Incorporation Of Tritiated Leucine Research Articles

Activities and Mechanism Based Combinations Of Omacetaxine In Acute Myeloid Leukemia

Omacetaxine, an inhibitor of translation, was recently granted accelerated approval for the treatment of chronic myeloid leukemia (CML). Omacetaxine blocks translation elongation by competing with the incoming aminoacyl-tRNAs for binding to the A-site cleft in the peptidyl-transferase center. Our previous studies showed that by transiently inhibiting translation, omacetaxine reduced the expression of the key, short-lived oncoproteins Bcr/Abl and Mcl-1, leading to cell death in the CML cells. This action sensitized the cells to the Abl kinase inhibitor and killed the CML cells synergistically. Further, as omacetaxine acts in a different mechanism than the Abl kinase inhibitors, it overcame resistance to TKI that was associated with kinase domain mutations. These studies paved the foundation for the clinical development of omacetaxine in CML. We also demonstrated that omacetaxine was active in chronic lymphocytic leukemia by translational inhibition of Mcl-1 expression. In contrast to normal tissues, the fact that the leukemia cells are critically dependent on the oncogene activity for survival provided a biologic context for a positive therapeutic index. As the biological features of acute myeloid leukemia (AML) rely largely on the overexpressed oncoproteins or constitutively activated kinases, we hypothesized that omacetaxine would have therapeutic benefit in AML either alone or in mechanism based combinations.To test this hypothesis, first, we compared omacetaxine to AC220, a potent FLT3 inhibitor, in AML cell lines OCI-AML3 and MV4-11. OCI-AML3 cells harbor the signature mutation of NPM1, whereas MV4-11 is a widely used model for the internal tandem duplications of FLT3 (FLT3-ITD), a common FLT3 mutation that constitutively activates the receptor tyrosine kinase. AC220 was selectively toxic to the MV4-11 cells, but had no effect on the viability of OCI-AML3. This is consistent with the biological context of MV4-11, but not OCI-AML3, that is addicted to the sustained activity of FLT3 for survival. In contrast, omacetaxine induced apoptosis in both cell lines with IC50s less than 100 nM. Protein synthesis was inhibited in both lines, measured by the incorporation of tritiated leucine. Apoptosis was induced rapidly within 24 h by omacetaxine, whereas AC220 required 72 h to kill the leukemia cells. These results indicated a common dependence on the continued protein synthesis in the AML lines, suggesting a potentially broad application of omacetaxine in AML patients with diverse genetic backgrounds. Over-expression of the anti-apoptotic protein Mcl-1 is associated with AML disease maintenance and resistant to therapy. Both Mcl-1 and FLT3 turn-over rapidly and are vulnerable targets of transient translation inhibition. Immunoblots showed that omacetaxine reduced the levels of both FLT3 and Mcl-1 in the MV4-11 cells. This activity augmented the effect of AC220 on FLT3 kinase, and induced synergistic apoptosis. Same synergistic combination was observed with omacetaxine and sunitinib, an inhibitor of FLT3, KIT and PDGF-R. Dose reduction index derived from these analyses showed that omacetaxine greatly potentiated the activity of both AC220 and sunitinib, resulting in profound apoptosis.Both Bcl-2 and Mcl-1 are pro-survival proteins that regulate apoptosis by interacting with the BH3 motifs of their pro-apoptotic partners. BH3 mimetics, such as ABT-199, bind with high affinity to Bcl-2 and block this interaction, but not to Mcl-1. Resistance to BH3 mimetics in AML cells is associated with upregulation of Mcl-1. Since ABT-199 inhibits Bcl-2 but spares Mcl-1, and omacetaxine reduces Mcl-1 without affecting Bcl-2 expression, we hypothesized that their combination would target the two parallel arms of apoptosis control and kill the AML cells synergistically. Indeed, omacetaxine reduced Mcl-1 in the OCI-AML3 cells, leading to loss of mitochondrial membrane potential and apoptosis. ABT-199 blocked Bcl-2 function and also induced the intrinsic pathway of apoptosis. Their combination induced greater mitochondrial damage and apoptosis than either drug alone. The median effect analysis showed that they potentiate each other and exhibited strong synergy. Taken together, these results demonstrated that omacetaxine is active in AML cells alone and in mechanism based combinations. These actions provide rationale that warrants investigation in the clinic. Disclosures:No relevant conflicts of interest to declare.

ASSA13-03-54 Adenosine A1 Agonist Inhibit the Hypertrophy of Cardiomyocyte Induced by Angiotensin II Through Calcineurin Pathway

<h3>Objective</h3> We aimed to study the mechanism and pathway of the adenosine A1 receptor agonist 2-chloro-N6 cyclopentyladenosine (CCPA) in the inhibition of the cardiomyocyte hypertrophy induced by angiotensin II (AngII) in vitro. <h3>Methods</h3> Cardiomyocytes from neonatal SD rats were treated with AngII to generate a cardiomyocyte hypertrophy model. The cell cultures were randomised into four groups: (1) control; (2) AngII; (3) AngII + cyclosporin A (CsA); and (4) AngII + CCPA. The MTT method was used to measure cardiomyocyte viability. Protein synthesis was monitored by measuring the incorporation of tritiated leucine (3H-Leu), and the expression of β-myosin heavy chain (β-MHC) and calcineurin Aβ (CnAβ) mRNA was measured by semi-quantitative RT-PCR. <h3>Results</h3> Forty-eight hours after AngII treatment, surface area, viability and 3H-Leu incorporation of the cardiomyocytes were significantly increased in a dose-dependent manner. Both CCPA and the calcineurin (CaN)-specific inhibitor CsA significantly inhibited AngII-induced cardiomyocyte 3H-Leu incorporation. These compounds also inhibited the mRNA expression of CnAβ and β-MHC in AngII-induced cardiomyocytes but had no significant effect on 3H-Leu incorporation in the control group. The inhibition by CCPA was dose-dependent within the range of 1 × 10–8 mol/L to 1 × 10–6 mol/L. <h3>Conclusions</h3> The adenosine A1 receptor agonist CCPA can significantly inhibit AngII-induced cardiomyocyte hypertrophy in a manner related to the CaN signalling pathway.

Bispecific targeting of EGFR and uPAR in a mouse model of head and neck squamous cell carcinoma

To investigate the efficacy of the bispecific targeted toxin, dEGFATFKDEL, on head and neck carcinoma cell lines in vitro and in vivo. A deimmunized bispecific anti-cancer agent was constructed to simultaneously target both the overexpressed EGF receptor on carcinomas and the urokinase receptor (uPAR), that is found on the endothelial cells of the neovasculature within tumors. Flow cytometry assays were performed to determine the level of EGFR expressed on a variety of carcinoma lines. These lines were then tested in tritiated leucine incorporation assays to determine the efficacy of dEGFATFKDEL. Human vein endothelial primary cells were also tested to determine the effectiveness of the ATF portion of the molecule that binds uPAR. Furthermore, mouse studies were performed to determine whether dEGFATFKDEL was effective at inhibiting tumor growth in vivo. UMSCC-11B and NA, two head and neck squamous cell carcinomas, highly expressed EGFR. Both the carcinoma lines and the human vein endothelial cells were inhibited at sub-nanomolar concentrations by dEGFATFKDEL. The tumor studies showed that the tumors treated with dEGFATFKDEL were significantly inhibited whereas the negative control and untreated tumors progressed. In a separate in vivo study involving another carcinoma line, MDA-MB-231, the effectiveness of dEGFATFKDEL was confirmed. No toxicity was seen at the doses used in either of these mouse studies. This bispecific agent is effective in a mouse model of head and neck squamous cell carcinoma. Further study of this reagent for use in the treatment of carcinomas is warranted.

In vitro effect of methyl farnesoate on the vitellogenin content of ovary and hepatopancreas, in the crayfish Cherax quadricarinatus

Vitellogenin levels were determined in pieces of either ovary or hepatopancreas taken from females of the crayfish Cherax quadricarinatus during both early pre-reproductive and reproductive periods, and exposed in vitro to 0.15, 1.5, or 15 µM of the juvenoid methyl farnesoate (MF). A significant accumulation of vitellogenin was seen in ovaries treated with 15 µM of MF during the early pre-reproductive period. Besides, protein synthesis, measured as tritiated leucine incorporation, was correspondingly increased in ovaries taken during both pre- and post-reproductive periods and exposed to 15 µM of MF. On the other hand, no stimulating effect of MF on the vitellogenin content of the hepatopancreas was seen in any period. These results, taken together, support the hypothesis of the endogenous vitellogenin synthesis stimulated by MF during the early ovarian maturation. However, this hormone was not able to stimulate vitellogenin synthesis in the ovary during the reproductive period.

Reducing Mcl-1 by Homoharringtonine Induces Apoptosis In Chronic Lymphocytic Leukemia Cells

AbstractAbstract 2896Homoharringtonine (HHT, omacetaxine) is an inhibitor of translation elongation that is currently in clinical evaluation in chronic myelogenous leukemia. We hypothesized that this compound would be active in inducing apoptosis in chronic lymphocytic leukemia (CLL) based on the following rationale: 1): CLL cells are characterized by their dependence on the overexpression of anti-apoptotic proteins to maintain their survival; 2): one of the key anti-apoptotic proteins, Mcl-1, turns over rapidly. Thus, upon transient exposure to a translation inhibitor, Mcl-1 expression would decrease rapidly and allow the pro-apoptotic proteins to activate the mitochondria-dependent apoptosis. Using tritiated leucine incorporation as an indicator of new protein synthesis, our results in primary CLL cells demonstrated that HHT was more potent at inhibiting translation than cycloheximide. A polysome profiling analysis confirmed that Mcl-1 translation was blocked by HHT. Mcl-1 protein levels were reduced significantly after HHT exposure, measured by an immunoblotting analysis. This led to mitochondrial membrane depolarization and initiation of apoptosis, analyzed by flow cytometry. Sensitivity to HHT varied in different CLL samples. Further studies showed that this heterogeneous response was independent of the CLL prognostic factors such as the mutation status of the IgVH or ZAP70 expression. Rather, it correlated significantly to the baseline Mcl-1 expression in each CLL sample and the extent of reduction of Mcl-1 protein. A detailed time course study showed that Mcl-1 reduction was evident as early as 2 h and continue to decrease in the next 6 to 8 h; cell death started in 2 h and continued to increase for 24-h. The reduction of Mcl-1 was not affected by the pan caspase inhibitor zVAD, indicating that reduction of Mcl-1 was the initiation event of apoptosis, rather than the results of caspase cleavage. On the contrary, the proteosome inhibitor MG-132 caused retention of the Mcl-1 level in the presence of HHT, suggesting that when Mcl-1 synthesis was blocked by HHT, the remaining Mcl-1 in the cells was degraded rapidly in a proteosome-dependent mechanism. As the action of HHT relied largely on the rapid turn-over of Mcl-1 protein, blocking Mcl-1 degradation by the proteosome inhibitor would counteract the effect of HHT. This explained the observed antagonistic reaction of HHT and MG-132 when they were used together. Further, interaction of CLL cells with the microenvironment has been shown to protect CLL cells from chemotherapy. Incubating CLL cells on either a murine or human stromal cell layer in vitro clearly protected CLL cells from the toxicity of fludarabine. There was an induction of both Mcl-1 transcript and protein levels, which could explain this protection. However, this induction was reversed by HHT, overcoming stromal cell-mediated protection. Finally, because of the important role of Mcl-1 in the pathogenesis CLL, we hypothesized a sequential blockade strategy that inhibits both Mcl-1 transcription and translation to completely block Mcl-1 synthesis. Median effect analysis showed that combining the transcription inhibitor SNS-032 together with HHT killed the CLL cells synergistically. An immunoblotting analysis demonstrated that the combination of the two drugs depleted Mcl-1 more completely than either single drug at the same concentration, which correlated to a greater induction of apoptosis than either drug alone. Taken together, our data demonstrated that transiently inhibiting translation and depleting Mcl-1 was the major mechanism of HHT-induced apoptosis in CLL cells. This action overcame stromal cell-mediated protection and was synergistic with inhibitors of transcription. Thus, these data provided rationale for the clinical development of HHT in CLL, and for the design of combination strategies. Disclosures:No relevant conflicts of interest to declare.

In vitro effects of both dopaminergic and enkephalinergic antagonists on the ovarian growth of Cherax quadricarinatus (Decapoda, Parastacidae), at different periods of the reproductive cycle

The in vitro effect of both spiperone (dopaminergic antagonist) and naloxone (enkephalinergic antagonist), was assayed on small pieces of ovary dissected from C. quadricarinatus females, with the eventual addition of some neuroendocrine organs, such as thoracic ganglion or eyestalk tissue. The incorporation of tritiated leucine by the ovary was measured in order to estimate the ovarian growth. During the post-reproductive period, both mentioned antagonists were able to significantly stimulate the ovary in the presence of thoracic ganglion, but did not produce any significant effect in the preparation containing ovary and eyestalk tissue, or only ovary. No significant effects of the assayed antagonists were noted during the pre-reproductive period. These results were in accordance with previous models describing the neuroendocrine control of crustacean reproduction, and represent new findings about the hormonal context in different periods of the reproductive cycle of crayfish. Besides, by means of the experimental combination of the tested antagonists with dopamine or met-enkephalin, a new model dealing with the interaction of these two neurotransmitters on the hormonal secretion of thoracic ganglion has been proposed.

AT1 receptor knockdown prevents the T3‐induced cardiomyocyte hypertrophy: modulation of the Akt Signaling Pathway

ObjectiveThe Renin Angiotensin System plays an important role in TH‐induced cardiac hypertrophy. We recently demonstrated the in vivo participation of Angiotensin II Type 1 receptor (AT1R) in TH‐induced cardiac hypertrophy. The aim of this study was to analyze the contribution of AT1R to the development of T3‐induced cardiomyocyte hypertrophy, as well as to evaluate the role that AT1R plays in the activation of the Akt/GSK‐3β/mTOR signaling pathway promoted by T3.MethodsCardiomyocytes cultures were prepared from ventricles of neonatal Wistar rats by colagenase/pancreatin method. The cells were transfected with a selective AT1R small interfering RNA (AT1R siRNA). The AT1R knockdown was evaluated by Real Time PCR and Western Blot. Protein synthesis was quantified by tritiated leucine incorporation. The Akt/GSK‐3β/mTOR signaling pathway was analyzed by Western Blot. The results were expressed as mean±SD and P0.05 (n=3).ResultsThe cells transfected with the AT1R siRNA presented reduced mRNA and protein expression of AT1R (P&lt;0.01). AT1R knockdown prevented the T3‐induced cardiomyocyte hypertrophy, as evidenced by the reduction in protein synthesis (P&lt;0.01). In addition, the AT1R siRNA diminished the T3‐induced activation of the Akt/GSK‐3β/mTOR (P&lt;0.01). These data demonstrate for the first time that the AT1R contributes to the trophic effect that T3 has on cardiomyocytes, as well as in the nongenomic T3 effect on the Akt/GSK‐3β/mTOR signaling pathway in cardiomyocytes.Financial Support: FAPESP.

Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death

Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Grégori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 microg ml(-1) of propidium iodide, simultaneous to a 10x concentration of Sybr green I, are best for detecting two separated populations of "live" (green cells) and "dead" (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.

Parathion effects on protein synthesis in the seminiferous tubules of mice

Agropesticides impart high risks of exposure for animals including humans. This work analysed the effect of parathion upon protein synthesis in seminiferous tubules cultured in vitro from adult male CF1 mice. They were incubated with parathion solutions (0.8, 0.4, 0.04, 0.004, 0.0004 mM) for 5 h. One hour after ending incubation tritiated leucine was added to each sample. In addition, groups treated with 0.8 mM parathion were incubated for 5 h, and thereafter transferred to a parathion-free medium; tritiated leucine incorporation was measured 1, 2, or 3 h afterwards. Results of protein synthesis denote an inhibitory dose-dependent effect of parathion. This is reversed after pesticide-free incubation. Testicular protein synthesis inhibition induced by parathion may compromise male fertility in view of the fact that, in spermatogenesis, proliferation (mitosis, meiosis) and cell differentiation occurs.

Mitochondrial regulation of insulin production in rat pancreatic islets

The study was designed to identify the key metabolic signals of glucose-stimulated proinsulin gene transcription and translation, focusing on the mechanism of succinate stimulation of insulin production. Wistar rat islets were incubated in 3.3 mmol/l glucose with and without esters of different mitochondrial metabolites or with 16.7 mmol/l glucose. Proinsulin biosynthesis was analysed by tritiated leucine incorporation into newly synthesised proinsulin. Preproinsulin gene transcription was evaluated following transduction with adenoviral vectors expressing the luciferase reporter gene under the control of the rat I preproinsulin promoter. Steady-state preproinsulin mRNA was determined using relative quantitative PCR. The mitochondrial membrane potential was measured by microspectrofluorimetry using rhodamine-123. Succinic acid monomethyl ester, but not other mitochondrial metabolites, stimulated preproinsulin gene transcription and translation. Similarly to glucose, succinate increased specific preproinsulin gene transcription and biosynthesis. The inhibitor of succinate dehydrogenase (SDH), 3-nitropropionate, abolished glucose- and succinate-stimulated mitochondrial membrane hyperpolarisation and proinsulin biosynthesis, indicating that stimulation of proinsulin translation depends on SDH activity. Partial inhibition of SDH activity by exposure to fumaric acid monomethyl ester abolished the stimulation of preproinsulin gene transcription, but only partially inhibited the stimulation of proinsulin biosynthesis by glucose and succinate, suggesting that SDH activity is particularly important for the transcriptional response to glucose. Succinate is a key metabolic mediator of glucose-stimulated preproinsulin gene transcription and translation. Moreover, succinate stimulation of insulin production depends on its metabolism via SDH. The differential effect of fumarate on preproinsulin gene transcription and translation suggests that these processes have different sensitivities to metabolic signals.

Effects of age on morphology, protein synthesis and secretagogue-evoked secretory responses in the rat lacrimal gland.

This study investigated changes in the morphology and protein synthesis and protein and peroxidase secretion due to peptidergic and aminergic stimulation from rat lacrimal gland acinar cells of 3-5, 9, 12, 20 and 24 month old rats. There was a marked reduction in the presence of Golgi apparatus in the acinar cells of glands from the 24 month old rats coupled to dilatation and degeneration of rough endoplasmic reticulum, when compared to that in the acinar cells of glands from 3-5 and 12 month old rats. Following incorporation of tritiated leucine for 360 min (6 h), the amount of newly synthesised protein in acinar cells of the 12 month old rats was significantly (p < 0.01) higher than that in the acinar cells of 3-5 month old animals. However, at 20 months the amount of newly synthesised protein in these acinar cells was significantly (p < 0.01) reduced to less than that in acinar cells of both the 3-5 and 12 month old animals. Immunohistochemical and immunofluorescence studies identified the presence of substance P (SP), vasoactive intestinal peptide (VIP), histamine and 5-hydroxytryptamine (5-HT) in the lacrimal glands of 3-5 month old rats. Stimulation by either SP, VIP, histamine or 5-HT resulted in significant increases in total protein output and peroxidase release from acinar cells of the 3-5 month old rats. However, all responses to the secretagogues were reduced with ageing from 3-5 to 24 months of age. The results indicate that ageing is associated with alteration in the ability of acinar cells to synthesise and secrete proteins.

Carbon flux in the northwest Mediterranean estimated from microbial production

Spring profiles of microbial production derived from the dark incorporation of tritiated leucine and tritiated thymidine in the northwest Mediterranean show an exponential decline with depth. Assuming this to represent a steady-state balance between microbial respiration and the downward flux of carbon, the downward flux is estimated as (1− ε/ ε) p/ b, where p is the microbial production, ε their gross growth efficiency and b the coefficient of exponential decline with depth. Summer profiles, ranging over about 3° of latitude and 4° of longitude, were well fitted by a two-component exponential decline, suggesting two distinct microbial substrates. Values of b for the more rapidly declining component varied between 0.01 and 0.06 m −1 according to location. In the case of the slower component, b was estimated as 0.002 m −1, and did not vary significantly over the region. Estimated fluxes of carbon at the surface are 123–335 mg m −2 d −1 for the fast and 95 mg m −2 d −1 for the slow component. Below about 200 m, carbon flux is dominated by the slow component. Flux estimates are compatible with flux estimates from sediment traps in the same region. The observed changes between the spring and summer profiles, combined with the horizontal homogeneity of the summer profiles below 200 m, are consistent with a downward transport of about 5–10 m d –1, implying a significant dispersive component to the observed fluxes.

Evidence of protein synthesis during resuscitation of ischemically damaged canine pancreas by the two-layer method.

The two-layer method, which supplies sufficient oxygen to the canine pancreas graft and allows adenotriphosphate synthesis within the graft, resuscitates the ischemically damaged pancreas graft during mild hypothermic (20 degrees C) preservation, but the mechanisms are unknown. The purpose of this study was to determine whether protein synthesis was performed on ischemically damaged pancreas graft during preservation by the two-layer method in a canine autotransplantation model. The pancreas grafts subjected to 90 minutes of warm ischemia were preserved by the two-layer method (perfluorochemical/University of Wisconsin solution) at 20 degrees C for 5 hours. Graft viability was judged from graft survival after autotransplantation. DNA, RNA, and protein synthesis was quantitated by determining incorporation of tritiated thymidine, tritiated uridine, and tritiated leucine, respectively, during preservation. Significant increases in RNA and subsequent protein syntheses were observed during preservation by the two-layer method. In contrast, DNA synthesis did not follow RNA and protein synthesis. We conclude that protein is synthesized in the process of postischemic cellular recovery of the pancreas graft during mild hypothermic preservation by the two-layer method.

A NONTOXIC DIPHTHERIA TOXIN ANALOGUE INHIBITS NEONATAL BLADDER SMOOTH MUSCLE CELL PROLIFERATION

The response of the neonatal bladder to infravesical obstruction, such as posterior urethral valves or detrusor-sphincter dyssynergia, may result in structural and functional changes that persist well after obstruction is treated. A pharmacological means of inhibiting smooth muscle cell proliferation would likely serve to halt or reverse this deleterious process. Heparin binding (HB) epidermal growth factor (EGF) is a known smooth muscle cell mitogen, while its membrane bound precursor serves as the diphtheria toxin receptor. We report the effects of the nontoxic diphtheria toxin analogue cross reacting material (CRM) 197 on neonatal sheep bladder smooth muscle cell proliferation. Neonatal sheep smooth muscle cell cultures were obtained from whole bladder explants. Immunohistochemical staining was performed for desmin and alpha-smooth muscle actin. HB-EGF messenger RNA was detected by reverse transcriptase polymerase chain reaction using primers to the human sequence, while pro-HB-EGF peptide was confirmed using a diphtheria toxin sensitivity assay with incorporation of tritiated leucine. Cells grown in 96 well plates were exposed to 1, 10 and 100 microg./ml. CRM 197 for 5 days, after which relative cell number was determined using an 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; thiazolyl blue based assay read at a wavelength of 550 nm. Statistical analysis was performed using Student's t test. Primary cell cultures stained positive for desmin and alpha-smooth muscle actin, confirming a smooth muscle origin. Reverse transcriptase polymerase chain reaction yielded a 453 bp product with 88% homology to human HB-EGF. Total protein synthesis significantly decreased when cells were incubated with diphtheria toxin, confirming the presence of membrane bound pro-HB-EGF. CRM 197 inhibited bladder smooth muscle cell growth in a dose dependent fashion at a concentration of 10 microg./ml., resulting in a 40% decrease in proliferation (p <0.0001). CRM 197 inhibits bladder smooth muscle cell proliferation in a dose dependent, nontoxic fashion through its interaction with HB-EGF. These data suggest that molecular strategies designed to inhibit HB-EGF mediated cell growth may prove beneficial for the prevention and/or treatment of detrusor hypertrophy secondary to anatomical or functional bladder outlet obstruction.

Carbamylated proteins activate glomerular mesangial cells and stimulate collagen deposition

Carbamylated proteins formed in renal insufficiency from the spontaneous decomposition of urea exert a variety of metabolic effects. Here we examined the effects of carbamylated proteins on glomerular mesangial cells to determine whether urea retention in early renal insufficiency may itself promote glomerular sclerosis and hasten the progression to kidney failure. To this effect we carbamylated fetal bovine serum proteins in vitro and tested their effect on mesangial cell proliferation (by tritiated thymidine uptake), de novo protein synthesis (by tritiated leucine uptake), collagen I and collagen IV accumulation (by avidin-biotin enzyme immunoassay), and gelatinase levels in the medium (by zymography and quantitative fluorescence assay). Carbamylated fetal bovine serum at concentrations present in uremia increased tritiated thymidine incorporation by 50% without altering tritiated leucine incorporation, and it increased collagens I and IV in the monolayer by 150% to 300%. Gelatinase activity was unchanged. We conclude that carbamylated proteins can activate mesangial cells to a profibrogenic phenotype. From a clinical perspective, the carbamylation of proteins by elevated urea levels may accelerate the progression to kidney failure and thus set up a vicious cycle in which the nitrogen retention itself would cause further progression of fibrosis and deterioration of kidney function.

Malaria circumsporozoite protein inhibits protein synthesis in mammalian cells.

Native Plasmodium circumsporozoite (CS) protein, translocated by sporozoites into the cytosol of host cells, as well as recombinant CS constructs introduced into the cytoplasm by liposome fusion or transient transfection, all lead to inhibition of protein synthesis in mammalian cells. The following findings suggest that this inhibition of translation is caused by a binding of the CS protein to ribosomes. (i) The distribution of native CS protein translocated by sporozoites into the cytoplasm as well as microinjected recombinant CS protein suggests association with ribosomes. (ii) Recombinant CS protein binds to RNase-sensitive sites on rough microsomes. (iii) Synthetic peptides representing the conserved regions I and II-plus of the P.falciparum CS protein displace recombinant CS protein from rough microsomes with dissociation constants in the nanomolar range. (iv) Synthetic peptides representing region I from the P.falciparum CS protein and region II-plus from the P.falciparum, P.berghei or P.vivax CS protein inhibit in vitro translation. We propose that Plasmodium manipulates hepatocyte protein synthesis to meet the requirements of a rapidly developing schizont. Since macrophages appear to be particularly sensitive to the presence of CS protein in the cytosol, inhibition of translation may represent a novel immune evasion mechanism of Plasmodium.

Decorin and suramin inhibit ocular fibroblast collagen production.

The process of ocular wound healing with respect to glaucomatous filtering procedures is of current interest. Delaying this response in patients could possibly lead to more favorable surgical results. So far, only highly toxic antimetabolites have come into frequent clinical use. The possible efficacy of other groups of substances such as growth factor inhibitors has not yet been examined in vitro. We exposed Tenon's capsule fibroblasts in tissue culture to various concentrations of decorin and suramin. The dose responses of type I and type III collagen to these inhibitors were measured using an ELISA-type dot blot assay. Total cellular protein production was assayed by measuring the incorporation of tritiated leucine. At a concentration of 10 micrograms/ml, suramin reduced the collagen production by more than 80%. Decorin, at a concentration of 100 micrograms/ml, reduced type I collagen production by about 50% while type III collagen was reduced by 80%. At these concentrations, the total cellular protein production was not inhibited. Both suramin and decorin, which specifically inhibit the action of growth factors on target cells, reduce the production of collagen synthesis by Tenon's capsule fibroblasts. This is a specific effect, because total protein production is not influenced. This sets these substances apart from antimetabolites. Decorin and suramin may have clinical relevance in that they appear to interfere with ocular wound healing more specifically than the substances so far frequently used.

Protein synthesis in developing ovaries of mealworm under in vivo and in vitro conditions: Effects of diflubenzuron

The effect of diflubenzuron (DFB) on protein synthesis in the ovaries of Tenebrio molitor was evaluated during oocyte maturation using in vivo and in vitro assays. When incorporated into the diet (5 and 10 mg g −1), DFB was found to affect both the weight, the protein levels and the incorporation of tritiated leucine into proteins of ovaries. In addition, electrophoretic separation of ovarian proteins by sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAGE) showed that DFB applied in vivo did not have a significant effect on the number of protein bands. When added to the culture medium (5 and 10 μg ml −1), DFB resulted in a slight but significant decrease in the rate of incorporation of tritiated leucine into proteins of ovaries cultured for 5 d compared to controls. These in vitro effects of DFB on protein synthesis were less marked than those observed in orally treated females.

Protein synthesis in the fat body of Tenebrio molitor (L.) during oocyte maturation: Effect of diflubenzuron, cycloheximide and starvation

Diflubenzuron (DFB), incorporated into the diet (5 and 10mg g −1) was tested on protein synthesis in the fat body of adult females of Tenebrio molitor (L.) during the first 6 days of adult life. DFB had no significant effect on fat body protein during the period examined, as evidenced both by the electrophoretic pattern and the incorporation of tritiated leucine. In contrast, cycloheximide (2.5 μg per insect) injected into newly emerged females significantly affected both the level of protein and the incorporation of tritiated leucine into proteins, as did starvation.

Suppression of proliferation of a human B-cell leukaemic cell line derived from acute lymphoblastic leukaemia by soluble factor(s) from Campylobacter rectus

Soluble sonic extracts of several strains were examined for their ability to alter proliferation of a cell line derived from acute lymphoblastic leukaemia (BALL-1). Extracts of all strains tested caused dose-dependent suppression of proliferation when assessed by DNA (tritiated thymidine incorporation), RNA (tritiated uridine incorporation) and protein (tritiated leucine incorporation) synthesis. There was no effect on the viability of BALL-1 as measured by either trypan-blue exclusion or extracellular release of the cytoplasmic enzyme lactate dehydrogenase. The suppressive factor(s) was separated in a well-defined peak by high-pressure liquid DEAE ion-exchange chromatography, which revealed a single active peak with a molecular mass of 48 kDa. Characterization of the peak indicated that the suppressive factor(s) was heat labile (activity destroyed at 80 °C) and sensitive to the proteolytic enzyme pronase P. The soluble suppressive factor(s) from Campylobacter rectus thus has protein-like properties and no cytotoxicity to a human B-cell leukaemic cell line.