluations of larvicidal activity of medicinal plant extracts to Aedes aegypti （Diptera： Culicidae） and other effects on a non target fish

A preliminary study was conducted to investigate the effects of the extracts of 112 medicinal plant species, collected from the southern part of Thailand, on Aedes aegypti. Studies on larvicidal properties of plant extracts against the fourth instar larvae revealed that extracts of 14 species showed evidence of larvicidal activity. Eight out of the 14 plant species showed 100% mosquito larvae mortality. The LC50 values were less than 100μg/mL （4.1μg/ mL-89.4μg/mL）. Six plant species were comparatively more effective against the fourth instar larvae at very low concentrations. These extracts demonstrated no or very low toxicity to guppy fish （Poecilia reticulata）, which was selected to represent most common non-target organism found in habitats ofAe. aegypti, at concentrations active to mosquito larvae. Three medicinal plants with promising larvicidal activity, having LC50 and LC50 values being 4.1 and 16.4 μg/mL for Mammea siamensis, 20.2 and 34.7 μg/mL forAnethum graveolens and 67.4 and 110.3μg/mL forAnnona muricata, respectively, were used to study the impact of the extracts on the life cycle ofAe. aegypti. These plants affected pupal and adult mortality and also affected the reproductive potential of surviving adults by reducing the number of eggs laid and the percentage of egg hatchability. When each larval stage was treated with successive extracts at the LC50 value, the first instar larvae were found to be very susceptible to A. muricata and the second instar larvae were found to be susceptible to A. graveolens, while the third and fourth instar larvae were found to be susceptible to M. siamensis. These extracts delayed larval development and inhibited adult emergence and had no adverse effects on P. reticulata at LC50 and LC50 values, except for the M. siamensis extract at its LC50 value.     

Defective maintenance of intracellular Ca^2＋ homeostasis is linked to increasedmuscle fatigability in the MG29 null mice

Mitsugumin 29 (MG29) is a transmembrane protein that is normally found in the triad junction of skeletal muscle. Our previous studies have shown that targeted deletion of rag29 from the skeletal muscle resulted in abnormality of the triad junction structure, and also increased susceptibility to muscle fatigue. To elucidate the basis of these effects, we investigated the properties of Ca^2 uptake and -release in toxin-skinned Extensor Digitorium Longus (EDL) muscle fibers from control and rag29 knockout mice. Compared with the control muscle, submaximal Ca^2 uptake into the sarcoplasmic reticulum (SR) was slower and the storage of Ca^2 inside the SR was less in the mutant muscle, due to increased leakage process of Ca^2 movement across the SR. The leakage pathway is associated with the increased sensitivity of Ca^2 /caffeine -induced Ca^2 release to myoplasmic Ca^2 . Therefore, the increased fatigability of mutant EDL muscles can result from a combination of a slowing of Ca^2 uptake, modification of Ca^2 -induced Ca^2 release (CICR), and a reduction in total SR Ca^2 content.