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PURPOSE: To evaluate the possible role of cytokines interleukin (IL)-1beta, IL-1alpha and IL-6 in patients with urolithiasis. MATERIALS AND METHODS: Fifty-six patients currently with stone disease, 63 patients with bacterial cystitis, and 66 normal individuals were evaluated for urinary IL-1alpha, IL-1beta and IL-6. Clean catch urine samples were obtained and evaluated for cytokine levels using enzyme immunoassays for the respective cytokines. Statistical analysis of the results was carried out using the Kruskal-Wallis test followed by Newman-Keuls test and the chi-squared test. RESULTS: The patients with stone disease had significant elevations in IL-6 (p value< 10(-7)) relative to normal subjects. The levels of IL-6 in stone patients were lower than those of patients with bacterial cystitis. Neither IL-1beta nor IL-1alpha was elevated in stone patients relative to normals. By contrast, bacterial cystitis patients showed significant elevations in all three cytokines relative to normal subjects. Chi-squared analysis confirmed that stone patients had elevated IL-6 without elevation in either IL-1alpha or IL-1beta relative to normal subjects. CONCLUSIONS: Stone patients show significant elevations in IL-6 without marked increases in either IL-1beta or alpha relative to normal subjects. This elevation in IL-6 is not from infection as is seen in bacterial cystitis subjects. The elevation in IL-6 may be useful in the understanding of the pathogenesis of urolithiasis or as a potential marker for stone disease and we are currently investigating these possibilities.

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A variety of drugs have been used to treat B-lymphocyte neoplasms, including both cell cycle-specific (CCS) and non-cell-cycle-specific drugs. Although the therapy for such cancers is complex and can include both types of drugs, the efficacy of these drugs in inducing cell death remains unclear. In this paper we have concentrated on specific CCS drugs and have examined their ability to induce programmed cell death (apoptosis) in Burkitt's lymphoma cell lines derived from patients. The CCS drugs chosen were hydroxyurea and aphidicolin (active in late G1, early S phase), the topoisomerase poisons camptothecin and etoposide (S, early G2 phase) and vincristine and Taxol (late G2, M phase). These choices allow comparison of two drugs with differing modes of action for each of the various phases of the cell cycle. Our results indicate that the variation in apoptosis between drugs that act at the same phase of the cell cycle is negligible. Both S/G2 and G2/M blockers are very potent at inducing apoptosis whereas G1/S blockers are ineffective in the induction of apoptosis. In addition, marked kinetic variations in the rate of apoptosis induction were observed, etoposide and camptothecin being more rapid in their action than the other agents. The order of effectiveness in inducing apoptosis on a kinetic basis was S/G2 agents >> G2/M agents >> G1/S agents. In this study we have also found that growth inhibition was induced by all the CCS agents chosen and by anti-IgM in various Burkitt's lymphoma lines. Furthermore c-myc was down-regulated under similar conditions. Since apoptosis was only selectively induced by some of the CCS agents, it implies c-myc expression is associated with growth regulation and c-myc down-regulation is an insufficient condition for the induction of apoptosis. In addition, cotreatments using the CCS and other agents revealed the following: Cotreatment using two CCS drugs which act at the same stage in the cell cycle showed either no change or only additivity to the effects seen with either agent alone. However, cotreatment with CCS drugs showed that an inhibitory effect is found between G1/S and G2/M drugs or S/G2 and G2/M drugs. No effect was found between G1/S and S/G2 drugs. Anti-IgM, which by itself was capable of inducing apoptosis, was observed to augment apoptosis induced by very low concentrations of G2/M-acting drugs but it has little effect on G1/S or the S/G2 drugs. The inhibitory effect of anti-CD40 or TNF-alpha on anti-IgM-induced apoptosis did not carry over to an effect on apoptosis induction by the CCS agents. Thus specific combinations of agents may lead to either enhancement, inhibition, or no interactive effect on apoptosis.

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OBJECTIVES: to determine IL-1alpha and IL-1beta levels in patients with bacterial cystitis, microscopic hematuria, and gravid females relative to a control group of normal subjects. METHODS: enzyme immunoassays were used to measure concomitantly urinary IL-1alpha and IL-1beta in clean catch urine samples from normal subjects (n = 31) and study patients (n = 46). All normal subjects and patients underwent urinalysis, urine culture, and urine creatinine level determination. Since the IL-1alpha assay was developed for serum, the utility of the assay for urine specimens was unknown. The key parameters of urine collection, processing and sample storage for IL-1alpha were evaluated in detail. RESULTS: mean values +/- SEM (pg/mg) for IL-1alpha/ Cr and IL-1beta/Cr were control group (0.25 +/- 0.10 and 0.17 +/- 0.06), bacterial cystitis (9.97 +/- 1.15 and 42.45 +/- 1.86), and microscopic hematuria (2.81 +/- 0.65 and 2.82 +/- 0.70). Differences in cytokine levels between the control group and patients with either bacterial cystitis or microscopic hematuria were statistically significant for both IL-1alpha/Cr (P < 0.026; P < 0.007, respectively) and IL-1beta /Cr (P < 0.0004; P < 0.014, respectively). IL-1beta/Cr correlates better with pyuria than IL-1alpha/ Cr (P = 0.02 vs P = 0.44). In gravid females, only IL-1alpha was significantly elevated relative to non-pregnant females (IL-1beta elevation approached statistical significance). Gravid females with positive urine cultures could not be distinguished from those with negative cultures based on either interleukin (P > 0.05). CONCLUSIONS: Significant elevations of IL-1alpha and IL-1beta occur in patients with bacterial cystitis and microscopic hematuria. Correlation between pyuria and cytokine elevation was stronger for IL-1beta than for IL-1alpha. Changes in IL-1alpha may reflect changes in the bladder epithelium rather than in the inflammatory leukocytes. The ability of IL-1alpha and IL-1beta to serve as markers for bacterial cystitis in gravid females is diminished due to high basal levels during pregnancy.

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PURPOSE: We have examined urinary cytokine levels to define the inflammatory response in patients with bacterial cystitis or microhematuria relative to normal subjects. Cytokines examined include interleukin-1beta (IL-1beta), IL-1alpha, tumor necrosis factor (TNF alpha), IL-6 and IL-4. Unique features of this study include a) a simultaneous study of several relevant cytokines b) a study of the inflammatory response at both low and high counts of bacterial infection and c) an assessment of whether microhematuria without bacterial cystitis or pyuria is associated with cytokine elevation compared to normals. MATERIALS AND METHODS: Enzyme immunoassays were utilized for each cytokine. Patients studied include those with bacterial cystitis (n = 49), patients with microhematuria (n = 11), and normal subjects (n = 36). Cytokine levels were also determined for patients with low count bacterial cystitis (1,000-50,000 organisms; n = 15) and compared to high count bacterial cystitis (>100,000 organisms; n = 34) and normal subjects. Statistical analysis was carried out using the Kruskal-Wallis test followed by pairwise testing with Newman-Keuls test. RESULTS: a) The means for normal, microhematuria and bacterial cystitis groups were significantly different (p <0.05) for IL-1beta, IL-1alpha, TNF alpha and IL-6, but not for IL-4. b) Except for IL-4, all cytokines were found to be significantly elevated in low count bacterial cystitis compared to normals. No statistically significant difference was observed between low and high count bacterial cystitis groups for any of the cytokines tested. CONCLUSIONS: a) Significant and similar inflammatory responses are present in both low and high count bacterial cystitis groups as compared with the normal group. b) IL-6 and TNF alpha are significantly elevated in patients with microhematuria compared to normals. c) The potential clinical utility of the assays lies in identifying the specific cytokines elevated, understanding the pathways that give rise to their production, and in defining potential virulence factors that may produce significant inflammation at low count bacterial infections.

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Ornithine decarboxylase (ODC) is a key enzyme involved in polyamine production and is thought to regulate growth and apoptosis in multiple cell systems. A potential link between ODC and growth may involve the action of an oncogene c-myc which is thought to transcriptionally regulate ODC. We have examined the involvement of ODC in anti-IgM-induced growth inhibition and apoptosis in Burkitt's lymphoma cells. Inhibitors of ODC such as difluoromethylornithine (DFMO) completely blocked ODC activity, resulting in growth inhibition but not apoptosis. Addition of putrescine, the product of ODC enzymatic action, to Ramos cells had only a minor effect on growth, did not cause apoptosis, did not augment or block anti-IgM-mediated growth inhibition and apoptosis, but did reverse DFMO-mediated growth inhibition. Anti-IgM treatment of Ramos cells, which markedly decreased c-myc mRNA and protein, caused a paradoxical increase in ODC mRNA level as well as ODC enzymatic activity and increased cellular levels of putrescine. DFMO and putrescine did not alter c-myc mRNA levels directly, nor did they have any affects on anti-IgM-mediated down-regulation of c-myc mRNA. TNF-alpha, which inhibited anti-IgM-mediated apoptosis, did not inhibit either anti-IgM or DFMO-mediated inhibition of growth. These agents were without effect on ODC activity itself or on the anti-IgM-mediated increase in ODC activity. From these studies we conclude that ODC inhibition affects growth but is unrelated to the induction of apoptosis. Both anti-IgM-mediated inhibition of growth and induction of apoptosis are independent of ODC. Thus two distinct pathways for growth regulation are present: one in which ODC and polyamines are important and the other cell surface receptor-mediated (sIg) which is independent of ODC and polyamines.

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Anti-IgM treatment of Burkitt's lymphoma cells is followed by either growth arrest or induction of apoptosis. In this study we have explored the role of c-myc in these events. Our results in Ramos cells indicate the following. (a) The decline in c-myc mRNA occurs at about 4 h; inhibition of about 80% being observed. (b) The stability of c-myc message is involved since the half-life of c-myc mRNA is decreased from about 30 min in untreated cells to about 15 min following treatment with anti-IgM. In the presence of cycloheximide, a protein synthesis inhibitor, the half-life is increased to about 50 min and was unaltered by treatment with anti-IgM. (c) By contrast, nuclear run-on experiments indicated no change in transcription rates for c-myc message due to treatment with anti-IgM. (d) A decrease in c-myc causes apoptosis since specific repression of c-myc with antisense oligonucleotides decreases the levels of c-Myc, inhibits growth rate, decreases viability, and induces apoptosis. (e) Anti-CD40 inhibition of apoptosis occurs without alteration in anti-IgM-induced down-regulation of c-myc mRNA, suggesting that it acts distally to c-myc down-regulation. Other cell lines were also investigated. In Epstein-Barr virus (EBV)-positive cell lines (Daudi, Raji, and Namalwa), anti-IgM treatment for 24 h results in growth inhibition without induction of apoptosis. In EBV-negative cell lines (ST486 and CA46, as well as Ramos), a more heterogeneous pattern of responses to anti-IgM are observed. Ramos and ST486 cells both show growth inhibition and apoptosis upon anti-IgM treatment; CA46 cells shown only growth inhibition but not apoptosis. Anti-IgM causes a decline in c-myc mRNA levels in all of these lines, as well as in c-Myc protein level in the two lines investigated, Daudi and Ramos, regardless of apoptosis. Addition of antisense c-myc oligonucleotides to the cells reduced growth in both Daudi and Ramos cells lines, however it resulted in substantial apoptosis only in Ramos cells. These results suggest that anti-IgM destabilizes c-myc mRNA by a process that involves mRNA turnover, rather than transcription rates. However anti-IgM exerts differential effects in EBV-positive and EBV-negative cell lines. EBV-positive cells are uniformly resistant to apoptosis, while EBV-negative cell lines show a tendency to apoptosis but with exceptions. Growth inhibition can be uncoupled from apoptosis in EBV-positive cell lines, but not in those EBV-negative cell lines prone to apoptosis. Furthermore, down-regulation of c-myc message correlates with growth inhibition in these cells, but is an insufficient link to apoptosis. By contrast inhibition of apoptosis by anti-CD40 occurs even though c-myc mRNA is decreased.

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We have examined the effects of tumor necrosis factor-alpha (TNF-alpha) as an inducer or modulator of necrosis and/or apoptosis in B cell lines. TNF-alpha does not induce either necrosis or apoptosis in EBV-positive or -negative cell lines, regardless of the culture conditions of the cells or the presence or absence of cytokines. By contrast anti-IgM induces apoptosis in two EBV-negative cell lines (Ramos and ST486) but not in EBV-positive cell lines. Since TNF receptor and CD40 belong to the TNF receptor superfamily and anti-CD40 is a known inhibitor of apoptosis, we tested for TNF-alpha's effects on the inhibition of apoptosis induced by anti-IgM. Our results indicate that TNF-alpha inhibits apoptosis induced by anti-IgM in Ramos cells but not in ST486. The effects are dose and time dependent; the degree of apoptosis achieved and the selectivity of the effect among cell lines are strikingly similar for both TNF-alpha and anti-CD40. Furthermore when both agents are tested together no additivity in the inhibition is observed. The inhibition of apoptosis is a direct effect of TNF-alpha and not a permissive effect of another cytokine, since it is observed in defined medium. Although anti-IgM induces both TNF-alpha secretion and TNF receptors in Ramos cells, the concentration of secreted TNF-alpha is too low to affect apoptosis. Inhibition does not involve perturbation of the cell cycle distribution of Ramos cells. Furthermore rapid induction of c-fos and the decrease in c-myc observed after anti-IgM treatment are both unaltered by TNF-alpha. Our results suggest that TNF-alpha is an inhibitor of apoptosis in Ramos cells, that its overall pattern of inhibition is similar to that of anti-CD40, and that both agents act at some point distal to the alteration of c-fos and c-myc by anti-IgM.

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Four tandem subtilisin-like protease genes were found on a 6,854-bp DNA fragment cloned from the alkalophilic Bacillus sp. strain LG12. The two downstream genes (sprC and sprD) appear to be transcribed independently, while the two upstream genes (sprA and sprB) seem to be part of the same transcript.

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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.

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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.

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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.

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Previous reports have suggested that dimethyl sulfoxide (DMSO) may be a useful reversible G1 arresting agent for synchronizing Raji Burkitt's lymphoma cells (K. Takase et al. (1992) Cell Growth Differ. 3, 515-521; M. Sawai et al. (1990) Exp. Cell Res. 187, 4-10). We have therefore critically evaluated several aspects of DMSO's effects using Daudi and Ramos Burkitt's lymphoma (BL) cells. In BL cells starved in the presence or absence of DMSO for 4 to 6 days (approximately four to six doubling times), the following observations were noted: (A) Both Daudi and Ramos cells show increased cell synchrony accompanied by apoptosis when starved in RPMI 1640 supplemented with 10% fetal calf serum (FCS). Inclusion of 1.5% DMSO causes a diminution in apoptosis with minimal effects on synchrony. (B) Lowering the FCS concentration to 5% induces apoptosis. DMSO-mediated protection from apoptosis is observed in Daudi but not in Ramos. (C) When human serum (10%) is used instead of FCS, Daudi cells show no apoptosis and DMSO is without effect on cell cycle distribution. By contrast, Ramos cells show significant apoptosis, which is prevented by the inclusion of DMSO. (D) When starved in a chemically defined medium (AIM-V), both Daudi and Ramos cells show significant apoptosis. DMSO protects Ramos from apoptosis under these conditions. (E) Upon removal of DMSO, both Daudi and Ramos cells reenter the cell cycle but with significant apoptosis. (F) The protective effect of DMSO from apoptosis is observed in a narrow range of concentration between 1 and 2%. At higher concentration, DMSO itself induces apoptosis. These results suggest that DMSO itself prevents apoptosis, an effect which may present as an apparent effect on cell synchrony.

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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.

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In order to identify groups essential for the activity of endo-beta-N-acetylglucosaminidase H (Endo H), all 8 glutamate residues, all 19 aspartates, and both tryptophans were individually substituted with glutamines, asparagines, and phenylalanines, respectively, by oligonucleotide site-directed mutagenesis. Only variants D170N, D172N, and E174Q were found to have specific activities significantly less than wild-type Endo H. Another variant, D173N, did not produce detectable amounts of protein. Wild-type enzyme was found to have a bell-shaped pH activity profile, which was retained in the essential aspartate mutants, but E174Q lost the basic pH limb of the curve, indicating that E174 is good candidate for the proton donating group necessary for catalysis. The general base needed for activity could not be unambiguously identified; although, of the essential aspartates, D172 is the only one conserved in other related glucosidases.

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Urinary cytokines as markers for the intravesical inflammatory response have become an active area of research. Interleukin-1b, a well studied and early produced cytokine in the immune recognition cascade, was evaluated. After extensive analysis of 56 control and study group urine samples, a simplified and reliable protocol for the preparation of urine before cytokine analysis was devised. The application of an available serum interleukin-1b enzyme-linked immunosorbent assay kit for urinary interleukin-1b analysis was then tested. Finally, a reference value for interleukin-1b in normal human urine was established and urinary interleukin-1b was measured in various bladder diseases. The normal and interstitial cystitis groups showed no interleukin-1b elevation. Significant elevation of urinary interleukin-1b was found in patients with bacterial cystitis compared to the interstitial cystitis and control groups (p < 0.001). Of the patients with bladder tumors 58% showed elevation of urinary interleukin-1b (p < 0.001). Urinary interleukin-1b may be used as a marker to distinguish between bacterial and interstitial cystitis. The absence of urinary interleukin-1b in interstitial cystitis argues against an immunological or autoimmune etiology of the disorder. This study represents an important methodological approach to cytokine subtyping of bladder diseases.

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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.

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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.

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To investigate the possible similarities between the reactive oxygen generating systems of the leukocyte and the spermatozoa, and to identify possible biochemical differences between fertile and infertile patients, the effect of various stimulants on the production of reactive oxygen in fertile, fertile with varicocele and infertile patients was examined. Generation of reactive oxygen species by human spermatozoa was examined in 2 patient groups: a fertile group (14), which included 7 patients with a palpable varicocele, and an infertile group (16) composed of 7 patients with a palpable varicocele and 9 who remained infertile 6 months to 3 years (mean 20 months) after internal spermatic vein ligation. Various known stimulants of reactive oxygen generation in leukocytes were used to assess spermatozoal reactive oxygen production. Reactive oxygen species generation was measured by the technique of chemiluminescence. In the infertile group reactive oxygen generation was markedly increased by the calcium ionophore A23187 and the chemoattractants N-formyl-methionyl-leucyl-phenylalanine and complement 5a (p < 0.05). In addition, fertile patients with varicoceles showed significantly enhanced chemoattractant stimulated reactive oxygen generation compared to fertile donors without varicoceles. This finding of a biochemical alteration in the sperm of infertile patients and patients with varicoceles may lend support to the argument for early varicocele repair.

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