Lipase activity in the hemolymph of Biomphalaria glabrata (Mollusca) challenged with bacterial lipids

The levels of lipase activity in both the cellular and serum constituents of the hemolymph of Biomphalaria glabrata that had been challenged in vitro to beat-killed and sonicated Bacillus megaterium as well as samples challenged with live B. megaterium were ascertained. There were no significant alterations in the levels of enzyme activity in both cells and serum of the samples that had been challenged with sonicated bacteria; however, there was a signficant elevation in the enzyme activity associated with both the cells and serum of hemolymph that had been challenged with live bacteria. It has been concluded that live B. megaterium can stimulate hypersynthesis of lipase, a lysosomal enzyme, in phagocytes of B. glabrata and that this enzyme subsequently is released into serum. Consequently, the hydrolysis of lipid constituents of bacteria could theoretically occur in serum as well as within phagocytes.     

Release of lysozyme from hemolymph cells of Mercenaria mercenaria during phagocytosis

Activity of the lysosomal enzyme, lysozyme, has been quantitatively determined in the serum and cells of the hemolymph of Mercenaria mercenaria which had been exposed to known quantities of Bacillus megaterium and also in the serum and cells of hemolymph which had not been exposed to bacteria. The results indicate that the level of enzyme activity is greater in serum of hemolymph that had been exposed to B. megaterium and concurrently, there is an equivalent decrease in the level of activity in the cells. This evidence indicates that the amount of lysozyme released from cells into serum is enhanced during phagocytosis of the bacteria.It has also been demonstrated that the release of lysozyme from cells occurs during the process of phagocytosis and is not a delayed phenomenon.Enzyme release by secondary phagosomes is reflected morphologically by what is commonly referred to as degranulation. This process does not involve the rupture of the plasma membrane of the hemolymph cells since biochemical studies have revealed that there is no release of the cytoplasmic enzyme, lactate dehydrogenase.     

Aminopeptidase and lysozyme activity levels and serum protein concentrations in Biomphalaria glabrata (Mollusca) challenged with bacteria.

The total serum protein concentrations and levels of aminopeptidase and lysozyme activities in the sera of the gastropod Biomphalaria glabrata have been determined. The groups of snails from which hemolymph samples were taken for study included (1) untampered controls, (2) sham-injected snails, (3) heat-killed Bacillus megaterium-injected, and (4) live B. megaterium-injected ones. Our results indicate that there are significant elevations in the levels of aminopeptidase activity in 2 hr in the sera of snails that had been sham-, dead bacteria-, and liver bacteria-injected. The levels of lysozyme activity were not altered in sham-, dead bacteria-, and live bacteria-injected snails. This is contrary to an earlier finding (T. C. Cheng, M. J. Chorney, and T. P. Yoshino, 1977. J. Invertebr. Pathol., 29, 170–174), and the difference is believed to be due to the age of the snails employed. Comparisons of total serum proteins have revealed that the concentration in snails injected with live B. megaterium is significantly higher than in sham-injected ones. This may be due to increase of some yet undetermined serum protein fraction.     

Aminopeptidase activity in the hemolymph and body tissues of the pulmonate gastropod Biomphalaria glabrata

The levels of aminopeptidase activity in the whole hemolymph, serum, hemocytes, headfoot, and visceral mass of Biomphalaria glabrata were determined. The highest enzyme level occurs in the serum and the lowest in the hemocytes. Both the headfoot and visceral mass include subequal levels of aminopeptidase activity. From our data, it now appears possible that the serum aminopeptidase in B. glabrata, which has an open circulatory system, could have originated in hemocytes as well as in other tissues. The biologic function of serum aminopeptides is uncertain; however, because of the known chemical function of this enzyme, it could serve to degrade foreign proteins in serum prior to phagocytosis.     

Release pattern of aminopeptidase from Biomphalaria glabrata hemocytes subjected to high-level bacterial challenge

Specimens of Biomphalaria glabrata, NIH albino strain, challenged with 2 μl of heat-killed Bacillus megaterium at a concentration of 9 × 10⁹ bacteria/ml, showed significant elevation of serum aminopeptidase activity level at 2 hr postchallenge when compared with the serum activity levels of the enzyme in snails of the other experimental groups at four time intervals postchallenge. It was found that challenge with a heavy dosage of heat-killed bacteria did not cause a shift in the highest level of enzyme activity from 2 hr postchallenge. Also, the activity of aminopeptidase was higher at 2 hr postchallenge with a greater dosage of heat-killed bacteria.     

Lipase activity in the serum and hemolymph cells of the soft-shelled clam, Mya arenaria, during phagocytosis.

Quantitative determinations of lipase activity in the sera and hemolymph cell homogenates of Mya arenaria that had been challenged with heat-killed Bacillus megaterium and those of control clams have revealed that there is an increase in both fractions of the hemolymph during phagocytosis.The occurrence of lipase in serum and cells suggests that suitable substrates could be degraded by hydrolysis extracellularly as well as within phagocytes.     

Lysozymelike activity in the hemolymph of Biomphalaria glabrata challenged with bacteria.

Levels of lysozyme activity have been determined in the serum and cells of untreated Biomphalaria glabrata and in snails that had been challenged with heat-killed Bacillus megaterium and water at 1, 2, and 4 hr postinjection. Lysozyme activities have also been ascertained in sham-injected snails at 1, 2, and 4 hr postchallenge. Our results indicate significant alterations in the serum lysozyme activity levels at 2 and 4 hr postchallenge with bacteria and at 1 hr postinjection of water. Also, there is a significant increase in cell lysozyme activity at 1 hr postchallenge with B. megaterium. It is concluded that lysozyme is released from phagocytes into serum as a result of challenge with B. megaterium. Although the exact role of the released enzyme is uncertain, it is hypothesized that it may serve as a humoral defense molecule.     

Determination of the strength of the cell walls of vegetative cells ofBacillus megaterium andBacillus stearothermophilus

The tensile strength of the cell walls ofBacillus megaterium andBacillus stearothermophilus was found to be about 2.4×10⁷ N/m². The internal pressure and water activity of the cells were 14 atm, 0.99 a_w forB. megaterium and 28 atm, 0.98 a_w forB. stearothermophilus. The greater strength ofB. stearothermophilus cells, considered as pressure vessels, restricts absorption of water by the protoplasm so that the water content on a dry weight basis is 3.4 g/g forB. megaterium cells in water but only 1.8 g/g forB. stearothermophilus.     

Elevation of aminopeptidase activity in Biomphalaria glabrata (Mollusca) parasitized by Echinostoma lindoense (Trematoda)

Comparisons of the levels of aminopeptidase activity in the hemocytes and serum of Biomphalaria glabrata at 20 and 30 days postexposure to irradiated Echinostoma lindoense miracidia with enzyme levels in control snails have revealed that there are significant elevations in the serum of snails at both time periods postexposure. Furthermore, there is a significantly higher level of aminopeptidase activity in the serum of snails at 30 days than at 20 days postexposure. Although the biologic function of the elevated levels of serum aminopeptidase in sensitized snails remains uncertain, it is possible that this lysosomal enzyme may degrade the surface proteins of secondarily introduced parasites and thus act as a form of acquired humoral immunity.     

A quantitative study of phagocytosis by hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria

Fresh hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria were exposed to known concentrations of Bacillus megaterium, Escherichia coli, and Staphylococcus aureus in vitro and it was ascertained that all four types of cells of C. virginica and all three types of M. mercenaria became associated with the bacteria. Association is defined as either the first, i.e., contact and adherence, or second, i.e., engulfment, phase of phagocytosis. However, when the surfaces of each type of cell, as well as the percentages of each type in whole hemolymph, from both species of molluscs are taken into consideration, it is concluded that the granulocytes are the most important from the standpoint of phagocytosis.When hemocytes of M. mercenaria were exposed to Bacillus megaterium at 4°, 22°, and 37°C, it was found that the association indices were higher at the latter two temperatures. It is postulated, because of the results of Feng and Feng (1974), that nonself materials adhere with less frequency at 4°C and hence are not phagocytosed at this lower temperature.     

Experimentally induced elevations in acid phosphatase activity in hemolymph of Biomphalaria glabrata (Mollusca).

The levels of acid phosphatase activity in the hemocytes and serum of the following four groups of the pulmonate gastropod Biomphalaria glabrata were ascertained: (1) those injected with heat-killed Bacillus megaterium, (2) those challenged with sterile distilled water, (3) those shaminjected, and (4) those left untampered. It was determined that challenge with heat-killed bacteria sesulted in significant elevations in acid phosphatase activity in both cells and serum at 1, 2, and 4 hr postinjection, with the level being highest at 2 hr. Injection with water resulted in a significant elevation in cellular enzyme level at 1 hr postinjection but not at 2 and 4 hr; however, there were significant elevations at 2 and 4 hr in serum. This is interpreted to indicate that the elevated intracellular enzyme was subsequently released into serum. Sham injection resulted in significant elevations in acid phosphatase levels in hemocytes at 2 and 4 hr postinjection but no increase of enzyme activity in serum during the course of the experiment. This is interpreted to mean that this hydrolase was not released as a result of sham injection. The source of acid phosphatase was apparently the cytoplasmic granules of granulocytes, which are true lysosomes.     

Induced thermal stress on serotonin levels in the blue swimmer crab,Portunus pelagicus

The temperature of habitat water has a drastic influence on the behavioral, physiological and biochemical mechanisms of crustaceans. Hyperglycemia is a typical response of many aquatic animals to harmful physical and chemical environmental changes. In crustaceans increased circulating crustacean hyperglycemic hormone (CHH) and hyperglycemia are reported to occur following exposure to several environmental stress. The biogenic amine, serotonin has been found to modulate the CHH levels and oxidation of serotonin into its metabolites is catalysed by monoamine oxidase. The blue swimmer crab, Portunus pelagicus is a dominant intertidal species utilized throughout the indo-pacific region and is a particularly important species of Palk bay. It has high nutritional value and delicious taste and hence their requirements of capture and cultivation of this species are constantly increasing. This species experiences varying and increasing temperature levels as it resides in an higher intertidal zone of Thondi coast. The present study examines the effect of thermal stress on the levels of serotonin and crustacean hyperglycemic hormone in the hemolymph of P. pelagicus and analyzes the effect of the monoamine oxidase inhibitor, pargyline on serotonin and CHH level after thermal stress. The results showed increased levels of glucose, CHH and serotonin on exposure to 26 °C in control animals. Pargyline injected crabs showed highly significant increase in the levels of CHH and serotonin on every 2 °C increase or decrease in temperature. A greater CHH level of 268.86±2.87 fmol/ml and a greater serotonin level of 177.69±10.10 ng/ml was observed at 24 °C. This could be due to the effect of in maintaining the level of serotonin in the hemolymph and preventing its oxidation, which in turn induces hyperglycemia by releasing CHH into hemolymph. Thus, the study demonstrates the effect of thermal stress on the hemolymph metabolites studied and the role of pargyline in elevating the levels of serotonin and CHH on thermal stress in the blue swimmer crab, P. pelagicus.     

Leukocytes of Biomphalaria glabrata: Morphology and behavior of granulocytic cells in vitro

Blood cells from Biomphalaria glabrata hemolymph were studied by phase microscopy in vitro. The most common cell is a granulocytic leukocyte, present in the hemolymph at a concentration of 334 ± 105 cells/mm³. This cell ranges in length from 14.1 ± 2.8 μm (including pseudopodia), when suspended in hemolymph, to 77.2 ± 15.1 μm, when allowed to spread on a glass substrate. Cytoplasmic inclusions and other organelles are described. Observations on the behavior of these granulocytic leukocytes in vitro confirm that a strong phagocytic response develops toward a variety of particles in the virtual absence of snail hemolymph.     

Respiratory and osmoregulatory responses of the hermit crab, Clibanarius vittatus (Bosc), to salinity changes

Intertidal hermit crabs were stepwise acclimated to 10, 20, and 30‰ salinity (S) and 21 ± 1 °C. Hemolymph osmolality, sodium, chloride, and magnesium were isosmotic (isoionic) to ambient sea water at 30‰ and hyperosmotic (hyperionic) at 20 and 10‰ S, while hemolymph potassium was significantly hyperionic in all acclimation salinities. Total body water did not differ significantly at any acclimation salinity. Oxygen uptake rates were higher in summer-than winter-adapted crabs. No salinity effect on oxygen consumption occurred in winter-adapted individuals. Summer-adapted, 30‰ acclimated crabs had a significantly lower oxygen consumption rate than those acclimated 10 and 20‰ S. Crabs exposed to 30 10 30‰ and 10 30 10‰ semidiurnal (12 h) and diurnal (24.8 h) fluctuating salinity regimes showed variable osmoregulatory and respiratory responses. Hemolymph osmolality followed the osmolality of the fluctuating ambient sea water in all cases, but was regulated hyperosmotically. Hemolymph sodium, chloride, and magnesium concentrations were similar to hemolymph osmolality changes. Sodium levels fluctuated the least. Hemolymph potassium was regulated hyperionically during all fluctuation patters, but corresponded to sea water potassium only under diurnal conditions. The osmoregulatory ability of Clibanarius vittatus (Bosc) resembles that reported for several euryhaline brachyuran species. The time course of normalized oxygen consumption rate changed inversely with salinity under semidiurnal and diurnal 10 30 10‰ S fluctuations. Patterns of 30 10 30‰ S cycles had no effect on oxygen consumption rate time course changes. The average hourly oxygen consumption rates during both semidiurnal fluctuations were significantly lower than respective control rates, but no statistical difference was observed under diurnal conditions.     

Biolytic extraction of poly(3-hydroxybutyrate) from Bacillus megaterium Ti3 using the lytic enzyme of Streptomyces albus Tia1

The applicability of Streptomyces sp. cell lytic enzymes for devising a simple and competent biological polyhydroxyalkanoate (PHA) recovery approach from Bacillus megaterium cells was investigated. B. megaterium strain Ti3 produced 50% (w/w) PHA using glucose as carbon source. The intracellular PHA was recovered employing a non-PHA accumulating actinomycetes (Tia1) identified as Streptomyces albus, having potent lytic activity against living and heat inactivated B. megaterium. Interestingly, maximum biomass (2.53?±?0.6?g/L by 24?h) of the lytic actinomycete was obtained in PHA production medium itself thus circumventing the prior actinomycete acclimatization just by co-inoculation with B. megaterium as an inducer. Maximum lytic activity was observed at pH 6.0, 40?°C, 220?mg of biomass and 33.3?mL of concentrated culture filtrate in a 100?mL reaction mixture. Preliminary biochemical investigations confirmed the proteolytic and caseinolytic nature of the lytic enzyme. PHA yield of 0.55?g/g by co-inoculation extraction approach was comparable with the conventional sodium hypochlorite based extraction method. Interestingly, S. albus also demonstrated a broad spectrum lytic potential against varied Gram-negative and Gram-positive PHA producers highlighting the extensive applicability of this biolytic PHA recovery approach. The lytic enzyme retained almost 100% relative activity on storage at ?20?°C upto two months. ¹H Nuclear magnetic resonance analysis of the extracted polymer confirmed it as a homopolymer composed of 3-hydroxybutyrate monomeric units. This is the first report on Streptomyces sp. based biological and eco-friendly, intracellular PHA recovery from Bacillus spp.     

Aminopeptidase Activity in Marine Chroococcoid Cyanobacteria

Synechococci are important primary producers in the ocean and can also utilize some components of the dissolved organic matter (DOM). The readily utilizable DOM in seawater is mainly polymeric (e.g., protein, polysaccharide) or phosphorylated and requires hydrolysis prior to uptake. We examined whether synechococci express ectoenzymes to hydrolyze DOM components and considered the possible significance of ectohydrolases for Synechococcus ecology and organic matter cycling in the sea. Five strains of non-nitrogen-fixing synechococci in axenic cultures were tested for enzyme activities with fluorogenic substrates. All strains show ectocellular aminopeptidase activity, but other enzymes were undetectable. The aminopeptidase level was in the range determined for five marine heterotrophic bacterial isolates tested for comparison. Aminopeptidase was not secreted into the medium; the majority (74%; tested in WH 7803) was cell surface bound, and a small fraction was periplasmic. The periplasmic activity was not released by cold osmotic shock of WH 7803. Phenylmethylsulfonyl fluoride and EDTA, inhibitors of serine and metalloproteases, strongly or completely inhibited WH 7803 aminopeptidase. The enzyme seemed constitutive; per-cell activity did not change during incubations in unenriched seawater, bovine serum albumin, or nitrate-replete mineral medium. In natural planktonic assemblages in the Southern California Bight, aminopeptidase activity was correlated with Synechococcus abundance as well as the abundance of other bacteria. Ectocellular aminopeptidase may be common in marine synechococci and play roles in their nitrogen nutrition, particularly in low-nitrate and low-light environments. Since synechococci are much less abundant than heterotrophic bacteria in seawater, the impact of Synechococcus aminopeptidase on proteolysis in the sea is likely to be episodic and restricted to specialized microenvironments.     

Effect of nitrate and l-arginine therapy on nitric oxide levels in serum, heart, and aorta of fetal hypothyroid rats

Reduced nitric oxide availability and a heterogeneous pattern of nitric oxide synthase activity in some tissues have been reported in hypothyroidism. This study aimed at determining the effects of oral nitrate and l-arginine administration on serum, heart, and aorta nitric oxide metabolite concentrations in fetal hypothyroid rats. In an experimental study, pregnant Wistar rats were administrated tap water or 0.02 % of 6-propyl-2-thiouracil in drinking water during pregnancy and their male pups were followed (n?=?8/group). In adult progeny, serum, heart, and aorta nitric oxide metabolite concentrations were measured by the Griess method after 1-week administration of sodium nitrate (500 mg/L) or l-arginine (2 %) in drinking water. Serum thyroid hormone and thyroid-stimulating hormone levels were also measured. Compared to controls, fetal hypothyroid progeny had significantly lower nitric oxide metabolite concentrations in heart (0.32?±?0.07 vs. 0.90?±?0.14 nmol/mg protein, p?=?0.004) and aorta (2.98±0.56 vs. 6.15±0.74 nmol/mg protein, p?=?0.011) tissues. Nitrate therapy restored heart nitric oxide metabolite levels decreased by fetal hypothyroidism, while l-arginine administration further decreased aorta nitric oxide metabolite levels. Sodium nitrate increased and l-arginine decreased serum nitric oxide metabolite levels in both control and fetal hypothyroid animals. In conclusion, nitrate therapy restores decreased heart nitric oxide metabolite levels, whereas l-arginine decreases aorta nitric oxide metabolite levels even further in fetal hypothyroid rats, findings relevant to the cardiovascular consequences of congenital hypothyroidism in adulthood.     

Reduced Levels of Brain Derived Neurotrophic Factor (BDNF) in the Serum of Diabetic Retinopathy Patients and in the Retina of Diabetic Rats

Diabetic retinopathy (DR) is widely recognized as a neurovascular disease. Retina, being a neuronal tissue of the eye, produces neurotrophic factors for its maintenance. However, diabetes dysregulates their levels and thereby may damage the retina. Among neurotrophins, brain derived neurotrophic factor (BDNF) is the most abundant in the retina. In this study, we investigated the level of BDNF in the serum of patients with DR and also in the serum and retina of streptozotocin-induced diabetic rats. The level of BDNF was significantly decreased in the serum of proliferative diabetic retinopathy patients as compared to that of non-diabetic healthy controls (25.5 ± 8.5–10.0 ± 8.1 ng/ml, p < 0.001) as well as compared to that of diabetic patients with no retinopathy (21.8 ± 4.7–10.0 ± 8.1 ng/ml, p < 0.001), as measured by ELISA techniques. The levels of BDNF in the serum and retina of diabetic rats were also significantly reduced compared to that of non-diabetic controls (p < 0.05). In addition, the expression level of tropomyosin-related kinase B (TrkB) was significantly decreased in diabetic rat retina compared to that of non-diabetic controls as determined by Western blotting technique. Caspase-3 activity was increased in diabetic rat retina after 3 weeks of diabetes and remained elevated until 10 weeks, which negatively correlated with the level of BDNF (r = ?0.544, p = 0.013). Our results indicate that reduced levels of BDNF in diabetes may cause apoptosis and neurodegeneration early in diabetic retina, which may lead to neuro-vascular damage later in DR.     

Mechanism of lysosomal enzyme release from Mercenaria mercenaria granulocytes: a scanning electron microscope study

A scanning electron microscope study of Mercenaria mercenaria granulocytes at 1 and 2 hr postchallenge with a 0.02-ml Millipore-filtered, sterile sea water suspension of heat-killed Bacillus megaterium at a concentration of 4 × 10⁶ bacteria/ml revealed that (1) granulocytes challenged with bacteria revealed more and larger lysosomes protruding from their surfaces than those of the control groups; (2) release of intact lysosomes from granulocytes into serum occurred concurrently with phagocytosis; (3) enzymes released from discharged lysosomes acted on bacterial cell walls causing their partial degradation, thereby enhancing endocytosis; and (4) degranulation is a normal process but is greatly enhanced when stimulated by phagocytosis of bacteria. This study also demonstrated that lysosomes budded off from the plasma membrane of granulocytes into serum, although the actual biochemical mechanism(s) that causes the detachment of lysosomes from the plasma membrane and the subsequent lysis of the double-membraned lysosomes to release the hydrolases remains unresolved.     

Selenium and Glutathione Peroxidase Status in Adult Egyptian Patients with Acute Myeloid Leukemia

This study was undertaken to evaluate selenium (Se) and glutathione peroxidase (GPX) status in patients with newly diagnosed acute myeloid leukemia (AML) before and after induction therapy. Twenty-five patients with newly diagnosed AML and 15 healthy age- and sex-matched control subjects were included in this study. Serum Se level by the graphite furnace atomic absorption spectrometric technique and GPX activity by an adaptation of Beutler method was performed for the patients before and after receiving the induction therapy. Serum Se level was significantly lower in patients with AML versus control subjects (63.1?±?8.8 versus 77?±?8.8 µg/L before therapy with a P value <0.01 and 69?±?6.8 versus 77?±?8.8 µg/L after therapy with a P value <0.01).GPX activity was significantly lower in patients with AML versus control subjects (1.6?±?0.4 versus 3.4?±?0.7 µ/g protein pretreatment with a P value <0.01and 1.9?±?0.6 versus 3.4?±?0.7 µ/g protein post induction treatment with P value <0.01).Se level and GPX activity significantly increased in AML patients after treatment. Patients who accomplished complete remission after induction harbored significantly higher Se levels than resistant patients before and after treatment. There was no significant correlation between serum Se level and GPX activity. Decreased Se level and reduced GPX activity in AML patients support the association of carcinogenesis and subnormal Se states.