Anti-bacterial activity of Achatina CRP and its mechanism of action.

The physiological role of C-reactive protein (CRP), the classical acute-phase protein, is not well documented, despite many reports on biological effects of CRP in vitro and in model systems in vivo. It has been suggested that CRP protects mice against lethal toxicity of bacterial infections by implementing immunological responses. In Achatina fulica CRP is a constitutive multifunctional protein in haemolymph and considered responsible for their survival in the environment for millions of years. The efficacy of Achatina CRP (ACRP) was tested against both Salmonella typhimurium and Bacillus subtilis infections in mice where endogenous CRP level is negligible even after inflammatory stimulus. Further, growth curves of the bacteria revealed that ACRP (50 µg/mL) is bacteriostatic against gram negative salmonellae and bactericidal against gram positive bacilli. ACRP induced energy crises in bacterial cells, inhibited key carbohydrate metabolic enzymes such as phosphofructokinase in glycolysis, isocitrate dehydrogenase in TCA cycle, isocitrate lyase in glyoxylate cycle and fructose-1,6-bisphosphatase in gluconeogenesis. ACRP disturbed the homeostasis of cellular redox potential as well as reduced glutathione status, which is accompanied by an enhanced rate of lipid peroxidation. Annexin V-Cy3/CFDA dual staining clearly showed ACRP induced apoptosis-like death in bacterial cell population. Moreover, immunoblot analyses also indicated apoptosis-like death in ACRP treated bacterial cells, where activation of poly (ADP-ribose) polymerase-1 (PARP) and caspase-3 was noteworthy. It is concluded that metabolic impairment by ACRP in bacterial cells is primarily due to generation of reactive oxygen species and ACRP induced anti-bacterial effect is mediated by metabolic impairment leading to apoptosis-like death in bacterial cells.

Estimation of amino acids, urea and uric acid in tasar silkworm, Antheraea mylitta Drury.

The tasar silkworm, Antheraea mylitta Drury, Andhra local ecorace is an exclusive race of Andhra Pradesh. It is on the verge of extinction due to difficulty of acclimatisation at breeding and rearing stages. As an attempt to protect this race, a method of total indoor rearing has been done. In this context, the estimation of free amino acids, excretory products- urea and uric acid were compared during the fourth and fifth instars of tasar silkworm, reared under outdoor and indoor conditions. The study has revealed that amino acids decreased in the fat body in outdoor and indoor reared larvae in contrast to that in the haemolymph where it has gradually increased from first to third crops. This is an important finding as it reveals that indoor worms seem to adopt proteolytic activity in the haemolymph. Secondly, in the fifth instar the excretory products are more compared to fourth instar in the indoor reared worms. During fifth instar, formation of nitrogenous products lessens as silk synthesis enhances. The present study reveals that decrease in uric acid in fifth instar implies increase in growth rate and silk synthesis in both outdoor and indoor worms. The findings of the present investigation is helpful in the conservation and protection of the A. mylitta, Andhra local ecorace.

Immune haemolymph proteins in response to bacterial infection and identification of a putative bacteria binding protein in malaria vector Anopheles stephensi.

Induction of haemolymph proteins in mosquito A. stephensi due to wounding or bacterial infection (E. coli) was analyzed using SDS-PAGE. Wounding response of pupa revealed subsequent induction of two polypeptides (21 and 74 kDa). Two other polypeptides (44 and 57 kDa) were induced commonly in both pupa and adult female haemolymph upon bacterial infection. In vitro binding assay revealed identification of 44 kDa, a putative bacterial binding protein, a more relevant protein for further elucidation of molecular mechanism involved in host parasite interactions.

Carbaryl-induced alterations in biochemical metabolism of the prawn, Macrobrachium malcolmsonii.

The present study was performed to investigate the toxic impact of carbaryl on biochemical metabolism in the hemolymph, brain, hepatopancreas, gills and muscle of intermoult juveniles of the economically important prawn, Macrobrachium malcolmsonii. The concentration of glutathione S-transferase (GST) was found to be higher in test prawns when compared with controls. This suggests that a mechanism of detoxification was in operation to neutralise carbaryl toxicity. However, the toxic effect of carbaryl was not fully neutralised, and hence, alterations were recorded in basic biochemical metabolism of test prawns. The concentration of acetylcholinestrase (AchE) was found to be lower in test prawns than that of controls. Carbaryl toxicity resulted in utilisation of major biochemical constituents, such as total carbohydrate, glycogen, protein and lipid to generate required energy as an attempt to withstand the toxic stress. Glycogenolysis resulted in elevation of total free sugar level in the hemolymph of test prawns. While proteolysis led to elevation of total free amino acid level in test prawns. The content of total lipid have also been found lower in test prawns than that of controls. This suggests that carbaryl toxicity resulted in severe energy crises in test prawns. In the present study, toxic effects of carbaryl impair basic metabolic functions and hence pose a threat to the life of M. malcolmsonii.

Protein mediated cholesterol absorption in locusts Schistocerca gregaria (Forskal) and Locusta migratoria (Linn).

Absorption and transport of 3H cholesterol from the midgut to hemolymph and other tissues was studied in the locusts Schistocerca gregaria and Locusta migratoria. S. gregaria are able to absorb dietary cholesterol in the midgut and release into the hemolymph in vivo and into the incubation medium in virto. Certain proteins of midgut origin are involved in the absorption and release of cholesterol. The proteins designated as cholesterol binding proteins (CBP's) were fractionated by gel filtration chromatography using Sepharose CL-6B-200 column. Presence of a protein and its binding with cholesterol is confirmed by TCA precipitation after subsequent incubation of midgut in the incubation medium. Cholesterol binding with the proteins was also confirmed in native polyacrylamide gel electrophoresis. Biosynthesis of this protein takes place in the midgut which is inhibited by a protein synthesis inhibitor, cycloheximide. It also inhibits absorption and release of cholesterol from the midgut. The cholesterol binding activity was associated with a peak containing proteins ranging from molecular weights of 17-32 kDa in SDS-PAGE gels. Treatment of midgut with cycloheximide resulted in reduced cholesterol binding activity. Dilipidation of mucin and transport in presence of bile salts yielded a higher cholesterol binding activity. Although the absorption and release of cholesterol was observed in the hemolymph of both sexes, the ovary exhibited higher cholesterol binding as compared to testis.

Acute and chronic effects of cadmium on blood homeostasis of an estuarine crab, Chasmagnathus granulata, and the modifying effect of salinity

Whole body oxygen consumption and some hemolymph parameters such as pH, partial pressure of gases, level of ions and lactate were measured in the estuarine crab Chasmagnathus granulata after both acute (96 h) and chronic (2 weeks) exposure to cadmium at concentrations ranging from 0.4 to 6.3 mg/l. In all instances, the crabs developed hemolymph acidosis, but no respiratory (increased PCO2) or lactate increases were evident. Hemolymph levels of sodium and calcium were always increased by cadmium exposure. The chronic toxicity of cadmium was enhanced at 12 0/00 salinity, even causing a significantly higher mortality in comparison with the higher salinity (30 0/00) used. A general metabolic arrest took place at 12 0/00 salinity in the crabs chronically exposed to cadmium, as indicated by decreases of oxygen consumption and PCO2, an increase of PO2, along with no changes in lactate levels. These imbalances were associated with severe necrosis and telangiectasia in the respiratory gills, probably leading to respiratory impairment and finally histotoxic hypoxia and death of the animals

Changes of haemolymph protein profile in the larva of Pericallia ricini (Fabricius) parasitised by the Braconid Wasp, Apanteles taragamae Viereck (Hymenoptera: Braconidae).

Parasitism by the braconid wasp, A. taragamae caused alterations in the haemolymph polypeptides of woolly bear larvae of P. ricini. Analysis of haemolymph proteins by SDS-PAGE and densitometry showed that the quantities of haemolymph proteins were reduced dramatically in the parasitised larvae. Simultaneously, parasitism induced large amount of 95 kDa polypeptides in the haemolymph of the parasitised larvae. Also, a remarkable induction of 43 and 45 kDa polypeptides which are not detectable in non-parasitised larvae appeared in the parasitised larvae.

Profile of organic acid concentrations in the digestive gland and hemolymph of Biomphalaria glabrata under estivation

Using high performance liquid chromatography (HPLC) analysis it was possible to determine simultaneously the concentration of organic acids (pyruvate, lactate, succinate, fumarate, malate, acetate, propionate, acetoacetate, and ß-hydroxybutyrate) in the digestive gland and the extracellular concentration of these same acids in the hemolymph of estivating Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. After a 7 day period of estivation, there was a significant increase in the tissue levels of lactate, succinate, malate and acetate compared to non-estivating snails. After 14 days of estivation, the levels of lactate and acetate were also significantly elevated. The hemolymph concentrations of pyruvate and acetate increased significantly after 7 days and acetate concentrations continued to be significantly increased up to 14 days of estivation. The other organic acids studied, such as ketone body acetoacetate and ß-hydroxybutyrate or the volatile acid propionate, did not accumulate. Their tissue concentrations, however, increased on the 7th day of estivation and reached normal levels within two weeks of estivation for some of them. One should take into consideration how the reduction in metabolism can be handled under aerobic conditions, and what role anaerobic pathways may play in both energy formation and redox balance processes

Metabolic post-feeding changes in flat body and hemolymph of Dipetalogaster maximus (Hemiptera: Reduviidae)

Lipids and glycogen in fat body as well as the modifications in the wet weight of this organ were evaluated in an unfed insect, Dipetalogaster maximus, on day 5 after adult acdysis (time 0) and during a 30-day period after ingestion of blood meal. Total lipids, high density lipophorin (HDLp), carbohydrates, total protein and uric acid were determined in the hemolymph during the same period. Fat body wet weight was maximum on day 10 post-feeding and represented on day 30 only 42 per cent of the maximum weight. Lipids stored in the fat body increased up to day 15 reaching 24 per cent of the total weight of tissue. Glycogen was maximum on day 20, representing approximately 3 per cent of the fat body weight. HDLp represented at all times between 17-24 per cent of the total proteins, whose levels ranged between 35 and 47 mg/ml. Uric acid showed at 20, 25 and 30 days similar levels and significantly higher than the ones shown at days 10 an 15. Hemolymphatic lipids fluctuated during starvation between 3-4.4 mg/ml and carbohydrates showed a maximum on day 15 after a blood meal, decreasing up to 0.26 mg/ml on day 25. The above results suggest that during physiological events such as starvation, the availability of nutrients is affected, involving principally the fat body reserves.

The hemolymph of chasmagnathus granulata dana,1851 (Decapoda - grapsidae) as a target tissue ofthe crustacean hyperglycemic hormone

The effect of crustacean hyperglycemic hormone (CHH) was investigated on the hemolymph of Chasmagnathus granulata, a meso-supralitoral crab from southern Brazil. Serum glucose increased significantly (P®0.05) after incubation of total hemolymph in the presence of the eyestalk extract of a member of the same species. Also glucose uptake from blood serum, not affected by eyestalk extract (P¼0.05) was observed after incubation of total hemolymph in the presence of glucose.The results that the hemolymph may be a target tissue of CHH and that this hormone may act by mobilizing carbohydrate reserves possibly from hematocytes.

Natural lectin activity in the haemolymph of Panstrogylus megistus (Heteroptera: Reduvidae)

A hemolinfa de Panstrongylus megistus mostrou uma atividade lectínica natural para eritrócitos de vários vertebrados e näo mostrou especificidade para os diversos tipos de eritrócitos testados (humano ABO, pato, coelho,c amundongo,carneiro, galinha e boi). Com relaçäo aos eritrócitos humanos a atividade lectínica foi similar nos tipos A+, B+ e AB+ enquanto a atividade mais alta foi observada no tipo O+. O título de aglutinaçäo entre eritrócitos animais näo mostrou diferença apreciável, excluindo eritrócitos de boi, que apresentaram o título mmais baixo. A determinaçäo da concentraçäo mínima de inibiçäo foi realizada com eritrócitos humanos O+. A aglutinaçäo foi inibida por vários carboidratos (ramnose, D-dalactose, rafinose, D-lactose e D-fucose). A ramnose foi o inibidor mais potente (0,78 mM). Os resultados sugerem a presença de mais de uma lectina na hemolinfa de P. megistus

Bioquímica del ciclo evolutivo del Triatoma infestans (Vinchuca): VIII. Estudio preliminar de las apolipoproteinas hemolinfáticas de machos adutos / Biochemistry of the evoluation of Triatoma infestans (Vinchuca): VIII. Preliminary study of hemolymph apolipoproteins of adult males

Las tres lipoproteínas de alta (HDL) y muy alta densidad (VHDL-I y VHDL-II) fueron separadas de la hemolinfa de machos adultos de T. infestans. Ellas fueron deslipidizadas y las correspondientes apolipoproteínas se examinaron por electroforesis en gel de dodecil sulfato sodio en poliacrilamida en presencia de 2-mercapto-etanol. Luego, fueron separadas por enfoque isoelétrico en un gradiente de pH de 5 a 8. Los tres grupos de apolipoproteínas fueron separados en varias cadenas polipeptídicas, las que tenían en común una unidad glicoproteica de peso molecular 86 000. Las VHDL-II presentaron la composición más simple, con predominio de una banda de 86 000. La apo-HDL fue la más complicada y mostró bandas intensas con peso molecular tan alto como 210 000 y tan bajo como 17 000, conjuntamente con bandas de 44 000 y 86 000 y otras de menor intensidad. La apo-VHDL-I se separó en cadenas polipeptídicas en el rango de 86 000 a 17 000. Aparentemente, las distintas apoproteínas están formadas por la asociación, al menos en parte, de polipéptidos comunes