Pramod M. Lad

Anti-IgM-mediated Regulation of c-myc and Its Possible Relationship to Apoptosis

Anti-IgM treatment of Burkitts 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.

Stimulated Reactive Oxygen Species Generation in the Spermatozoa of Infertile Men

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.

Stimulated release of urine histamine in interstitial cystitis.

The diagnosis of interstitial cystitis is primarily made based on clinical and cystoscopic findings with exclusion of other bladder diseases. Despite all of the efforts at definitive identification, interstitial cystitis lacks universal objective findings. Mast cell activation with associated histamine release has been postulated as an etiological factor leading to the symptom complex associated with interstitial cystitis. To investigate this hypothesis, a 3-step controlled prospective study was conducted. In step 1 reliability of urine histamine assay was critically examined, and the assay was established to be simple, reliable and valid. In step 2 random spot urine histamine levels (basal state) were measured in 25 noninterstitial cystitis and 15 interstitial cystitis patients (22.1 +/- 0.95 ng./ml. versus 19.2 +/- 1.19 ng./ml.). There was no significant difference in the random urine histamine levels between the 2 groups (p greater than 0.05). In step 3 urine histamine levels were measured before and after hydrodistention (acute stimulation) in 7 noninterstitial cystitis controls and 6 newly diagnosed interstitial cystitis patients under general anesthesia. The urine histamine-to-creatinine ratio was used to correct for the dilutional effect of normal saline used during hydrodistention. The urine histamine-to-creatinine ratios of the control group showed no significant difference before and after hydrodistention. However, the difference in the urine histamine-to-creatinine ratios of the interstitial cystitis group compared to the controls before and after hydrodistention was highly significant (p less than 0.001). Although measurement of random spot urine histamine alone (basal state) was not found useful to make the diagnosis of interstitial cystitis, measurement of urine histamine before and immediately after hydrodistention (acute stimulation) may become an important objective parameter to assist in the diagnosis of interstitial cystitis.

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.

Multiple Urinary Cytokine Levels of Bacterial Cystitis

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.

Interleukin-1B: a clinically relevant urinary marker.

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.

URINARY IL-6 IS ELEVATED IN PATIENTS WITH UROLITHIASIS

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.

The characteristics of lubrol-solubilized adenylate cyclase from rat liver plasma membranes

Abstract Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) was solubilized from rat hepatic plasma membranes with Lubrol PX. The enzyme could be stabilized during solubilization by pretreating the membrane with glucagon and 5′-guanylimidodiphosphate (Gpp(MH)p) or Gpp(NH)p alone but not by glucagon alone or in the presence of GTP or other guanine nucleotides. The solubilized enzyme was purified 3–4-fold by molecular exclusion chromatography on Ultrogel AcA22; the presence both 0.01% Lubrol and 25% sucrose were required to maintain both the solubility as well as stability of the enzyme. The enzyme was purified from 70% of the other solubilized membrane proteins and 85% of the solubilized phospholipids, and was completely separated from 5′-nucleotidase. In addition, the enzyme was purified 2-fold with respect to non-specific nucleotide phosphohydrolase and pyrophosphohydrolase activities but not with respect to ‘specific’ GTPase activity. No perceptible change in molecular size of partially purified adenylate cyclase was observed after it had been pre-activated in its membrane-bound form with hormone and Gpp-(NH)p. The solubilized enzyme also retained its ability to be activated by the guanine nucleotide analogues Gpp(NH)p and Gp(CH2)pp with similar concentration and time-dependent characteristics displayed by the membrane-bound form of the enzyme. Attempts to further purify the solubilized enzyme by a variety of size- and charge-based chromatography techniques proved unsuccessful. As an explanation for this phenomenon we suggest that, after solubilization with Lubrol, the enzyme is probably but a fraction of the total protein in the complex. That is, judging from the highest specific activity obtained for the partially purified liver enzyme (1.2 nmol./min per mg) and assuming that its turnover number is that of the highly purified bacterial adenylate cyclase [54], the enzyme comprises less than 0.1% of the protein present in the adenylate cyclase-containing fraction from the Ultrogel column. The idea of determining the physical properties of Lubrol-solubilized adenylate cyclase becomes even more difficult when one considers that properties, such as Stokes radii on Ultrogel columns depend upon the concentrationof detergent used in the column elution buffer. For example, in the absence of detergent we have observed that Lubrol-solubilized adenylate cyclase aggregates and elutes in the void volume of the column. Thus, knowledge of the aggregation characteristics of the enzyme further suggests that caution must be used in assigning values to the intrinsic physical properties of the enzyme.

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.

Proteolysis activates adenylate cyclase in rat liver and AC− lymphoma cell independently of the guanine nucleotide regulatory site

Adenylate cyclase activity is normally under the control of modulating factors such as various hormones, neutrotransmitters, cations and adenosine. Guanine nucleotides seem to be necessary for the expression of stimulatory as well as inhibitory regulations, probably via binding to two distinct nucleotides binding proteins (Ns and Ni, respectively) [l-3]. Mild proteolysis can also produce an activation of the adenylate cyclase of rat liver plasma membranes [4-71, cultured fibroblasts [8,9], kidney cells [lo], rat ovarian membranes [ 11 ,121 and rat cerebral cortical membranes [ 131. In some cases, the stimulatory effect of proteolysis was more marked, or uniquely observed,in the presence ofGTP [8-10,141. Here, we demonstrate that proteolysis activates cyclase in rat liver plasma membrane and in the ACmutant of the S49 lymphoma cell line, independently of the GTP regulatory component (which will be refered to as N).