Identification of structural and secretory lectin-binding glycoproteins of normal and cancerous human prostate.

We have utilized the technique of lectin-loading of SDS gels with iodinated concanavalin A and wheat germ agglutinin to identify glycoproteins in prostatic and seminal fluids as well as in prostate tissue fractions. The following subunits which bound both lectins were detected: (a) 50, 43 and 38 kDa subunits common to prostatic and seminal fluids, and an additional 55 kDa subunit which predominates only in prostatic fluid; (b) 78, 55, 50 and 43 kDa subunits in prostatic tissue cytosol and (c) 195, 170, 135, 116 and 95 kDa subunits present in the particulate fractions of prostatic tissue. Immunoblotting using specific rabbit antibodies revealed the 50 kDa band to be prostatic acid phosphatase and the 38 kDa band to be prostate-specific antigen. Interestingly, antibodies directed toward prostatic acid phosphatase were found to cross-react with the 43 kDa band. Fractionation on sucrose gradients showed that several of these particulate glycoproteins were associated with a vesicle fraction enriched in adenylate cyclase activity, implying that they are plasma membrane glycoproteins. Comparison of soluble and particulate fractions of normal and cancerous tissue homogenates was made by densitometric scanning of autoradiograms of lectin-loaded gels. Similar relative intensities of lectin-binding were obtained for corresponding proteins in normal and cancerous tissue fractions. Also, immunoblotting showed no differences in prostatic acid phosphatase or prostate-specific antigen between normal and cancerous soluble homogenate fractions. Our results suggest that major lectin-binding proteins are conserved in the transition from normal to cancerous tissue. These results may be useful in developing a multiple-marker profile of metastatic prostate cancer and for the design of imaging agents, such as monoclonal antibodies, to prominent soluble and particulate prostate glycoproteins.

Characteristics of the guanine nucleotide regulatory component of adenylate cyclase in human erythrocyte membranes.

This study probes the structure and mutual interactions of the components of adenylate cyclase. We use a complementation assay which involves the addition of an adenylate cyclase-related guanine nucleotide-binding protein component to a membrane lacking this component to measure guanine nucleotide-stimulated-adenylate cyclase. Instead of using detergent extracts we were able to achieve full complementation by mixing intact membrane preparations in the presence of the nucleotide component. Of particular interest was the human erythrocyte membrane which contains very low amounts of catalytic activity and no measurable beta-adrenergic receptor but has normal amounts of the nucleotide component. This component appears to be the same, by several criteria, as components found in pigeon and turkey erythrocytes and in rat liver plasma membrane. The component confers Gpp(NH)p, fluoride, and GTP stimulation of adenylate cyclase along a single reconstitution curve. It is labeled with NAD by cholera toxin, and has an apparent molecular weight of 39 000 upon sodium dodecyl sulfate gel electrophoresis. The presence of the nucleotide unit in the virtual absence of the active catalytic unit allowed us to determine those properties intrinsic to each unit and those conferred by the association of the units. The nucleotide component binds guanine nucleotides weakly in the human erythrocyte membrane, yet produces persistent activation of adenylate cyclase and tight binding (of Gpp(NH)p) upon combination with the catalytic unit. Treatment of the human erythrocyte membrane with N-ethylmaleimide causes a simultaneous diminution in both Gpp(NH)p and fluoride stimulation in reconstituted activities, suggesting that both activities are conferred by the same component.

Human platelet adenylate cyclase: persistent binding of guanine nucleotide is central to the regulation of both hormone-stimulated and basal activities.

Prostaglandin E1 stimulation of human platelet adenylate cyclase, in purified plasma membranes, occurs without the addition of exogenous GTP. Possible contamination of the adenylate cyclase assay mixture by GTP either from nonspecifically bound nucleotide in the plasma membrane or from the substrate ATP was ruled out as follows: (a) variation of the membrane concentration, repeated washing, inclusion of EDTA, GDP beta S, or GMP in the wash step, or UDP in the assay, are all without effect, and (b) analysis of the substrate by high-performance liquid chromatography revealed no contaminating GTP. Other prostaglandins (I2, E2, D2) also activate cyclase without the addition of GTP. In sharp contrast, stimulation of adenylate cyclase in the human neutrophil plasma membrane by prostaglandin E1 shows an obligatory requirement for GTP, under identical assay conditions. GDP beta S pretreatment amplifies the fold cyclase stimulation by GTP in the presence and absence of prostaglandin E1, by lowering the basal activity. This alteration occurs without lowering the GTP-independent prostaglandin E1 activation, and is specific for inhibitory guanine nucleotides (GDP beta S, GMP, GDP) in the pretreatment. Extensive washing with buffer or incubation with other nucleotides, epinephrine, or prostaglandin E1 prior to the assay, is without effect. GTP gamma S treatment of the membrane induces a high-activity state and abolishes the GDP beta S effect on basal activity as well as prostaglandin E1 activation of cyclase. The results suggest distinct patterns of prostaglandin stimulation in platelet and neutrophil cyclase systems, and further imply that guanine nucleotide, prebound to specific sites within the GTP-regulatory proteins, may modify the kinetic characteristics of platelet adenylate cyclase.

Receptor-specific threshold effects of cyclic AMP are involved in the regulation of enzyme release and superoxide production from human neutrophils.

We have investigated the sequence of events leading from the activation of adenylate cyclase and increases in intracellular cyclic AMP to the modulation of enzyme release and superoxide production in human neutrophils. In the isolated plasma membrane, adenylate cyclase is activated by both prostaglandin E1 and isoproterenol. In the whole cell only a small increase in cyclic AMP is observed, though in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine a substantial amplification in intracellular cyclic AMP is observed with both isoproterenol and prostaglandin E1. These conditions are relevant to the regulation of cell function, since fMet-Leu-Phe-stimulated superoxide production is inhibited by either prostaglandin E1 or isoproterenol in the absence of methylisobutylxanthine, while enzyme release is inhibited only via the prostaglandin E1 receptor and then only in the presence of methylisobutylxanthine. For enzyme release and superoxide production, the order of potency for three prostaglandins tested was prostaglandin E1 greater than prostaglandin D2 much greater than prostaglandin F2 alpha. Our results suggest that (a) superoxide production is more sensitive to regulation by cyclic AMP than enzyme release, (b) the type of receptor occupied as well as the threshold level of cyclic AMP attained are important to the regulation of enzyme release, and (c) although elevation in cyclic AMP is inhibitory to neutrophil function, phosphodiesterase inhibition is required in addition to adenylate cyclase activation to effect maximal inhibition.

Effects of phospholipase A2 and filipin on the activation of adenylate cyclase.

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.

Regulation of low-density lipoprotein receptors and assessment of their functional role in Burkitt's lymphoma cells.

The status of low-density lipoprotein receptors (LDLR) in Daudi Burkitt's lymphoma (BL) cells was examined using a flow cytometric assay employing the fluorescent ligand DiI-LDL, and a radioligand-binding assay using [125I]LDL. The binding is concentration-and time-dependent; and is specific, as judged by its competitive displacement in the presence of unlabeled LDL, and inhibition by heparin, EGTA, and 4 degrees C incubation. The regulation of the receptor and its functional role were then explored. Our results suggest the following: (a) In sharp contrast to normal peripheral blood lymphocytes, the LDLR levels in BL cells are basally elevated when cultured in fetal bovine serum (FBS) medium. (b) In accord with normal peripheral blood lymphocytes, incubation in lipoprotein-deficient serum (LPDS) medium further up-regulates the level of the receptor in BL cells, and co-incubation with LDL or 25-hydroxycholesterol down-regulates the receptor level. The magnitude of the up-regulation is significantly smaller than in normal peripheral blood lymphocytes. (c) Northern blots using a plasmid-DNA probe for LDLR mRNA point to a similar pattern for message regulation as is observed in direct binding studies. (d) Although the LDLR level is constitutively high in BL cells, availability of LDL, unlike transferrin, is not a growth requirement since incubation of cells in LPDS medium does not prevent proliferation of these cells. (e) In contrast to anti-transferrin receptor antibody which results in apoptosis upon binding, anti-LDLR antibody does not inhibit growth or induce apoptosis. Our results suggest LDLR is expressed at a significantly higher level in BL cells than in normal peripheral blood lymphocytes. Although up-regulation and down-regulation of LDLR are observed, this applies only to a small population of LDLR. The bulk receptor population is significantly resistant to down-regulation. Furthermore, notable differences in the functional role of the LDLR are found relative to the transferrin receptor which is also up-regulated in the BL cells.

Peptide mapping studies of the pertussis toxin substrate in human neutrophils, platelets and erythrocytes.

Protease digestion of the ADP-ribosylated pertussis toxin substrate (PTS) protein was carried out after solubilization with SDS (Cleveland gels) and in the intact membrane. Cleveland gel analysis showed substantial similarities in the maps for the PTS component in neutrophils, platelets and erythrocytes and also in the S49 AC-lymphoma cell line. In the intact membrane ADP-ribosylation followed by digestion showed limited access of proteases to the PTS component. Of eight proteases tested, only papain and Staphylococcus aureus gave substantial digestion. This pattern was observed in the human platelet, erythrocyte and neutrophil plasma membranes. When the sequence was reversed and ADP-ribosylation was carried out after protease digestion, a very different pattern was observed with much greater susceptibility to digestion being noted with several proteases. By contrast, analysis of the murine AC-membrane showed some minor variations in the digest patterns. In addition, under all three conditions tested, maps of the cholera toxin substrate for the human platelet showed remarkable similarities to those obtained with the pertussis toxin substrate. Our results indicate that the protease sensitive sites of the alpha subunit of PTS and protection from proteolysis after ADP-ribosylation are properties which are shared by the PTS components of human platelets, erythrocytes and neutrophils.

Regulation of human neutrophil guanylate cyclase by metal ions, free radicals and the muscarinic cholinergic receptor.

We have examined the properties of soluble guanylate cyclase activity in the human neutrophil. The enzyme showed complex regulation by metal ions. A 10-fold higher activity was observed in the presence of Mn2+ than Mg2+, while Ca2+ caused an increase in activity only in the presence of Mg2+ ion. Sodium nitroprusside (SNP), azide and hydrogen peroxide were activators of the enzyme. Dithiothreitol blocked the activation by SNP, suggesting the involvement of thiol groups in the activation process. Carbachol acting through the muscarinic cholinergic receptor caused a dose-dependent activation, which was blocked by atropine. Higher concns of carbachol were required to activate guanylate cyclase than were required for the modulation of enzyme release elicited by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Nordihydroguaracetic acid inhibited carbachol stimulation of guanylate cyclase. By contrast, trifluoperazine (TFP), a calmodulin antagonist, caused a biphasic modulation of basal activity in the presence or absence of carbachol. Our results indicate that: allosteric interactions of metal ions are important to the regulation of the enzyme, the free radical nitroxide as well as hydrogen peroxide enhances enzyme activity, agonist occupancy of the muscarinic cholinergic receptor activates neutrophil guanylate cyclase probably through a mechanism involving calcium influx and the activation of the lipoxygenase pathway, and a TFP-sensitive site (possibly calmodulin) is involved in the selective regulation of basal enzyme activity.

Identification of receptor regulatory proteins, membrane glycoproteins, and functional characteristics of adenylate cyclase in vesicles derived from the human neutrophil.

Human neutrophils were disrupted by brief sonication under conditions which preserve the hormone sensitivity of adenylate cyclase and yield minimal granule lysis. Fractions enriched in adenylate cyclase were analysed for hormonal and guanine nucleotide regulation of the enzyme as well as structural proteins. Adenylate cyclase was activated by PGE1 and isoproterenol in a GTP-dependent fashion, while f-met-leu-phe and C5a gave no stimulation. Cholera toxin treatment, which specifically modifies cyclase-related GTP-binding proteins, caused a dose-dependent enhancement of GTP activation, in which GTP alone activated maximally and PGE1 was without further effect. The following proteins were detected in the cyclase-containing vesicles: a 42 K mol. wt protein labeled selectively by cholera toxin; protein subunits observed in SDS gels at 214, 165, 105 and 47 K, of which the 47 K band was the most prominent and comigrated with actin; prominent lectin-binding activities at 165 K (concanavalin A and wheat germ agglutinin) as well as at 100 K (wheat germ agglutinin); and a set of proteins and lectin-binding activities in fractions containing beta-glucuronidase activity distinct from adenylate cyclase containing vesicles. The identification of receptor-controlled cyclase, GTP-binding regulatory proteins, cytoskeletal elements and unique lectin-binding activities in a single vesicle preparation should contribute to an understanding of receptor-mediated control of neutrophil function.

A step sensitive to pertussis toxin and phorbol ester in human neutrophils regulates chemotaxis and capping but not phagocytosis.

Treatment of human neutrophils with pertussis toxin (PT) abolishes chemotaxis in response to either platelet-activating factor (PAF) or f-Met-Leu-Phe (FMLP), and capping induced via the concanavalin A (Con A) receptor. These functional effects are accompanied by the inhibition of calcium mobilization by PAF, FMLP and Con A. The agent phorbol 12-myristate-13-acetate (PMA) also inhibits chemotaxis and capping as well as calcium mobilization by these receptors. In sharp contrast, neither PT, cholera toxin (CT), nor PMA, inhibits the phagocytosis of non-opsonized and opsonized Candida albicans, sheep erythrocytes or fluorescent latex beads. Our results suggest that receptor-initiated chemotaxis and capping involve a step that is sensitive to PT and PMA, and that phagocytosis is not regulated in a similar fashion.

Flow cytometric evaluation of LDL receptors using DiI-LDL uptake and its application to B and T lymphocytic cell lines.

Low density lipoprotein receptors (LDL-R) on Daudi Burkitt's lymphoma (BL) cells were assessed using fluorescent DiI (3,3'-dioctadecylindocarbocyanine iodide)-LDL and flow cytometric analyses. Receptor-specific binding of DiI-LDL is followed by internalization of the bound complex and lysosomal hydrolysis of the ligand. Increase in the fluorescence intensity per cell is hence used as a measure of DiI-LDL uptake and, implicitly, as an indication of LDL-R presence. Our results show that uptake was observed in > 98% of the Daudi cells, and the level of uptake was significant and clearly distinguishable from autofluorescence, suggesting that: (a) this assay is comparable to the iodinated LDL uptake assay, although the ED50 values for the ligands are different; (b) this assay is comparable to the flow-cytometric detection of LDL-R using a commercial antibody directed against the receptor itself, and superior to a similar assay based on an antibody directed against membrane-bound LDL; (c) LDL uptake could be monitored along with transferrin uptake, suggesting that multiple endocytic receptor activities can be concurrently studied; (d) DiI-LDL uptake can be examined along with fluorescein-conjugated anti-CD10, -CD19, and -CD71, with little cross-interference, offering the added advantage that endocytic uptake and phenotyping can be simultaneously monitored; (e) the expression of LDL-R is intrinsically elevated in diverse cell lines such as Daudi, Raji, Ramos, Jurkat, and WIL2-NS, but not in normal lymphocytes. Our results therefore indicate that flow cytometric analysis of DiI-LDL uptake has potentially useful applications in the detection and study of endocytic receptor LDL-R in B and T lymphocytic cell lines.

A method for the preparation of mononuclear cells devoid of platelet contamination and its application to the evaluation of putative alpha-receptors in normal and asthmatic subjects.

Receptor studies of human mononuclear leukocytes (MNLs) are complicated by the presence of contaminating platelets which have common receptors. A method was devised to produce MNLs free of platelets (less than 1%) and consists of sequential Ficoll-Hypaque gradients, a BSA gradient and a washing step. Lack of platelet contamination was confirmed by the following criteria: (a) microscopic evaluation using fluorescent dyes showed less than 1% platelets; (b) PGE1 stimulation of the leukocyte membrane adenylate cyclase required addition of exogenous GTP while the platelet cyclase did not; (c) immunoblots of the cells and membranes using antibodies strongly reactive against platelet membranes showed no reactivity against MNL membranes; (d) [3H]yohimbine showed no binding in MNL membranes under conditions where substantial binding to platelets was detected. MNLs were viable as judged by dye exclusion. PHA stimulation of lymphocytes was unimpaired. Plasma membranes of MNLs were prepared by brief sonication and fractionation on a sucrose step gradient. Binding studies using 3H-DHE, an alpha-receptor ligand, revealed no binding in MNLs from normal subjects (n = 6). By contrast, studies on cells from subjects with mild asthma with medication appropriately withheld (n = 8) showed low levels of binding (60-300 fmol/10(6) cells). The subtype and functionality of the putative alpha-receptors are being further evaluated.

Multiple forms of the G protein-related beta subunit in Daudi lymphoblastoid cells.

We have explored the forms of the G protein-related beta subunit which are present in Daudi lymphoblastoid cells. Northern blotting with labeled beta-1 and beta-2 probes indicates that two messages of 3.3 kb and 1.7 kb are present for both beta-1 and beta-2, implying that multiple forms of the beta subunit are present. Antibodies were raised against two peptides of the beta subunit (residues 1-23 and 127-145). Both antibodies detected subunits at 35 kDa and 31 kDa, of which the 35-kDa form predominates in the membrane fraction and the 31-kDa one in the cell cytosol. Crosslinking of the membrane fraction with the cleavable crosslinker (DTSSP) caused a simultaneous diminution in the 31-kDa form while increasing the amount of the 35-kDa form--a pattern which was reversed upon the reduction of these crosslinks with DTT. Studies of the soluble form indicate that this is truly a soluble protein since centrifugation at 200,000 x g for 2 h did not diminish the levels of the protein in the soluble fraction. Sedimentation analysis indicates that the soluble beta-homologue is found in fractions which overlap with those which contain the mu chain of immunoglobulin at a position clearly distinct from the expected positions of free mu or free beta. Our results suggest that at least two forms of a subunit which is closely related to, or identical with, the beta subunit of G proteins are present in Daudi cells.

Induction of FMLP-mediated calcium mobilization requires crosslinking of surface immunoglobulin in Daudi cells.

We have explored the requirements for the induction of the N-formyl-methionyl-leucyl-phenylalanine (FMLP) response in Daudi cells after anti-immunoglobulin treatment. Our results indicate that (a) induction of responsiveness to FMLP was observed in Daudi only after crosslinking of surface immunoglobulin by anti-immunoglobulin; (b) this induced responsiveness was not observed in Ramos or Wil-2 cells; (c) the F(ab')2 fragment was sufficient for the induction of the FMLP response, but the Fab fragment and the Fc fragment were ineffective; (d) of the many agents active in B lymphocyte regulation which were tested, none were as effective as anti-immunoglobulin in the induction of the FMLP response; and (e) three inhibitors of calcium mobilization (W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide), PMA (phorbol 12-myristate 13-acetate), and colchicine), acting on distinct mechanisms, inhibited both the calcium mobilization due to anti-immunoglobulin and the induction of responsiveness to FMLP. Our results suggest important determinants in the induction of a calcium-mobilizing FMLP response in cells of B lymphocyte lineage include (a) the cell type, (b) a selective requirement for activation via surface immunoglobulin, and (c) crosslinking of the surface immunoglobulin.

Synergy between signal transduction pathways is obligatory for expression of c-fos in B and T cell lines: implication for c-fos control via surface immunoglobulin and T cell antigen receptors.

Expression of the protooncogene c-fos is controlled by three main regulatory pathways involving kinase C, cAMP, and calcium. Kinase C mediates its effects via phosphorylation of serum response factor (SRF) which interacts with the serum response element (SRE); cAMP and calcium mediate their effects via phosphorylation of CREB (cAMP regulatory element binding protein) presumably by activation of a protein kinase A or calmodulin-regulated kinase. We have examined the function of these elements in Burkitt's lymphoma cells (Ramos and Daudi) as well as a T lymphocytic cell line (Jurkat). We have found that stimulation of any one of these pathways alone has little or no effect on c-fos induction. However, kinase C activation (PMA stimulation) combined with either cAMP (forskolin plus MIX) or calcium stimulation (ionophore) leads to greatly enhanced c-fos induction. By contrast, cAMP in the presence of calcium shows no synergy in c-fos induction. Okadaic acid augments PMA- as well as calcium-mediated activation of c-fos, and has little or no effect when combined with cAMP. The main difference between Ramos (B cells) and Jurkat (T cells) in the regulation of c-fos is that cAMP plus calcium is strongly synergistic in Jurkat and is without effect in Ramos. Analysis of AP-1 activity using gel mobility shift assays confirms that the requirements for synergy in c-fos mRNA induction are paralleled by requirements for synergy in induction of AP-1 activity. Signaling in B cells due to anti-Ig stimulation involves both kinase C activation and release of intracellular calcium, and results in c-fos mRNA induction. Our results indicate that synergy between the kinase C activation and calcium is needed for efficient c-fos induction since neither of these two alone induces c-fos well. That synergy of signaling pathways is relevant for the anti-Ig induction of c-fos is supported by the fact that cAMP-inducing agents and okadaic acid further enhance anti-Ig induction of c-fos. These results suggest that cell-specific patterns of synergy are an essential feature for c-fos induction and may be relevant for c-fos control through B and T cell antigen receptors.

Dimethyl sulfoxide does not trigger urine histamine release in interstitial cystitis.

OBJECTIVES: Dimethyl sulfoxide (DMSO), an agent that provides symptomatic relief in patients with interstitial cystitis (IC) works via an unknown mechanism. We investigated whether DMSO acts as a chemical stimulant of mast cell degranulation. METHODS: A radioimmunoassay (RIA) specific for histamine was used to test this hypothesis. Twelve women with strictly diagnosed IC were treated with intravesical instillations of DMSO. Treatments were repeated at varying intervals, and each patient received three to six treatments. Urine histamine levels were measured before and after each intravesical instillation of DMSO. Dilutional effects of DMSO were corrected for by conversion of urine histamine concentration to urine histamine:creatinine ratio. RESULTS: The RIA was unaffected by the addition of DMSO to urine. No consistent change in the urine histamine:creatinine ratio following DMSO instillation was found. Trend analysis revealed no trend in the histamine:creatinine ratio with time. CONCLUSIONS: The relief of symptoms reported in 50% to 77% of patients treated with intravesical DMSO is not related to detectable mast cell release of histamine. Other mechanisms of action must be investigated to explain the beneficial effect of this agent.

Chemoattractant agents and nerve growth factor stimulate human spermatozoal reactive oxygen species generation.

OBJECTIVE: To investigate the ability of N-formyl-methionyl-leucyl-phenylalanine (f-MLP), complement 5a (C5a), and nerve growth factor (NGF) to stimulate human spermatozoal reactive oxygen species generation in fertile and infertile patients. DESIGN: Prospective, controlled study measuring human spermatozoal reactive oxygen species generation after addition of f-MLP, C5a, or NGF. SETTING: A large health maintenance organization. PATIENTS, PARTICIPANTS: The fertile group consisted of 14 men with established fertility and normal bulk semen parameters. The infertile group was comprised of 8 men who were infertile after > 18 months of unprotected sexual intercourse. INTERVENTIONS: The sperm samples were subjected to four test conditions: f-MLP stimulation, C5a stimulation, NGF stimulation, and no stimulation (control). MAIN OUTCOME MEASURE: Reactive oxygen generation was measured over a 15-minute period using the method of chemiluminescence. RESULTS: In both the fertile and infertile groups, reactive oxygen species generation was significantly enhanced by f-MLP, C5a, and NGF compared with controls. No significant difference in f-MLP- and C5a-stimulated reactive oxygen production was demonstrated between the infertile and fertile groups; however, there was a significant difference in reactive oxygen generation between infertile and fertile subjects when stimulated with NGF. CONCLUSIONS: The current study represents the first report of f-MLP-, C5a-, and NGF-stimulated reactive oxygen species generation by human spermatozoa. Nerve growth factor enhanced reactive oxygen species production to a greater extent in infertile subjects compared with fertile subjects. This points to a possible NGF-mediated biochemical defect in the sperm of infertile patients.

Distribution of prostatic acid phosphatase isoenzymes in normal and cancerous states.

We have studied the IEF (isoelectric focusing) profiles and the sedimentation characteristics of intracellular and secretory prostatic acid phosphatase (PAP) in normal and cancerous states. IEF studies show a similar relative distribution of tartrate inhibitable pI 4.9 (approximately 80%) and 5.6 (approximately 20%) forms of this enzyme in normal as well as cancerous prostate. The same IEF profile is obtained regardless of whether an enzymatic or RIA method is utilized for detection of PAP. Of these two isoenzymes, only the form of pI 4.9 predominates in prostatic and seminal fluids and in Stage IV serum. Sedimentation analysis shows that the purified enzyme is exceptionally stable since it retains an S020,w value of 5.7 at low concentrations (ng/ml). While only the 5.7S form is observed in normal and cancerous tissues as well as in prostatic fluid, analysis of Stage IV serum reveals an additional form at 8.7S. Control experiments suggest that the 8.7S form is not induced by non-specific association with normal serum proteins or by the inhibitor tartrate. Our results suggest that: (a) of the two major isoenzymes in tissue, only the pI 4.9 isoenzyme predominates in secretion, (b) this relationship of intracellular to secretory forms is unaltered in the transition from normal to cancerous tissue, and (c) the utility of PAP as a tumor marker is derived at least in part by the intrinsic stability of the 5.7S form. The significance of the 8.7S form is unknown at the present time, but it does not distort the clinical (RIA) measurement of PAP in serum.