Regeneration of midgut epithelial cell in the silkworm, Bombyx mori, infected with viruses

In the larvae of the silkworm, Bombyx mori, the regeneration of midgut cells infected with a cytoplasmic polyhedrosis virus (CPV), a flacherie virus (FV), and a small DNA virus (SDV) was studied. Large numbers of newly developed cells appeared in the CPV-infected part of the midgut epithelium just before larval molt, and along with their development, the CPV-infected old columnar cells were discharged into the midgut lumen during the molt. On the other hand, in the uninfected portion of the midgut only a few cells developed, and no columnar cells were discharged. Similarly, the marked replacement of midgut epithelial cells during larval molt was also observed in larvae infected with CPV + FV. In the larvae infected with CPV + SDV, the columnar cells lost their regenerative ability, and because of the exfoliation of infected columnar cells, the midgut epithelium consisted mainly of uninfected goblet cells at a late stage of infection. The degree of epithelial regeneration varied with the silkworm strain and the dosage of the virus.     

Screening of molecular markers for NPV resistance in Bombyx mori L. (Lep., Bombycidae)

Abstract: Silkworm ( Bombyx mori L.) is one of the important economic insects. Silkworm rearing and silk industry plays an important role in China, India and other developing countries. In the long history of sericultural practice, introduction of silkworm strains with high resistance to diseases has greatly improved cocoon and silk quality and productivity. However, current silkworm breeding is mainly based on traditional method that involves high input of time and labour. In order to increase the selection efficiency and accuracy for future silkworm breeding, it is necessary to establish a molecular marker-assisted selection system. In our study, three silkworm near isogenic lines that had different resistance to nuclear polyhedrosis virus (NPV) were established by means of different hybridization methods. A total of 150 random amplified polymorphic DNA (RAPD) random primers were used to screen molecular markers. Among them, two molecular markers OPA-18₇₀₀ and OPY-11₄₀₀ were found linked to major genes resistant and susceptible to NPV, respectively. Validity of the molecular markers was proved in F2 populations.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Effects of physico-chemical treatments on hemagglutination activity of Anopheles gambiae haemolymph and midgut extract

Abstract. Anopheles gambiae midgut extracts and haemolymph possessed agglutinins, titre 1:16 to 1:256, against human red blood cells (RBCs). Subjection of both tissues to protein precipitation reagents, organic chemical and selected protease, neuraminidase and other glycosidic hydrolase treatments revealed the haemagglutinins to be protein, most likely glycoprotein, in nature - not lipoprotein, lipid, glycolipid or nucleic acid. An.gambiae agglutinins were thermo-labile >40^oC, affected by freezing and thawing treatments, and contained disulphide and hydrogen bonds on the basis of sensitivity following exposure to dithio-threitol and urea respectively. Optimum haemagglutination depended generally on slightly acid to neutral pH conditions and agglutinin activity was Ca²⁺ ion, albeit to a lesser extent Mg²⁺ ion, dependent. The midgut extract agglutinin subunit molecule had a relative molecular weight (M_r) of 65kDa whilst that of haemolymph was 40kDa.
This study presents the first report on selected physico-chemical properties, the glycoproteinaceous nature and tentative subunit M_r of mosquito midgut extract and haemolymph anti-RBC agglutinin(s).     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

ENERGETICS OF AMINO ACID TRANSPORT INTO BRAIN SLICES: EFFECTS OF K+ DEPLETION AND Rb+ OR Cs+ SUBSTITUTION ON AMINO ACID UPTAKE

Abstract— Mouse brain slices were depleted of K⁺ by three 10-min incubations-in oxygenated HEPES-buffered medium lacking glucose and K⁺. Addition of K⁺ or Rb⁺ (or Cs⁺, to a smaller degree) with glucose, or with succinate, malate, and pyruvate (SMP) before incubation at 37°C with ¹⁴C-amino acids restored active low-affinity transport of d -Glu, α-aminoisobutyrate (AIB), GABA, Gly, His, Val, Leu, Lys, and Orn. Ouabain at 1–2μ m with Rb⁺ was more inhibitory with SMP than with glucose, suggesting that the glycoside may affect specific energy coupling to transport. Valinomycin, in contrast, showed no specificity of inhibition of amino acid uptake with glucose or SMP and K⁺ or Rb⁺. Cs⁺ partially restored amino acid uptake, but Li⁺ was less effective than Cs ⁺. NaF at 10 m m with SMP + Rb⁺, or SMP + K⁺ did not inhibit amino acid uptake. Therefore, it was possible to dissociate glycolysis and Na⁺, K ⁺ -ATPase activity from amino acid transport. The ion replacements for K ⁺ that supported active amino acid transport indicate that the specificity of ions in possible ionic gradients for transport energetics should be reexamined.     

Long-term effects of abscisic acid on K+ transport in young wheat plants of different K+ status

The effects of abscisic acid (ABA) on growth, uptake and translocation of potassium ions, K⁺,Mg²⁺-ATPase activity and transpiration were investigated in young wheat ( Triticum aestivum L. cv. Martonvásári-8) plants grown at different K⁺ supplies. Long-term treatment with ABA (10 μ M ) reduced growth in high-K⁺ plants, but had less effect under low-K⁺ conditions. K⁺(⁸⁶Rb) uptake was inhibited by about 70 and 40% in low- and high-K⁺ plants, respectively. The stimulation by K⁺ of the Mg²⁺-ATPase activity in the root microsomal fraction was lost with ABA treatment. It is suggested that the inhibitory effect of ABA on K⁺ uptake may be related to this effects on the K⁺,Mg²⁺-ATPase. Translocation of K⁺ to the shoot was inhibited in low-K⁺ plants only, and it was not affected in high-K⁺ plants. In parallel to this, ABA treatment reduced transpiration by about 50% in low-K⁺ plants, whereas a much smaller effect was seen in high-K⁺ plants. These observations suggest that the regulation by ABA of the stomatal movements is strongly counteracted by high-K⁺ status.     

Phytase activity of Selenomonas ruminantium: a preliminary characterization

Obligately anaerobic ruminal bacteria have been found to possess phytase activity, in particular, Selenomonas ruminantium . The phytase activity of S. ruminantium JY35 was produced late in growth and required neither phytate for induction nor phosphate limitation for derepression. The activity was completely cell-associated with a significant fraction extractable by a magnesium chloride solution. Zymogram analysis suggested that the activity was the result of a single gene product of a monomeric nature and approximately 46 kDa in size. The phytase had a temperature optimum of 50–55 °C, but activity dropped off sharply at 60 °C. Phytase activity was optimal over the pH range of 4·0–5·5, and dependent on the nature of the buffer used. Activity was inhibited by citric acid buffer and by the addition of 5 mmol l⁻¹ Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺ and Hg²⁺. The addition of 5 mmol l^–1 Pb²⁺ to the enzyme assay appeared to enhance activity of the enzyme.     

Antiviral activity of selected medicinal plants and marine seaweeds on the grasserie infected larvae of silkworm,Bombyx mori

Nuclear polyhedrosis virus (NPV) is the most harmful virus responsible for the manifestation of grasserie disease in the larvae of silkworm, Bombyx mori. It causes a huge economic loss in the sericulture industry. An attempt was made in the present investigation for the screening of antiviral activity using medicinal plants such as Lantana camara, Phyllanthus amarus and marine seaweeds such as Sargassum wightii, Turbinaria ornata against BmNPV. Crude extracts were prepared using different solvents, such as hexane, ethyl acetate, methanol and water. The silkworm feeding bioassay study was carried out with the crude extracts to investigate the presence of anti-BmNPV activity after inoculating fifth instar larvae of silkworm with occlusion bodies (OBs) of BmNPV. Each extract was tested for their anti-BmNPV activity using various concentrations of crude extracts ranging from 200 μg to 1000 μg. Among the crude extracts tested, methanol and aqueous extracts of P. amarus showed significant anti-BmNPV activity.     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Mycobiont-cyanobiont interactions during dark nitrogen fixation by the lichen Peltigera aphthosa

Acetylene reduction (nitrogenase activity) by excised cephalodia of Peltigera aphthosa Willd. slowly declined on transfer of the cephalodia from light to darkness. The decline was more rapid in the absence of CO₂ or when phosphoenolpyruvate carboxylase activity was inhibited by adding maleic acid or malonic acid. When glutamine synthetase (GS) activity was totally inhibited by adding l -methionine- dl -sulphoximine (MSX) the decline in nitrogenase activity in the absence of CO₂ still occurred. However, this loss of activity did not occur when the mycobiont was disrupted using digitonin (0.01 % w/v) and the fixed NH₄⁺ was released into the medium. The data suggest that dark CO₂ fixation by the fungus supplies carbon skeletons which remove newly fixed NH₄⁺ produced by the cyanobacterium. When such carbon skeletons are not available MH₄⁺ accumulates and inhibits nitrogenase activity even in the absence of GS activity. It is probable that NH₄⁺ and a product of GS exert independent inhibitory effects on nitrogenase activity.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

EFFECT OF CRY1A δ-ENDOTOXINS OF BACILLUS THURINGIENSIS ON Mg+-ATPASE IN THE COLUMNAR CELLS OF BOMBYX MORI MIDGUT

Abstract  The effects of Cry1A δendotoxins from Bacillus thringiensis subsp. kurstaki HD-1 on the activity of M²+-dependent ATPase in the columnar cells (CC) of Sombyx mori were examined. Cytochemical localization showed a strong M$ ' -ATPase activity on the apical brush border of CC and even on the peritrophic membrane, but not on the goblet cells except slight activity on their interdigitated valves. Although overdose toxins were imposed to the larvae and resulted in serious cellular pathological changes in the treated midgut in vim , no obvious reduction of the M²+-ATPase activity on the CC membrane was observed. This may exclude the M²+ ATPase on the CC brush border membrane to be the potential natural channel modulated by B-endotoxins of B. thuringiensis or a target of the toxin during larval intoxication.     

EFFECTS OF SULFHYDRYL REAGENTS ON BRAIN MICROTUBULE-ASSOCIATED ATPase ACTIVITY IN VITRO

Abstract— The effects of two sulfhydryl reagents, PCMBS ( p -chloromercuribenzene sulfonic acid) and NEM ( N -ethylmaleimide) on microtubule-associated Mg²⁺ -and Ca²⁺ -ATPase activity were studied in a MTP (microtubule proteins) preparation and in a MAP (microtubule-associated proteins) fraction. In the MTP preparation at pH 6.8, PCMBS stimulated the Mg²⁺ -ATPase activity at low concentrations and inhibited at higher, whereas the Ca²⁺ -ATPdse activity was only inhibited. NEM affected the activity in a similar way. At pH 8.0 PCMBS was only inhibitory. NEM showed stimulatory effects over a broader concentration range.
Preincubation in the presence of ATP counteracted the stimulatory effects of both PCMBS and NEM on Mg²⁺ -ATPase at pH 6.8.
In the MAP fraction at pH 6.8 PCMBS and NEM caused similar but less pronounced effects on the Mg²⁺ -and Ca²⁺ -ATPase.
The results show that brain microtubule-associated ATPase activity is similar to dynein and myosin ATPases with respect to biphasic alteration by sulfhydryl reagents.     

Effect of salicylic acid on nitrite reductase and glutamate dehydrogenase activities in maize roots

The effects of 0.01 to 5 m M salicyclic acid on the increase in nitrite reductase or glutamate dehydrogenase activities in maize roots by nitrate or ammonium respectively, were examined. Nitrite reductase activity was inhibited by the highest concentration of the acid. The activity of NADH-glutamate dehydrogenase was stimulated slightly (but consistently) by the lowest concentration and was inhibited by higher concentrations. Total protein content was also inhibited at high concentrations. When the crude enzyme extract was stored at 25°C in light, the glutamate dehydrogenase activity in the control decreased after 4 h of incubation. Low concentrations of the acid had no effect on this decrease but higher concentration accelerated the process. The divalent cations Caz²⁺, Mn²⁺, Mg²⁺ and Zn²⁺ protected against loss of enzyme activity during storage, both in the absence and presence of the acid. The inhibitory effect of 5 m M salicylic acid on glutamate dehydrogenase activity is apparent due to interference with the activity of the enzyme rather than with its synthesis.     

Early effects of excess cadmium uptake in Phaseolus vulgaris

Abstract. Seedlings of Phaseolus vulgaris were exposed to solutions containing Cd²⁺ in the range 0 to 1 molm⁻³. Ethylene formation started following 3 h of exposure to 10⁻², 10⁻¹ and 1 mol m⁻³ Cd²⁺, peaked at 18 h and returned to a relatively low rate after 24 h. Cadmium-induced ethylene formation depended on the formation of 1-aminocyclopropane-1-carboxylic acid (ACC). Aminoethoxyvinylglycine (AVG, 0.1 mol m⁻³) inhibited ACC accumulation and ethylene production during exposure to 0.2 mol m⁻³ Cd²⁺.
Activity of soluble and ionically-bound peroxidase increased after 18 h of exposure to Cd²⁺ concentrations above 10⁻³ mol m⁻³ due to an increase in activity of cathodic isoperoxidases. Stimulation of soluble and ionically-bound peroxidase by 0.2 mol m⁻³ Cd²⁺ was reduced in the presence of 0.1 mol m⁻³ AVG.
Accumulation of soluble and insoluble ('ligninlike') phenolics was found in plants exposed to Cd²⁺ (10⁻² mol m⁻³ or above) in the presence or absence of AVG. Deposition of insoluble (autofluorescing) material occurred in cell walls around vessels and was associated with reduced expansion and water content of leaves.     

Utilization of the polymerase chain reaction in the diagnosis of nuclear polyhedrosis virus infections of gypsy moth (Lymantria dispar, Lep., Lymantriidae) populations

Abstract:  Three specific DNA probes were used for the detection of the nuclear polyhedrosis (NPV) virus of Lymantria dispar ( Ld NPV) genome. Two of these probes, H2 and H3 were obtained by classical cloning method and one (TR6) by polymerase chain reaction (PCR). These probes, used individually or in a pool in the standard slot–blot hybridizations, were able to detect 10⁹ genome copies. By performing 35 cycles of PCR amplification before hybridization with primers specific to Ld NPV genome on DNA extracted from infected larvae, the sensitivity of the hybridization technique was increased, so that as little as 10 copies of the Ld NPV genome could be detected. Using these methods, L. dispar naturally infected by Ld NPV were identified among field populations in Canada and in the United States near the eastern Canadian border. Using a combination of PCR and hybridization, Ld NPV contamination of egg masses were also detected. By disinfecting the eggs with sodium hypochlorite prior to PCR amplification and hybridization, it was also demonstrated that transmission of viral infection in the natural populations is mainly caused by external contamination of the egg and is unlikely to occur through the transovarial route.     

Influence of exogenously supplied potassium and sodium salts on the abscisic acid-induced proline accumulation in barley leaf segments

A stimulation of the abscisic acid (ABA)-induced increase in proline was observed in leaf segments of barley ( Hordeum vulgare L. cv. Georgie) if K⁺ or Na⁺ were supplied in the external medium as salts of monovalent anions such as NO₃, Br, Cr and I, but not when sulphate or phosphate were used. To a lesser extent, the effect was evident also with RbCl, but it did not occur when chlorides of Li⁺. Cs⁺, NH₄⁺, Mg:⁺ and Ca²⁺ were used. Both KC1 and NaCl in the concentration range 2–100 m M influence the ABA-dependent proline accumulation to the same extent; the increase induced was about 100% at 10 m M , and reached a maximum between 60 and 100 m M. The effect is not due to the osmotic activity of the salts and does not seem to depend on changes in K⁺ and Na⁺ levels within the leaf tissue, but it is somehow linked to their external concentration. The existence of a specific interaction between ABA and K⁺ or Na⁺, possibly at the cell membrane level, is proposed.