Preferential distribution of group-II-like phospholipase A2 in mononuclear phagocytic cells in rat spleen and liver

The localization of calcium-dependent phospholipase A2, (PLA2) immunochemically closely related to the enzyme of the viperid and crotalid type (group II), in cells isolated from rat spleen and liver was examined using a polyclonal antibody directed against rat spleen group II, PLA2 (PLA2M). In isolated spleen cells, the monocyte/macrophage fraction had the highest PLA2 activity (1.28 +/- 0.35.min-1.10(6) cells-1) which was almost completely inhibited by the anti-PLA2M antibody. An immunoblot analysis confirmed the presence of the enzyme in this fraction. An immunocytochemical study revealed that the PLA2 was present in spleen macrophages. In the isolated liver cells, Kupffer cells (0.92 +/- 0.22 nmol.min-1.10(6) cells-1) contained higher anti-PLA2M-antibody-inhibitable PLA2 activity than parenchymal cells (0.26 +/- 0.06.min-1.10(6) cells-1). The immunocytochemical study showed that cells immunopositive with anti PLA2M antibody were Kupffer cells. These results suggest that the mononuclear phagocytic cells in rat spleen and liver have relatively high activity of group-II-like PLA2. Subcellular distribution patterns of the anti-PLA2M-antibody-inhibitable phospholipase A2 activity in different cell populations from spleen and liver were compared. A mode of the distribution of the enzyme in the spleen macrophages was essentially similar to that in the spleen lymphocytes. The distribution in Kupffer cells was similar to that in parenchymal cells.     

Segmental distribution and gestational changes of GABA-transaminase activity in the rat oviduct

The occurrence and the localization of 4-aminobutyrate:2-oxoglutarate transaminase (GABA-transaminase) in the non-pregnant and pregnant rat oviduct were examined using biochemical and enzyme histochemical techniques. Specific GABA-transaminase activity was detected in the ampullary and isthmic portions of the oviduct as well as in the utero-tubal junction. The enzymic activity was lower in the ampullary than in the isthmic or intramural segments of the oviduct. Pregnancy induced a significant increase of GABA-transaminase activity in each portion of the oviduct. Enzyme histochemistry showed the highest GABA-transaminase reactivity at the level of the epithelial cells of the oviduct irrespective of the portion of the tube examined. A faint specific activity was demonstrated in the smooth muscle of the oviduct while the serosa did not show specific staining. Our findings indicate that: the observed increase of GABA-transaminase activity in the oviduct of the pregnant rat may be responsible for the reduced GABA levels in the oviduct during gestation; and the extraneuronal localization of GABA-transaminase activity does not seem to support the suggestion of a possible GABAergic innervation of the oviduct.     

Glucocorticoids inhibit TNF alpha-induced cytosolic phospholipase A2 activity.

The cytosolic phospholipase A2 (PLA2) was characterized in the human epithelial carcinoma cell line HEp-2 by its apparent molecular mass (about 80 kDa); its in vitro activation by micromolar concentrations of calcium; and its calcium-dependent association with cellular membranes. The activity of this enzyme was induced by an overnight incubation with tumor necrosis factor alpha (TNF alpha). Glucocorticoids only moderately reduced PLA2 activity in control cells, but completely inhibited the TNF alpha-induced increase in the activity of the high-molecular-weight cytosolic PLA2.     

Phospholipid-deacylating enzymes of rat stomach mucosa

1. Rat stomach mucosa exhibited three distinguishable phospholipid-deacylating enzyme activities: lysophospholipase, phospholipase A1 and phospholipase A2. 2. The lysophospholipase hydrolyzed 1-palmitoyl lysophosphatidylcholine to free fatty acid and glycerophosphorylcholine. This enzyme had an optimum pH of 8.0, was heat labile, did not require Ca2+ for maximum activity and was not inhibited by bile salts or buffers of high ionic strength. 3. Phospholipase A2 and phospholipase A1 deacylated dipalmitoyl phophatidylcholine to the corresponding lyso compound and free fatty acid. The specific activity of phospholipase A2 was 2--4-fold higher than that of phospholipase A1 under all the conditions tested. Both activities were enhanced 4--7.5-fold in the presence of bile salts at alkaline pH and 11-18-fold at acidic pH. 4. In the absence of bile salts, phospholipase A1 exhibited pH optima at 6.5 and 9.5 and phospholipase A2 at pH 6.5, 8.0 and 9.5. The pH optima for phospholipase A1 were shifted to pH 3.0, 6.0 and 9.0 in presence of sodium taurocholate; the activity was detected only at a single pH of 9.5 in the presence of sodium deoxycholate and at pH 10.0 in the presence of sodium glycocholate. Phospholipase A2 optimum activity was displayed at pH 3.0, 6.0 and 8.0 in presence of taurocholage, pH 7.5 and 9.0, in presence of glycocholate and only at pH 9.0 in presence of deoxycholate. 5. Ca2+ was essential for optimum activity of phospholipases A1 and A2. But phospholipase A1 lost complete activity in presence of 0.5 mM ethyleneglycolbis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) at pH 6.0, whereas phospholipase A2 lost only 50%. 6. Phospholipases A1 and A2 retained about 50% of their activities by heating at 75 degrees for 10 min. At 100 degrees, phospholipase A1 retained 22% of its activity, whereas phospholipase A2 retained only 7%.     

Multiple phospholipase A2 activities in canine vascular smooth muscle.

Three phospholipase A2 activities from canine vascular smooth muscle were identified and characterized including: (1) a cytosolic calcium-independent phospholipase A2 which is activated by nucleotide di- and triphosphates; (2) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (3) a microsomal calcium-independent phospholipase A2 which was highly selective for plasmenylcholine substrate. Vascular smooth muscle cytosolic calcium-independent phospholipase A2 was activated 338% +/- 11 (X+S.E.; n = 15) by physiologic concentrations of ATP. Similar amounts of activation were also present utilizing other nucleotide di- and triphosphates (e.g., ADP, CTP, GDP and GTP) as well as non-hydrolyzable nucleotide triphosphate analogs (e.g., ATP-gamma-S, AMP-PNP and GTP-gamma-S). Vascular smooth muscle cytosolic calcium-dependent phospholipase A2 was purified 455-fold by sequential DEAE-Sephacel, Phenyl-Sepharose, Mono Q, hydroxyapatite and Superose 12 chromatographies. The partially purified calcium-dependent phospholipase A2 was activated by physiologic increments in calcium ion concentration (e.g., 1 microM) and possessed an apparent native molecular weight of 95 kDa, an acidic isoelectric point (pI = 4.8) and a neutral pH optimum (pH 7.0). Vascular smooth muscle microsomal phospholipase A2 activity was predominantly calcium-independent and was over six-fold selective for hydrolysis of plasmenylcholine substrate. Taken together, these results demonstrate the existence of three separate and distinct phospholipase A2 activities in vascular smooth muscle and identify ATP and calcium ion as independent modulators of discrete phospholipase A2 activities in vascular smooth muscle cells.     

Role of endogenous and exogenous prostaglandins on the contractile functioning of isolated sow (Sus scrofa) oviducts

The output prostaglandins (PHs) E₁, E₂ and F₂ α, from ampullary and isthmic portions of sow oviducts isolated during proestrus, estrus and metestrus, was explored. Moreover, $\text{in vitro}$ cumulative dose-response curves for the contractile effect of these three PGs, on identical oviductal segments, were constructed. Isthmic preparations form proestrous and metestrous animals released more PGE₁ and PGF₂ α than PGE₂ “like material”. During estrus, the outputs of PGE₁, PGE₂ and PGF₂ α were similar, whereas, oviducts from proestrous and metestrous sows released less PGE₁ and PGF₂ α than during estrus. Although the output of PGE₂ “like material” from isthmic and ampullary segments did not differ significantly during the three stages of the sex cycle, ampullary metestrous preparations released more PGE₁ and PGF₂ α, than estrous or proestrous ones. The addition of PGE₁, PGE₂ α, consistently stimulatedthe amplitude of contractions of isthmic oviductal segments isolated from proestrous and metestrous sows. Within the concentration-range explored, dose-response curves for PGE₂ and PGE₁ were to the left of those for PGF₂ α in the isthmus obtained before ovulation (proestrus) but not in segments isolated at later times (2–3 days) of the cycle (metestrus). The stimulatory dose-response curves for PGE₁, or PGE₂, in isthmic segments of metestrous preparations incubated with phentolamine (10^?6M) were shifted to the right of controls not exposed to the adrenoreceptor blocker, whereas, the curve for PGF₂ α without phentolamine, was identical to that obtained in its presence. PGE₁ and PGE₂ did not evoke significant contractile effects on oviductal ampullary protions from proestrous sows, wherea, PGF₂ α was clearly stimulatory at concentrations of 10^?9M and higher. In ampullary segments isolated after ovulation (metestrus) the threshold for contractile enhancement following PGF₂ α was greater than during proestrus, whereas, PGE₁ elicited a significant inhibition of contractions. The spontaneous contractile pattern exhibited by isthmic and ampullary oviductal regions, prior to and after ovulation, is discussed in terms of tissue PG generation and output and is compared with results regarding tubal motility following the exposure to exogenous PGs.     

Interleukin 1 and tumor necrosis factor synergistically stimulate prostaglandin synthesis and phospholipase A2 release from rat renal mesangial cells

Treatment of rat glomerular mesangial cells with recombinant human interleukin 1 alpha (rIL-1 alpha), recombinant human interleukin 1 beta (rIL-1 beta) or recombinant human tumor necrosis factor (rTNF) induces prostaglandin E2 (PGE2) synthesis and the release of a phospholipase A2 (PLA2) activity. rIL-1 beta is significantly more potent than rIL-1 alpha or rTNF in stimulating PGE2 as well as PLA2 release from mesangial cells. When given together, rTNF interacts in a synergistic fashion with rIL-1 alpha and rIL-1 beta to enhance both, PGE2 synthesis and PLA2 release. The released PLA2 has a neutral pH optimum and is calcium-dependent. Pretreatment of cells with actinomycin D or cycloheximide inhibits basal and cytokine-stimulated PGE2 and PLA2 release.     

Effects of 17beta-oestradiol or oestrous stage-specific cow serum on the ability of bovine oviductal epithelial cell monolayers to prolong the viability of bull spermatozoa

The effect of 17beta-oestradiol and oestrous stage-specific cow serum on bovine oviductal epithelial cell monolayers to extend the viability of co-cultured bull spermatozoa was examined. Monolayers of cells from ampullary and isthmic segments were pre-treated with medium containing either oestrous cow serum, luteal-phase cow serum, 1 microg/ml 17beta-oestradiol + foetal bovine serum or foetal bovine serum alone (control) before the addition of motile frozen/thawed spermatozoa. Motility was visually assessed throughout a 48 h co-incubation period, while fertilising ability of spermatozoa was evaluated by adding in vitro matured bovine oocytes. Pre-treatment with 17beta-oestradiol or oestrous cow serum resulted in a higher percentage of motile spermatozoa after 18 h in isthmic and after 36 h in ampullary cultures compared with the control, but pre-treatment did not affect fertilisation rates. Only at 42 h in ampullary cultures was motility higher in luteal serum pre-treated cultures compared to the control. Motility was also assessed in medium conditioned by pre-treated monolayers. Pre-treatment with 17beta-oestradiol enhanced the ability of conditioned medium to prolong motility and medium conditioned with oestrous cow serum was superior to medium conditioned by luteal-phase serum at maintaining motility. In conclusion, the ability of oviductal epithelium to prolong the motility of spermatozoa is enhanced by 17beta-oestradiol.     

Phospholipases A1 and A2 in bovine thyroid.

In both supernatant and sediment of thyroid tissue homogenate phospholipase and lysophospholipase activities were demonstrated. In the supernatant, using 1-acyl-2[1-14C]linoleoyl-sn-glycero-3-phosphorocholine in the presence of sodium taurocholate, phospholipase A1 activity with pH optima at 3.6 and 4.8 and phospholipase A2 activity with pH optima at 3.6 and 5.7 were found. The sediment showed mainly phospholipase A2 activity with a pH optimum at pH 6.5. Lysophospholipase activity (optimum pH 7--8), USING 1-[9,10-(3)H]stearyl-sn-glycero-3-phosphorocholine as a substrate was present in both supernatant and sediment. Enzyme assays performed on subcellular fractions suggest the soluble phospholipases to be of lysosomal origin and the solubilized phospholipase A2 activity of homogenate sediment to be of microsomal origin. Incubations with 3H-14C mixed labelled phosphatidylcholine further confirmed the above observations.     

Phospholipase activities of the P388D1 macrophage-like cell line

The murine macrophage (M phi) cell line, P388D1, was employed as a source of M phi phospholipases in order to characterize the enzymatic properties and subcellular localization of these enzymes because of their importance for prostaglandin biosynthesis. Phospholipase activity was assessed with dipalmitoylphosphatidylcholine (DPPC) as substrate. Phospholipases were characterized with respect to divalent cation dependence, pH optima, and localization in subcellular compartments using linear sucrose gradients. By these criteria a number of different phospholipases were identified. Most importantly, a single Ca2+-dependent activity with a pH optimum of 8.8 was identified in membrane-rich fractions (plasma membrane, mitochondria, and endoplasmic reticulum) and could be clearly separated from the remaining activities, which are Ca2+ independent and exhibit pH optima of 7.5, 5.1, and 4.2. The phospholipases with acidic pH optima may be associated with subcellular components containing lysosomal enzymes and both phospholipase A1 and phospholipase A2 activities are observed. In contrast, the phospholipase activity with a pH optimum of 7.5 sediments with the cytosolic proteins and is inhibited by 5 mM Ca2+. No significant phospholipase C activity was detected in assays performed with or without added Ca2+ at pH's 4.2, 5.1, 7.5, or 8.8 using DPPC as substrate. However, the P388D1 cells do contain a lysophospholipase that is at least 20 times more active than the phospholipase A activities identified. Its presence must be taken into account in evaluating the positional specificities and properties of the macrophage phospholipases.     

Components of oviduct physiology in eutherian mammals

Recalling the evolutionary sequence of development first of gonad and subsequently of oviducts, ovarian endocrine regulation of all known components of oviduct physiology is reviewed. Ovaries not only influence oviducts via the systemic blood circulation, but also locally by counter‐current transfer of relatively high concentrations of steroid hormones and prostaglandins between the ovarian vein and oviduct branch of the ovarian artery. The efficiency and impact of such counter‐current transfer is greatest around the time of ovulation, the transfer process receiving further inputs from hormones present in peritoneal fluid. Classical oviduct physiology is summarised, and the potential molecular consequences of temperature gradients within the duct lumen examined. At ovulation, an oocyte‐cumulus complex is displaced in minutes from the follicular surface to the site of fertilisation at the ampullary‐isthmic junction of the oviduct. This rapid initial phase is contrasted with the subsequent slow progression of embryos to the uterus in days, still encompassed within a zona pellucida. Regarding transport of spermatozoa, the formation of a pre‐ovulatory reservoir in the caudal portion of the oviduct isthmus is noted, with suppression of motility and sperm‐head binding to epithelial organelles acting to maintain fertilising ability. Completion of capacitation is prompted shortly before ovulation, predominantly by Ca²⁺ influx into bound spermatozoa. A controlled release of spermatozoa coupled with their hyperactivation results in initial sperm:egg ratios at the site of fertilisation close to unity, thereby avoiding the pathological condition of polyspermy. Both the oviduct milieu and embryonic development are influenced by paracrine activity of follicular granulosa cells released at ovulation and remaining in suspension in the vicinity of the oocyte or embryo. These cells may amplify early pregnancy signals from a zygote to the endosalpinx. Beneficial effects of the oviduct on domestic animal embryos are contrasted with anomalies arising as a consequence of in vitro culture. Primate embryos do not require exposure to an oviduct for normal development, perhaps due to overlapping compositions of endosalpingeal and endometrial secretions. Additionally, primate endometrial secretions may be modified by viable gametes or an embryo in the presence of a cumulus cell suspension.     

Ultrastructural changes in the oviductal epithelium of Merino ewes during the estrous cycle

Isthmic and ampullary oviductal epithelia sampled from Merino ewes at days -1, 1, 3, and 10 of the estrous cycle (estrus = day 0) were studied by scanning and transmission electron microscopy after fixation by vascular perfusion. Secretory cells, ciliated cells, and lymphocytelike basal cells were observed in both isthmic and ampullary epithelium at all stages of the estrous cycle studied and their ultrastructural features were analyzed. Synthesis of lamellated secretory granules occurred in the ampullary secretory cells during the follicular and early luteal phases, and their contents were released by exocytosis into the oviductal lumen during the luteal phase. Granule release was associated with nucleated apical protrusion of these cells into the oviductal lumen. No such secretory activity was displayed by isthmic secretory cells even though a few cells contained nonlamellated granules. Apocrine release of apical vesicles and accompanying cytoplasmic material from apical protrusions of ciliated cells occurred in the isthmus around estrus but not in the ampulla. This unexpected feature has not previously been reported in any other mammal. Dendritic basal cells were distinguished in the lower part of the epithelium by their heterochromatic nuclei, electron-lucent cytoplasm, and lack of attachment zones. No migration of basal cells was observed, and their ultrastructural features were similar in the ampulla and isthmus and at all stages of the estrous cycle examined. The function of these lymphocytelike cells in the epithelium is uncertain, but the presence of phagocytic bodies and lysosomes in 20% of them may indicate a phagocytic role.     

Trichomonas vaginalis: identification of soluble and membrane-associated phospholipase A1 and A2 activities with direct and indirect hemolytic effects

A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthal's inhibitor. The greatest effect was observed with 80 microM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.     

Enzymatic activity and distribution of phospholipase A2 in human cartilage

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.     

Microvascular architecture of the pregnant rabbit oviduct

Methyl-methacrylate vascular corrosion casts of the oviducts were prepared in 7 rabbits which were 2-3 weeks pregnant. Scanning electron microscopy of the acrylic casts revealed little change in tubal microvascular connections when compared with control oviducts. Venous distension in the isthmic subserosal venous plexus, ampullary subserosal vasculature and in the fimbrial core was substantially greater than that observed in controls. These changes are interpreted as indicating a sensitivity of tubal microvasculature to the increased levels of circulating placental hormones in pregnancy. The implications of this interpretation in the role of tubal microvasculature at the time of ovulation are discussed.     

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background

The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.

Methods

Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.

Results

Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.

Conclusion

Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.     

Modulation of post-thaw sperm functions with oviductal proteins in buffaloes

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at -20 degrees C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris-egg yolk-citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN(2) (-196 degrees C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1mg/ml of extended semen. The equilibrated and frozen-thawed (37 degrees C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 degrees C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher (P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher (P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.     

Phospholipase activities in clinical and environmental isolates of Acanthamoeba

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A(2) (PLA(2)) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA(2). Acanthamoeba exhibited optimal phospholipase activities at 37℃ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA(2)-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.     

Phospholipase and lysophospholipase activity of rat eosinophil leukocytes

Previous studies have shown the high lysophospholipase activity of rat eosinophilic leukocytes and used this enzyme to measure the rise in eosinophilic population of peripheral tissues caused by parasitic infections. This report details the methods and results of an investigation showing the presence in the same cells of high phospholipase (PLA) activity. Unfractionated and metrizamide-purified peritoneal eosinophil preparations were assayed using a mixed micelle substrate (6/15 mM lecithin/Triton X-100) at experimentally determined pH (6.4) and ionic strength (I=0.2) optima: the attendant reaction products included free fatty acids and organic P in a 2/1 molar proportion with a correspondent loss in the initial phospholipid concentration. The organic P fragment was further characterized as GPC (glycerylphosphorylcholine) by quantitative precipitation and acid hydrolysis. Estimates of PLA activity averaged 5 micromol/h/10(6) unfractionated eosinophils and metrizamide-purified eosinophil preparations. Paired tests for PLA and LysoPLA on unfractionated and enriched cell preparations, cytosolic extracts, and chromatographic fractions yielded similar activity ratios, supporting the inference of a close association of the two activities which could also be confirmed for the major tissues of eosinophil production and distribution.     

Interaction of surfactant protein A with peroxiredoxin 6 regulates phospholipase A2 activity

Peroxiredoxin 6 (Prdx6) is a "moonlighting" protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA(2) activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA(2) activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA(2) activity) showed K(m)0.35 mm and V(max) 138 nmol/min/mg of protein. SP-A inhibited PLA(2) activity non-competitively with K(i) 10 mug/ml and was Ca(2+) -independent. Activity at pH 7.4 was approximately 50% less, and inhibition by SP-A was partially dependent on Ca(2+). Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni(2) -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca(2+) dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA(2) activity of Prdx6 by SP-A.