Effect of natural toxins and adipokinetic hormones on the activity of digestive enzymes in the midgut of the cockroach Periplaneta americana.

This study examined the effect of two natural toxins (a venom from the parasitic wasp Habrobracon hebetor and destruxin A from the entomopathogenic fungus Metarhizium anisopliae), and one pathogen (the entomopathogenic fungus Isaria fumosorosea) on the activity of basic digestive enzymes in the midgut of the cockroach Periplaneta americana. Simultaneously, the role of adipokinetic hormones (AKH) in the digestive processes was evaluated. The results showed that all tested toxins/pathogens elicited stress responses when applied into the cockroach body, as documented by an increase of AKH level in the central nervous system. The venom from H. hebetor showed no effect on digestive enzyme activities in the ceca and midgut in vitro. In addition, infection by I. fumosorosea caused a decrease in activity of all enzymes in the midgut and a variable decrease in activity in the ceca; application of AKHs did not reverse the inhibition. Destruxin A inhibited the activity of all enzymes in the midgut but none in the ceca in vitro; application of AKHs did reverse this inhibition, and no differences between both cockroach AKHs were found. Overall, the results demonstrated the variable effect of the tested toxins/pathogens on the digestive processes of cockroaches as well as the variable ability of AKH to counteract these effects.

Systemic RNAi of V-ATPase subunit B causes molting defect and developmental abnormalities in Periplaneta fuliginosa.

The vacuolar (H+ )-ATPases (V-ATPases) are ATP-driven proton pumps with multiple functions in many organisms. In this study, we performed structural and functional analysis of vha55 gene that encodes V-ATPase subunit B in the smokybrown cockroach Periplaneta fuliginosa (Blattodea). We observed a high homology score of the deduced amino acid sequences between 10 species in seven orders. RNAi of the vha55 gene in P. fuliginosa caused nymphal/nymphal molting defects with incomplete shedding of old cuticles, growth inhibition, as well as bent and wrinkled cuticles of thoraxes and abdominal segments. Since growth inhibition caused by vha55 RNAi did not interfere in the commencement of cockroach molting, molting timing and body growth might be controlled by independent mechanism. Our study suggested V-ATPases might be a good candidate molecule for evolutionary and developmental studies of insect molting.

Glutathione S-transferase (GST) of American Cockroach, Periplaneta americana: Classes, Isoforms, and Allergenicity.

Insect glutathione S-transferases (GSTs) play important roles in insecticide/drug resistance and stress response. Medically, GSTs of house dust mites (Dermatophagoides pteronyssinus and Blomia tropicalis) and German cockroach (Blattella germanica) are human allergens. In this study, classes, isoforms and B-cell and allergenic epitopes of GST of American cockroach, Periplaneta americana, the predominant species in the tropics and subtropics were investigated for the first time. Enzymatically active native and recombinant P. americana-GSTs bound to IgE in sera of all P. americana allergic patients that were tested. By gel-based proteomics and multiple sequence alignments, the native GST comprises three isoforms of delta and sigma classes. All isoforms interacted with serum IgE of the cockroach allergic subjects. Molecularly, the protein contains six B-cell epitopes; two epitopes located at ß1-α1 and ß4-α3 regions bound to patients' serum IgE, indicating that they are allergenic. P. americana are ubiquitous and their GST can sensitize humans to allergic diseases; thus, the protein should be included in the allergen array for component resolved diagnosis (CRD) of allergic patients, either by skin prick test or specific IgE determination. The GST is suitable also as a target of environmental allergen detection and quantification for intervention of cockroach sensitization and allergic morbidity.

Biochemical efficacy, molecular docking and inhibitory effect of 2, 3-dimethylmaleic anhydride on insect acetylcholinesterase.

Evolution of resistance among insects to action of pesticides has led to the discovery of several insecticides (neonicotinoids and organophosphates) with new targets in insect nervous system. Present study evaluates the mode of inhibition of acetylchlonesterase (AChE), biochemical efficacy, and molecular docking of 2,3-dimethylmaleic anhydride, against Periplaneta americana and Sitophilus oryzae. The knockdown activity of 2,3-dimethylmaleic anhydride was associated with in vivo inhibition of AChE. At KD99 dosage, the 2,3-dimethylmaleic anhydride showed more than 90% inhibition of AChE activity in test insects. A significant impairment in antioxidant system was observed, characterized by alteration in superoxide dismutase and catalase activities along with increase in reduced glutathione levels. Computational docking programs provided insights in to the possible interaction between 2,3-dimethylmaleic anhydride and AChE of P. americana. Our study reveals that 2,3-dimethylmaeic anhydride elicits toxicity in S. oryzae and P. americana primarily by AChE inhibition along with oxidative stress.

Purification and functional characterization of lectin with phenoloxidase activity from the hemolymph of cockroach, Periplaneta americana.

Lectins also identified as hemagglutinins are multivalent proteins and on account of their fine sugar-binding specificity play an important role in immune system of invertebrates. The present study was carried out on the hemolymph lectin of cockroach, Periplaneta americana with appropriate screening and purification to understand its molecular as well as functional nature. The lectin from the hemolymph was purified using ion-exchange chromatography. The approximate molecular weight of purified lectin was 340 kDa as determined by FPLC analysis. Rabbit erythrocytes were highly agglutinated with purified lectin from the hemolymph of P. americana. The hemagglutination activity (HA) of lectin was specifically inhibited by fucose. Glycoproteins also inhibited the HA activity of lectin. The amino acid sequences of the purified lectin revealed homology with amino acid sequences of allergen proteins from P. americana. Purified lectin showed the highest phenoloxidase activity against dopamine. The activators such as exogenous proteases and LPS from Escherichia coli and Salmonella minnesota significantly enhanced the PO activity of the purified lectin. Besides, the presence of copper and hemocyanin conserved domain in the purified lectin provided a new facet that insects belonging to the ancient clade such as cockroaches retained some traces of evolutionary resemblance in possessing lectin of ancient origin.

Adipokinetic hormones control amylase activity in the cockroach (Periplaneta americana) gut.

This study examined the biochemical characteristics of α-amylase and hormonal (adipokinetic hormone: AKH) stimulation of α-amylase activity in the cockroach (Periplaneta americana) midgut. We applied two AKHs in vivo and in vitro, then measured resultant amylase activity and gene expression, as well as the expression of AKH receptor (AKHR). The results revealed that optimal amylase activity is characterized by the following: pH: 5.7, temperature: 38.4 °C, Km (Michaelis-Menten constant): 2.54 mg starch/mL, and Vmax (maximum reaction velocity): 0.185 µmol maltose/mL/min. In vivo application of AKHs resulted in significant increase of amylase activity: by two-fold in the gastric caeca and 4-7 fold in the rest of the midgut. In vitro experiments supported results seen in vivo: a 24-h incubation with the hormones resulted in the increase of amylase activity by 1.4 times in the caeca and 4-9 times in the midgut. Further, gene expression analyses reveal that AKHR is expressed in both the caeca and the rest of the midgut, although expression levels in the former were 23 times higher than levels in the latter. A similar pattern was found for the amylase (AMY) gene. Hormonal treatment did not affect the expression of either gene. This study is the first to provide evidence indicating direct AKH stimulation of digestive enzyme activity in the insect midgut, supported by specific AKHR gene expression in this organ.

Isolation and chemoenzymatic treatment of glycoalkaloids from green, sprouting and rotting Solanum tuberosum potatoes for solanidine recovery.

The estimation of glycoalkaloids in the flesh of different types of decayed potatoes was evaluated. The results showed that turned green and also sprouting or rotting potato flesh contain high amounts of toxic solanine and chaconine, exceeding by 2-5-fold the recommended limit, and ranging from 2578±86mg/kg to 5063±230mg/kg of dry weight potato flesh. For safety consideration, these decayed potatoes should be systematically set aside. To avoid a net economic loss and encourage the removal of this hazardous food, a recycling process was investigated to generate added-value compounds from the toxic glycoalkaloids. A simple chemo-enzymatic protocol comprising a partial acidic hydrolysis followed by an enzymatic treatment with the ß-glycosidase from Periplaneta americana allowed the efficient conversion of α-chaconine to solanidine.

Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three ß-glucosidases, one ß-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none ß-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single ß-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two ß-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of ß-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a discussion on Polyneoptera digestive physiology is provided.

Characterization of a chemostable serine alkaline protease from Periplaneta americana.

BACKGROUND: Proteases are important enzymes involved in numerous essential physiological processes and hold a strong potential for industrial applications. The proteolytic activity of insects' gut is endowed by many isoforms with diverse properties and specificities. Thus, insect proteases can act as a tool in industrial processes. RESULTS: In the present study, purification and properties of a serine alkaline protease from Periplaneta americana and its potential application as an additive in various bio-formulations are reported. The enzyme was purified near to homogeneity by using acetone precipitation and Sephadex G-100 gel filtration chromatography. Enzyme activity was increased up to 4.2 fold after gel filtration chromatography. The purified enzyme appeared as single protein-band with a molecular mass of ~ 27.8 kDa in SDS-PAGE. The optimum pH and temperature for the proteolytic activity for purified protein were found around pH 8.0 and 60°C respectively. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and compatibility towards detergents, oxidizing, reducing, and bleaching agents. In addition, enzyme also showed stability towards organic solvents and commercial detergents. CONCLUSION: Several important properties of a serine protease from P. Americana were revealed. Moreover, insects can serve as excellent and alternative source of industrially important proteases with unique properties, which can be utilized as additives in detergents, stain removers and other bio-formulations. Properties of the P. americana protease accounted in the present investigation can be exploited further in various industrial processes. As an industrial prospective, identification of enzymes with varying essential properties from different insect species might be good approach and bioresource.

Ca²âº/calmodulin-dependent protein kinase II in the cockroach Periplaneta americana: identification of five isoforms and their tissues distribution.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key kinase that transduces Ca²âº signals into downstream effects acting on a range of cellular processes in nervous system and muscular tissues. In insects, different CaMKII isoforms have been reported in Drosophila melanogaster, Apis florae, Bombus terrestris, and Bombus impatiens but little is known on the organization and tissue-specific expression of these isoforms with the exception of Drosophila. The present study reports the cloning of five CaMKII splice variants issued from a single gene and their tissue-specific expression in the cockroach Periplaneta americana. Each CaMKII isoform shared 82-90% identity with Drosophila CaMKII isoforms and accordingly were named PaCaMKII-A, PaCaMKII-B,PaCaMKII-C,PaCaMKII-D, and PaCaMKII-E. PaCaMKII-A and PaCaMKII-D isoforms are ubiquitously expressed in all tissues, but some such as PaCaMKII-B andPaCaMKII-C are preferentially expressed in the nerve cord and muscle. In addition, using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we found a tissue-specific expression of PaCaMKII-E in the dorsal unpaired median neurons. Alternative splicing of PaCaMKII transcripts is likely a common mechanism in insects to control the pattern of isoform expression in the different tissues.

The pars intercerebralis affects digestive activities of the American cockroach, Periplaneta Americana, via crustacean cardioactive peptide and allatostatin-A.

Our previous report showed that the pars intercerebralis (PI)-ablated cockroach, Periplaneta americana (PIX), exhibited hypertrophy and a significant increase in α-amylase and protease activities in the midgut under constant darkness (DD). Bath-applied crustacean cardioactive peptide (CCAP) and allatostatin (AST) stimulated α-amylase and protease activities in the dissected midgut cultured in medium. However, the functional relationship and regulatory mechanism between the brain, particularly the pars intercerebralis and the midgut digestive activity remain to be investigated. Here, we investigated the immunohistochemical reactivities (IHCr) against CCAP and AST in the midgut of cockroach subjected to the above operation (PIX-DD). Three types of IHCr cells were observed in both the muscle layer and the epithelium: (1) CCAP-ir only, (2) AST-ir only and (3) both reactivities are colocalized. The number of all three types increased intensively after PIX under DD compared with that of sham operated control that was kept under constant condition (CNT-DD), indicating that the PI suppresses the expression of CCAP and AST in the midgut epithelium. We also showed that co-administration of CCAP and AST to the midgut caused increases of 1.5-fold and 1.4-fold for α-amylase and protease activities, respectively, compared with application of either peptide above. On the other hand, CCAP-ir in the muscle layer was more strongly expressed but AST-ir was suppressed in PIX-DD. While these peptides showed opposite effects on spontaneous contraction, when epithelially released, these peptides both activated the digestive enzyme system. Overall, up-regulated AST-6 and down-regulated CCAP in the stomatogastric nerve in the muscle layer produce the same end result, that is, stimulation of digestive activity (hypertrophy) via both enzyme activation and the retarded peristalsis that leads to increased throughput time.

Brain-midgut short neuropeptide F mechanism that inhibits digestive activity of the American cockroach, Periplaneta americana upon starvation.

Immunohistochemical reactivity against short neuropeptide F (sNPF) was observed in the brain-corpus cardiacum and midgut paraneurons of the American cockroach, Periplaneta americana. Four weeks of starvation increased the number of sNPF-ir cells in the midgut epithelium but the refeeding decreased the number in 3h. Dramatic rises in sNPF contents in the midgut epithelium and hemolymph of roaches starved for 4 weeks were confirmed by ELISA. Starvation for 4 weeks reduced α-amylase, protease and lipase activities in the midgut of P. americana but refeeding restored these to high levels. Co-incubation of dissected midgut with sNPF at physiological concentrations inhibited α-amylase, protease and lipase activities. sNPF injection into the hemocoel led to a decrease in α-amylase, protease and lipase activities, whereas PBS injection had no effects. The injection of d-(+)-trehalose and l-proline into the hemocoel of decapitated adult male cockroaches that had been starved for 4 weeks had no effect on these digestive enzymes. However, injection into the hemocoel of head-intact starved cockroaches stimulated digestive activity. Injection of d-(+)-trehalose and l-proline into the lumen of decapitated cockroaches that had been starved for 4 weeks increased enzymes activities and suppressed sNPF in the midgut. Our data indicate that sNPF from the midgut paraneurons suppresses α-amylase, protease and lipase activities during starvation. Injection of d-(+)-trehalose/l-proline into the hemocoel of head-intact starved cockroach decreased the hemolymph sNPF content, which suggests that sNPF could be one of the brain factors, demonstrating brain-midgut interplay in the regulation of digestive activities and possibly nutrition-associated behavioral modifications.

Inhibition of protein kinase C decreases sensitivity of GABA receptor subtype to fipronil insecticide in insect neurosecretory cells.

Phosphorylation by serine/threonine kinases has been described as a new mechanism for regulating the effects of insecticides on insect neuronal receptors and channels. Although insect GABA receptors are commercially important targets for insecticides (e.g. fipronil), their modulation by kinases is poorly understood and the influence of phosphorylation on insecticide sensitivity is unknown. Using the whole-cell patch-clamp technique, we investigated the modulatory effect of PKC and CaMKinase II on GABA receptor subtypes (GABAR1 and GABAR2) in DUM neurons isolated from the terminal abdominal ganglion (TAG) of Periplaneta americana. Chloride currents through GABAR2 were selectively abolished by PMA and PDBu (the PKC activators) and potentiated by Gö6983, an inhibitor of PKC. Furthermore, using KN-62, a specific CaMKinase II inhibitor, we demonstrated that CaMKinase II activation was also involved in the regulation of GABAR2 function. In addition, using CdCl(2) (the calcium channel blocker) and LOE-908, a blocker of TRPγ, we revealed that calcium influx through TRPγ played an important role in kinase activations. Comparative studies performed with CACA, a selective agonist of GABAR1 in DUM neurons confirmed the involvement of these kinases in the specific regulation of GABAR2. Furthermore, our study reported that GABAR1 was less sensitive than GABAR2 to fipronil. This was demonstrated by the biphasic concentration-response curve and the current-voltage relationship established with both GABA and CACA. Finally, we demonstrated that GABAR2 was 10-fold less sensitive to fipronil following inhibition of PKC, whereas inhibition of CaMKinase II did not alter the effect of fipronil.

Insect-specific irreversible inhibitors of acetylcholinesterase in pests including the bed bug, the eastern yellowjacket, German and American cockroaches, and the confused flour beetle.

Insecticides directed against acetylcholinesterase (AChE) are facing increased resistance among target species as well as increasing concerns for human toxicity. The result has been a resurgence of disease vectors, insects destructive to agriculture, and residential pests. We previously reported a free cysteine (Cys) residue at the entrance to the AChE active site in some insects but not higher vertebrates. We also reported Cys-targeting methanethiosulfonate molecules (AMTSn), which, under conditions that spared human AChE, caused total irreversible inhibition of aphid AChE, 95% inhibition of AChE from the malaria vector mosquito (Anopheles gambia), and >80% inhibition of activity from the yellow fever mosquito (Aedes aegypti) and northern house mosquito (Culex pipiens). We now find the same compounds inhibit AChE from cockroaches (Blattella germanica and Periplaneta americana), the flour beetle (Tribolium confusum), the multi-colored Asian ladybird beetle (Harmonia axyridis), the bed bug (Cimex lectularius), and a wasp (Vespula maculifrons), with IC(50) values of approximately 1-11muM. Our results support further study of Cys-targeting inhibitors as conceptually novel insecticides that may be free of resistance in a range of insect pests and disease vectors and, compared with current compounds, should demonstrate much lower toxicity to mammals, birds, and fish.

Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of Periplaneta americana.

Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase (V-H(+)-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the active transport of protons across membranes, is commonly present in membranes of acidocalcisomes. In the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is mediated by proton-pumps present in their membranes. In this work, we describe an H(+)-PPase activity in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme activity was optimum at pH around 7.0, and was dependent on Mg(2+) and inhibited by NaF, as well as by IDP and Ca(2+). Immunolocalization of the yolk preparation using antibodies against a conserved sequence of V-H(+)-PPases showed labeling of small vesicles, which also showed the presence of high concentrations of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of P. americana present acidocalcisome-like characteristics. In addition, the possible role of these organelles during embryogenesis of this insect is discussed.

Identification of a serine protease as a major allergen (Per a 10) of Periplaneta americana.

BACKGROUND: Cockroach allergens are associated with the development of asthma, but none of these has been characterized for proteolytic activity. This study was undertaken to isolate and characterize a protease from Periplaneta americana and determine its allergenicity. METHODS: A serine protease was isolated from P. americana extract using benzamidine sepharose column and characterized by immunobiochemical methods. Allergenicity of the protease was assessed by enzyme-linked immunosorbent assay, immunoblot, intradermal testing, histamine release and peripheral blood mononuclear cells (PBMCs) proliferation. RESULTS: Affinity purified protein of approximately 28 kDa (Per a 10) showed a single band of activity in gelatin zymogram and agarose plate assay. N-terminal sequence (IVGGRPAQI) revealed similarity with mite serine protease allergens and insect trypsins. It demonstrated proteolytic activity with azocollagen > gelatin > defatted-milk > casein including serine protease specific substrate, N-benzoyl-arginine-ethyl-ester-hydrochloride. It was inhibited by serine protease inhibitors, namely aprotinin > pefabloc > AEBSF > PMSF > benzamidine > antipain > leupeptin and trypsin-specific inhibitor (tosyl-lysyl-chloromethyl-ketone) suggesting it to be a trypsin-like serine protease. Per a 10 was recognized as a major allergen, showing IgE reactivity with >80% of cockroach sensitized patients by skin tests and immunoblot. It could induce significant histamine release (P < 0.05) in blood and secretion of interleukin-4 (IL-4) (P < 0.05) and IL-5 (P < 0.05) in culture supernatant of PBMCs from cockroach hypersensitive patients, suggesting a strong allergenic potency. CONCLUSION: A serine protease isolated from P. americana was demonstrated to be a major allergen (Per a 10). It has a potential for component-based diagnosis of allergy and will be useful in elucidating the mechanism of allergy.

[Cloning, expression and purification of allergen arginine kinase from Periplaneta americana and its allergic activity].

OBJECTIVE: To clone the gene of arginine kinase (AK) from Periplaneta americana, produce its recombinant protein and investigate its allergenicity. METHOD: The cDNA of AK was cloned using specific primers from the total RNA of P. americana. The cloned gene was inserted into pMD18-T vector and digested by BamHI and HindIII. The cDNA was sequenced and subcloned into pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant AK (rAK) was purified by metal (Ni2+) chelating affinity chromatography. Its allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay (ELISA). RESULT: The cloned cDNA ORF sequence (Accession no. EU429466) contained 1068 bp and encoded 365 amino acids. Its sequence homology with the published one (Accession no. AY563004) was 99.9% at nucleotide level. The allergen rAK was highly expressed in E. coli BL21 (DE3) as a soluble protein mainly with the molecular weight of about Mr 45000 under induction of IPTG and purified by 6-His-tag purification system. Both in the non-denaturalization and denaturalization conditions, the recombinant allergen was identified as its affinity to IgE antibodies from the cockroach-allergic patient sera by Western blotting and ELISA. CONCLUSION: The recombinant cockroach arginine kinase has been obtained with proper allergenicity.

A vacuolar-type H+-ATPase and a Na+/H+ exchanger contribute to intracellular pH regulation in cockroach salivary ducts.

Cells of the dopaminergically innervated salivary ducts in the cockroach Periplaneta americana have a vacuolar-type H(+)-ATPase (V-ATPase) of unknown function in their apical membrane. We have studied whether dopamine affects intracellular pH (pH(i)) in duct cells and whether and to what extent the apical V-ATPase contributes to pH(i) regulation. pH(i) measurements with double-barrelled pH-sensitive microelectrodes and the fluorescent dye BCECF have revealed: (1) the steady-state pH(i) is 7.3+/-0.1; (2) dopamine induces a dose-dependent acidification up to pH 6.9+/-0.1 at 1 micromol l(-1) dopamine, EC(50) at 30 nmol l(-1) dopamine; (3) V-ATPase inhibition with concanamycin A or Na(+)-free physiological saline (PS) does not affect the steady-state pH(i); (4) concanamycin A, Na(+) -free PS and Na(+)/H(+) exchange inhibition with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) each reduce the rate of pH(i) recovery from a dopamine-induced acidification or an acidification induced by an NH(4)Cl pulse; (5) pH(i) recovery after NH(4)Cl-induced acidification is almost completely blocked by concanamycin A in Na(+)-free PS or by concanamycin A applied together with EIPA; (6) pH(i) recovery after dopamine-induced acidification is also completely blocked by concanamycin A in Na(+)-free PS but only partially blocked by concanamycin A applied together with EIPA. We therefore conclude that the apical V-ATPase and a basolateral Na(+)/H(+) exchange play a minor role in steady-state pH(i) regulation but contribute both to H(+) extrusion after an acute dopamine- or NH(4)Cl-induced acid load.

Stability of protease-rich periplaneta Americana allergen extract during storage: formulating preservatives to enhance shelf life.

Allergenic proteins in extracts degrade rapidly and lose potency on storage. Hence, formulation of optimum conditions is required to enhance shelf life of extracts for proper allergy diagnosis and immunotherapy. In the present study, allergenic potency of P. americana proteins was evaluated after storage with epsilon-aminocaproic acid (EACA), sucrose, glycerol, pepstatin A, and aprotinin, individually for 1, 3, 6, and 12 months at 4, 25, and 37 degrees C. P. americana extract stored with EACA and sucrose individually retained potency comparable to proteins in standard extract (freeze-dried extract, stored at-70 degrees C) upto 6 months at 4 degrees C. The extracts without preservatives or with glycerol, pepstatin A, aprotinin, or stored at 37/25 degrees C were severely degraded and lost potency by 3 months. A formulation containing a combination of EACA and sucrose enhanced the shelf life of P. americana proteins upto 12 months at 4 degrees C. Hence, EACA and sucrose together show better potential for stabilization of protease-rich extracts.

Use of the glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol as a probe to investigate hypertrehalosemic hormone-mediated change in carbohydrate and lipid metabolism in the cockroach, Periplaneta americana.

The glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DIA) was used to investigate the action of hypertrehalosemic hormone (HTH) on carbohydrate, neutral lipid, and phospholipid metabolism in the hemolymph and fat body of the cockroach, Periplaneta americana. DIA blocked the hypertrehalosemic and hyperglycemic effects of HTH, as well as the decrease in hemolymph neutral lipid and phospholipid normally induced by HTH. DIA diminished the accumulation of neutral lipid in the fat body formed under the influence of HTH and partly blocked the decrease in fat body phospholipid evoked by HTH. The increased incorporation of (14)C-glucose into fat body triacylglycerol in the presence of HTH was decreased by more than two-thirds when DIA was coinjected with the hormone. The results suggest that glucose derived from hemolymph trehalose is an important contributor to the formation of the glycerol backbone of newly formed triacylglycerol in the fat body.