Myeloperoxidase-catalyzed chlorination of histamine by stimulated neutrophils.

To investigate neutrophil interactions with mediators released by mast cells at sites of inflammation, stimulated neutrophils were incubated with histamine. No accumulation of chlorinated histamine derivatives was detected in the medium. Instead, histamine inhibited the formation of chloramine derivatives of other amines. Incubation with radiolabeled histamine resulted in rapid uptake of label into the cells, and most of the label could be extracted and recovered as histamine. About 3% of the label taken up was incorporated into acid-precipitable forms. Uptake depended on myeloperoxidase (MPO)-catalyzed formation of chlorinating agents. Uptake was promoted by adding MPO and blocked by the MPO inhibitor dapsone, catalase, scavengers for hypochlorous acid and chloramines, or in a low-chloride medium, but not by histamine receptor antagonists. Incubation of histamine with MPO, hydrogen peroxide, and chloride resulted in formation of mono- and dichloramine derivatives of the primary amino group. Above pH 7.0, the chloramines were primarily in uncharged, lipophilic forms as indicated by partitioning into organic solvents. Histamine is a cation at neutral pH, but chlorination eliminated the charge on the amino group and shifted the pKa of the imidazole ring, resulting in formation of neutral histamine-chloramines. Incubation of neutrophils or other blood cells with radiolabeled histamine-chloramines resulted in rapid uptake of label, indicating membrane permeation by the uncharged, lipid-soluble forms. Incubation with labeled histamine-dichloramine also resulted in acid-precipitable incorporation. The results indicate that MPO-catalyzed chlorination of histamine could modulate histamine activity, tissue distribution, and metabolism at sites of inflammation.

Ultraviolet spectral energy differences affect the ability of sunscreen lotions to prevent ultraviolet-radiation-induced immunosuppression.

Acute exposure to UV radiation causes immunosuppression of contact hypersensitivity (CH) responses. Past studies conducted with unfiltered sunlamps emitting nonsolar spectrum UV power (wavelengths below 295 nm) or using excessive UV doses have suggested sunscreens may not prevent UV-induced immunosuppression in mice. This study was thus designed to evaluate critically the effects of different UV energy spectra on the immune protection capacity of sunscreen lotions. Minimum immune suppression doses (MISD), i.e. the lowest UV dose to cause approximately 50% suppression of the CH response to dinitrofluorobenzene in C3H mice, were established for three artificial UV sources. The MISD for each UV source was 0.25 kJ/m2 for unfiltered FS20 sunlamps (FS), 0.90 kJ/m2 for Kodacel-filtered FS20 sunlamps (KFS), which do not emit UV power at wavelengths < 290 nm, and 1.35 kJ/m2 for a 1000 W filtered xenon arc lamp solar simulator. Using MISD as baseline, sunscreens with labeled sun protection factors (SPF) of 4, 8, 15 and 30 were tested with each UV source to establish their relative immune protection factors. The immune protection factor of each sunscreen exceeded its labeled SPF in tests conducted with the solar simulator, which has a UV power spectrum (295-400 nm) similar to that of sunlight. Conversely, sunscreen immune protection factors were significantly less than the labeled SPF in tests conducted with FS and KFS. Comparison of the immunosuppression effectiveness spectra showed that relatively small amounts of nonsolar spectrum UV energy, i.e. UVC (200-290 nm) and/or shorter wavelength UVB (between 290 and 295 nm), produced by FS and KFS contributes significantly to the induction of immunosuppression. For example, 36.3% and 3.5% of the total immunosuppressive UV energy from FS and KFS, respectively, lies below 295 nm. Sunscreen absorption spectra showed that transmission of immunosuppressive UV energy below 295 nm for FS was at least eight-fold higher than that for KFS. Compared to the solar simulator UV spectrum the transmission of nonsolar immunosuppressive UV energy through sunscreens was > 15-fold higher for FS and > or = 1.5-fold higher for KFS. These data demonstrate that relevant evaluations of sunscreen immune protection can only be obtained when tests are conducted with UV sources that produce UV power spectra similar to that of sunlight and UV doses are employed that are based on established MISD.

Minimum doses of ultraviolet radiation required to induce murine skin edema and immunosuppression are different and depend on the ultraviolet emission spectrum of the source.

Many photo immunological studies have used UV radiation sources that emit nonsolar UV spectral energy and UV doses based on nonimmunological endpoints, e.g. erythema and skin edema. Interpretation of these data has led to misunderstanding when extrapolated to hypothetical effects in humans exposed to solar UV. The purpose of this study was to: (1) establish UV dose response relationships for murine skin edema and immunosuppression, and (2) determine how different UV spectra affect these relationships. Back skin and ear minimum edema doses (MEdD) for Kodacel-filtered FS20 sunlamp UV (290-400 nm) were greater than two-fold higher than those for unfiltered FS20 sunlamp UV (250-400 nm). Xenon are solar simulator UV (295-400 nm) MEdD were > 10-fold higher than those for unfiltered sunlamp UV. Back skin and ear MEdD differed two- to five-fold between C3H/HeN, SWR/J and HRA/Skh-1 mice. The minimum immunosuppression doses (MISD) in C3H mice showed similar UV source spectrum dependence. The solar simulator UV MISD was 5.4- and 1.5-fold higher than for unfiltered and Kodacel-filtered sunlamp UV MISD, respectively. Furthermore, MISD were from 3- to 50-fold higher than the MEdD for the three UV sources. The UV bioeffectiveness spectra indicated that UVC energy (250-290 nm) contributed 12% and 18%, respectively, of the total skin edema and immunosuppression UV energy. These data demonstrate the variability in UV sensitivity among mouse strains, the significant differences between murine MEdD and MISD and how these differences are influenced by nonsolar regions (below 295 nm) of the UV spectrum.

The ultraviolet C energy emitted from FS lamps contributes significantly to the induction of human erythema and murine ear edema.

FS ultraviolet (UV) lamps are used extensively to study the biological effects of ultraviolet radiation. Using published data, we investigated the relative contributions of the UVC (250-289 nm), UVB (290-319 nm), and UVA (320-400 nm) portions of the FS lamp emission spectrum to the induction of human erythema and murine ear edema. Many investigators contend that the biological activity of the spectrum emitted from these lamps resides primarily in the UVB region, based on the proportions of the power emission spectrum in this region (53.6%) versus the small relative contribution of UVC (3.2%) and the low biological activity of UVA (43.2%). However, if the biological effectiveness spectrum of FS lamps is calculated by multiplying the power spectrum with different action spectra, the biological effects of UVC are readily observed. For example, 10.4% of the murine ear edema activity and 11.1% (Parrish) or 16.7% (McKinlay-Diffey) of the human erythemal activity is due to the energy emitted in the UVC region. Experimental determination of human erythema and murine ear edema demonstrated that, for an equal amount of energy delivered, radiation from the unfiltered lamps was more potent in causing these responses than radiation from filtered lamps, and the ratio of effectiveness could be predicted by the effectiveness spectra. Thus, the contribution of UVC emitted from FS lamps to biological effectiveness spectra should not be ignored.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of systemic cyclosporin A on a hairless mouse model of photoaging.

The mechanisms that cause skin wrinkling in response to chronic exposure to sunlight are unknown. We investigated the possibility that wrinkling of Skh-1 hairless mice is associated with an ultraviolet (UV) radiation-induced immunologic alteration. Exposing Skh-1 hairless mice to a regimen of nonerythemal UV-B (290-320 nm) radiation induced skin wrinkles after 6-7 weeks. Concomitant treatment with cyclosporin A decreased the time to the onset of wrinkles to approximately 4 weeks. Exposing HRS/J hairless mice or athymic nude mice to a similar nonerythemal UV-B radiation regimen for 10 weeks failed to induce skin wrinkles. Concomitant administration of cyclosporin A and UV-B radiation for 7 weeks to HRS/J hairless mice induced no skin wrinkles. Ultraviolet-B or UV-B plus cyclosporin A exposure caused increased immunohistochemical staining for Ia and F4/80 antigens in the upper dermis of tissue from Skh-1 mice, as compared to controls. Treating Skh-1 mice with UV-B radiation plus cyclosporin A was also associated with a large increase in the number of CD3+ cells in the dermis. These staining patterns were absent in similarly treated HRS/J hairless mice. Dermal mast cell numbers in Skh-1 mice were 2-3-fold higher than in HRS/J, athymic nude or NSA mice. Treatment with cyclosporin A increased Skh-1 dermal mast cell numbers approximately 2-fold but had no effect on the dermal mast cell numbers in HRS/J or NSA mice. Based on these findings we postulate that UV-B light and cyclosporin A exacerbate an immunological condition in Skh-1 mice, one consequence of which is manifested as skin wrinkles. Thus, the induction of skin wrinkles in this mouse strain may have no relevance to the wrinkles observed in human skin after chronic exposure to sunlight.

The hairless mouse model of photoaging: evaluation of the relationship between dermal elastin, collagen, skin thickness and wrinkles.

Quantitative and qualitative changes in dermal collagen and elastin occur in response to chronic ultraviolet (UV) irradiation. These changes have been implicated in the genesis of the wrinkling seen in chronically irradiated, or photoaged skin. We examined the relationship between wrinkle formation and changes in dermal structural protein content and type. Skh-1 hairless mice were irradiated with suberythemal doses of UV-B three times a week for up to 20 wk. Visible wrinkling was present after 6-7 wk of irradiation. Dermal elastic fiber content was quantified by color image analysis of paraffin-embedded tissue. There was no significant difference in dermal elastic fiber content between irradiated and age-matched control mice after either 10 or 20 wk of irradiation. The effect of UV-B irradiation on total dermal collagen content, ratio of collagen type III-type I, and extent of glycosylation and crosslinking of collagen was no different in irradiated and age-matched control mice after 10 wk of irradiation. Increased epidermal thickness was evident in frozen sections after 6 wk of irradiation, and the thickness increased with continued irradiation. Dermal thickening was evident after 10 wk of irradiation. Sufficient UV-B irradiation will eventually cause changes in dermal elastin and collagen content; however, wrinkle formation precedes such changes. A causal relationship between wrinkle formation and dermal structural protein content changes in Skh-1 hairless mice could not be established in this study.

Inhibition of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase by dapsone.

Dapsone (4,4'-diaminodiphenylsulfone) is an antimicrobial substance that also has anti-inflammatory activity, which has been attributed to inhibition of the leukocyte enzyme myeloperoxidase (MPO). We observed that dapsone was a much better inhibitor of the eosinophil peroxidase (EPO) in an assay that measured peroxidase-catalyzed oxidation of tetramethylbenzidine at pH 5.4. To clarify the specificity and pH-dependence of dapsone inhibition of the purified enzymes under more physiologic conditions, we studied peroxidase-catalyzed oxidation of chloride to the antimicrobial and cytotoxic agent hypochlorous acid. Taurine was added as a trap for hypochlorous acid, to prevent inactivation of the enzymes or chlorination of dapsone by hypochlorous acid. Dapsone was much more effective as an inhibitor of both MPO and EPO when chloride rather than tetramethylbenzidine was the substrate. Inhibition of both enzymes was greater at neutral pH than at acid pH (pH 7 vs pH 5), but EPO was more sensitive to inhibition than MPO regardless of pH. Inhibition was increased by lowering chloride, raising hydrogen peroxide, or lowering the enzyme concentration. Inhibition was accompanied by irreversible loss of enzyme activity, which was correlated with loss of the heme absorption spectrum, indicating chemical modification of the enzyme active site. EPO, but not MPO, was partially protected against inactivation by adding physiologic levels of bromide along with chloride. The results suggest that dapsone could prevent MPO- and EPO-mediated tissue injury at sites where the peroxidase enzymes are secreted and diluted into the neutral pH environment of the tissue interstitial space. Dapsone might not inhibit peroxidase-mediated antimicrobial activity, which occurs at high enzyme concentrations in the acid environment of phagolysosomes.

Quantitative determination of murine dermal elastic fibers by color image analysis: comparison of three staining methods.

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Kligman's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.

Numbers of murine dermal mast cells remain unchanged during chronic ultraviolet B irradiation.

Dermal mast cell numbers reportedly increase in response to chronic ultraviolet irradiation in both humans and in the HRS/Skh-1 mouse model of human photoaging. It has been hypothesized that these increased numbers of mast cells are responsible, at least in part, for the damage in this chronically irradiated or photoaged skin. However, few actual quantitative data have been reported to support this claim of increased dermal mast cell numbers caused by chronic ultraviolet irradiation. We sought to quantify the numbers of dermal mast cells in the skin of chronic ultraviolet-irradiated and control HRS/Skh-1 hairless mice. Dermal mast cells from irradiated and age-matched control mice were quantified by digital image analysis during a 20-week period of exposure to ultraviolet B (UVB) radiation. During the entire course of irradiation, there was no difference in the numbers of dermal mast cells between the irradiated and nonirradiated age-matched control mice. Visible physical evidence of the effects of chronic UVB irradiation, i.e., skin wrinkling, was evident after 6 weeks of treatment. The numbers of dermal mast cells in unirradiated age-matched NSA (CF-1) haired mice were three- to four-fold lower than those in either ultraviolet-exposed or unexposed HRS/Skh-1 mice. These findings indicate that dermal mast cell numbers in HRS/Skh-1 mice are not increased by chronic exposure to UVB radiation.

Taurine and hypotaurine content of human leukocytes.

Taurine (T) was reported to be at high concentrations in human leukocytes. It was proposed that T is a scavenger for chlorinated oxidants produced by the myeloperoxidase system of monocytes and neutrophils. Hypotaurine (HT) would be a more effective scavenger, and HT could also detoxify products of bromide or iodide oxidation produced by the eosinophil peroxidase system. Methods previously used to measure T in leukocytes might oxidize HT to T or fail to separate T and HT. Therefore, we examined T and HT content, uptake, and biosynthesis in isolated blood cells and cultured tumor cells derived from hematopoietic/lymphoid cells. Platelets and all leukocytes including monocytes, lymphocytes, neutrophils, and eosinophils had high T levels (10-20 mM), and all except eosinophils had substantial HT levels (0.3-1 mM). Intracellular levels were 500-times higher than in plasma. Erythrocytes were the only blood cells with low levels of both T and HT. Tumor cells from lymphoid (CCRF-CEM) and myeloid (HL-60, K-562, RWLeu4, HEL) lineages took up and concentrated T and HT from the bovine calf serum in the culture medium, and intracellular levels were similar to those in leukocytes. When cells were cultured in HT-supplemented media, HT almost completely replaced T, and HT was not converted to T. Levels of T were not raised by culturing cells with possible precursors, but HT levels were raised when cysteine sulfinic acid was present. Washed tumor cells took up T and HT by way of a beta-amino acid transport system, but uptake by leukocytes was very low. Therefore, leukocytes may acquire T and HT by active uptake rather than biosynthesis, and uptake may be completed during differentiation in the bone marrow. Though HT is low relative to T, HT levels may be sufficient to protect leukocytes from toxic oxidants.

Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase.

Conditions were optimized for measuring the activity of myeloperoxidase (MPO) and the eosinophil peroxidase (EPO) with tetramethylbenzidine (TMB) as the substrate. Detergents caused a small increase in the measured activity of the purified enzymes and were required when isolated neutrophils or eosinophils were assayed. Sharp concentration optima were observed with both ionic and non-ionic detergents. Activity was also influenced by halide ions. Bromide or iodide caused up to a 7-fold increase in EPO activity and a 1.5-fold increase in MPO activity. The effect of bromide is notable because the bromide-containing detergent CETAB is often used to extract the enzymes for assay and purification. Stimulation by bromide or iodide was consistent with peroxidase-catalyzed oxidation of the halides to hypohalous acids (HOBr and HOI), which oxidized TMB. MPO catalyzes the oxidation of chloride to hypochlorus acid (HOCl), which also oxidized TMB, but chloride up to 20 mM had little effect on the assay. Both MPO and EPO catalyze thiocyanate oxidation, but the product (HOSCN) was a poor oxidant for TMB, and thiocyanate inhibited the measured activities. Stimulation by bromide or iodide could be used to facilitate detection of EPO and to distinguish between MPO and EPO. Activities could also be distinguished based on the greater sensitivity of EPO to inhibition by thiocyanate, azide, aminotriazole, and dapsone. Methods reported here may prove useful for measuring leukocyte influx into inflamed tissues, detecting MPO or EPO deficiencies, and measuring enzyme synthesis and secretion.

Inhibition of tumor cell glutamine uptake by isolated neutrophils.

Antitumor activity of phorbol myristate acetate-(PMA) stimulated neutrophils was measured against CCRF-CEM cells. Neutrophils and tumor cells were incubated (a) as a suspension with continuous mixing to maximize the availability of oxygen or (b) after centrifugation as a pellet to maximize cell-cell contact. The cells were then incubated briefly as a suspension with [14C]glutamine under conditions that blocked further damage to the tumor cells. When cells were incubated as a suspension, inhibition of tumor-cell glutamine uptake was mediated by the myeloperoxidase/hydrogen peroxide/chloride system of stimulated neutrophils. Inhibition was blocked by adding catalase, an inhibitor of myeloperoxidase, or compounds that scavenge hypochlorous acid or chloramines. When cells were incubated as a pellet, a portion of the inhibition could not be blocked in this way, indicating that a nonoxidative mechanism contributed to inhibition. In both systems, inhibition of glutamine uptake was rapid and was obtained at effector-cell/target-cell ratios as low as 0.5:1. This inhibition was obtained under conditions that did not result in 51Cr release from cells labeled with [51Cr]-chromate, indicating that inhibition of glutamine uptake measured cytotoxicity rather than cytolysis. 51Cr release was observed only when cells were incubated together for an hour or more as a pellet at high E/T ratios. This cytolysis was mediated by the myeloperoxidase system, and a nonoxidative contribution to cytolysis was not observed. The results indicate that stimulated neutrophils are potent antitumor effectors cells when cytotoxicity rather than cytolysis is the measure of activity. Because glutamine is required for growth of many tumor cells, inhibition of glutamine uptake may represent a significant tumoristatic or tumoricidal effect.

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.

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils.

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.

Hypochlorite scavenging by Pseudomonas aeruginosa alginate.

Alginic acid was purified from a mucoid clinical isolate of Pseudomonas aeruginosa. Luminol-dependent chemiluminescence of phorbol myristate acetate-stimulated neutrophils was inhibited by this alginate, but oxygen consumption was unaffected. Further studies indicated that this effect was due to the ability of the pseudomonal alginate to scavenge hypochlorite. A seaweed alginate was less effective and dextran T500 was ineffective in hypochlorite scavenging. It appears that the uronic acid core and the O-acetyl groups of pseudomonal alginate are involved in its hypochlorite-scavenging ability. The relevance of this phenomenon was demonstrated by the greater resistance to killing by hypochlorite of mucoid P. aeruginosa compared with a nonmucoid revertant, and the addition of purified alginate to the nonmucoid revertant protected the organism from hypochlorite. Thus, this extracellular polysaccharide may enhance the virulence of P. aeruginosa by scavenging the phagocyte-generated oxidant HOCl. This enhanced virulence may be involved in disease processes in which mucoid organisms predominate, such as cystic fibrosis.

Mutagenic activity of chloramines.

Mutagenesis by chloramines and hypochlorous acid (HOCl) was studied to determine whether these agents could contribute to the mutagenic and potentially carcinogenic activity of stimulated leukocytes and whether environmental exposure to these agents is a cause for concern. Mutagenic activity was measured using the S. typhimurium TA97a, TA100 and TA102 tester strains. Because chloramines and HOCl are bactericidal, react rapidly with cell components, and can destroy the histidine and biotin required for the mutagenesis assay, activity can't be compared directly with that of less toxic or reactive agents. Nevertheless, chloramines were mutagenic when tested under appropriate conditions. TA100 was the most sensitive strain, and the most active mutagens were lipophilic dichloramines (RNCl2) including derivatives of histamine, ethanolamine and putrescine. Lipophilic monochloramines (RNHCl) such as histamine-monochloramine and NH2Cl were less active. Hydrophilic chloramines such as taurine-chloramines had low activity, and HOCl was inactive. The metabolic state of the bacteria was critical. Chloramines were mutagenic when added to bacteria with glucose at 37 degrees C, but killing predominated when chloramines were added at 4 degrees C or 25 degrees C, or at 37 degrees C without glucose. Production of chloramines and HOCl by leukocytes in vivo could contribute to the association of chronic inflammation and cancer as a result of: (1) the entry of membrane-permeable chloramines into normal cells followed by attack on intracellular components including DNA, and (2) the production of secondary mutagens such as compounds with carbonyl groups or carbon-chlorine bonds. On the other hand, chlorination of water supplies is perhaps more likely to destroy than create mutagens, and chloramines from the environment are unlikely to penetrate the skin and mucous membranes.

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.

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.