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PubMed2026

The clinical landscape of POLE-mutant colorectal cancer: a retrospective analysis of real-world outcome.

BACKGROUND: Pathogenic mutations in the POLE gene disrupt its proofreading function during DNA replication, causing an accumulation of erroneous nucleotide incorporations. This defect leads to a significantly elevated tumor mutation burden (TMB) and increased generation of tumor neoantigens. These molecular characteristics suggest a potential association between POLE-mutant tumors and distinct prognostic outcomes in colorectal cancer (CRC); however, clinical evidence supporting this correlation remains limited. METHODS: We retrospectively collected a cohort of CRC patients harboring pathogenic POLE mutations. Comparative analyses were performed between POLE-mutant and POLE wild-type CRCs regarding their clinical characteristics, prognostic outcomes, and genomic profiles. Additionally, we evaluated the response to immunotherapy in metastatic POLE-mutant CRC cases. RESULTS: Among 35,108 CRC patients, pathogenic POLE mutations were identified in 261 individuals, accounting for 0.74% of the cohort. The median age at diagnosis for POLE-mutant patients was 48 years, with a male predominance (74.4%) and a substantial proportion (50.4%) of tumors localized in the right-sided colon. All patients with pathogenic POLE mutations exhibited hypermutated phenotypes, characterized by a median TMB of 235.26 mutations per megabase (range: 71.20-719.00 mutations/Mb). In stage II CRC, POLE mutations were significantly associated with a reduced risk of recurrence (hazard ratio [HR] 0.344, 95% confidence interval [CI] 0.157-0.754, p = 0.008) when compared to POLE wild-type, microsatellite stable CRC patients. However, this association was not evident in stage III patients (HR 1.004, 95% CI 0.490-2.057, p = 0.992). Importantly, the incorporation of immune checkpoint inhibitors in first-line treatment regimens significantly improved progression-free survival (HR = 0.247, 95% CI 0.117-0.552, p = 0.0002) and overall survival (HR = 0.317, 95% CI 0.103-1.143, p = 0.0832) in metastatic CRC patients with pathogenic POLE mutations. CONCLUSIONS: Pathogenic POLE-mutant CRC constitutes a relatively rare, yet clinically important, subtype. These cancers exhibit distinct clinicopathological and genomic features. Our results indicate that mutations in the POLE gene may serve as a valuable prognostic marker and a potential indicator of benefit to immunotherapy in CRC, offering promising avenues for personalized treatment strategies.

Humans↗

PubMed2026

Clinicopathologic Features, Treatment Patterns, and Outcomes of Microsatellite Instability-High Gastric and Gastroesophageal Junction Adenocarcinoma: A Single-Institution Retrospective Analysis.

PURPOSE: Microsatellite instability-high (MSI-H) and deficient mismatch repair (dMMR) gastric or gastroesophageal junction (GEJ) adenocarcinomas are biologically distinct tumors with established sensitivity to immune checkpoint inhibitors (ICIs). However, real-world treatment patterns, response heterogeneity, and predictors of durable benefit remain poorly defined. METHODS: We retrospectively analyzed patients with biopsy-confirmed MSI-H/dMMR gastric/GEJ adenocarcinoma treated at a single center. Clinicopathologic, genomic, treatment, and outcome data were collected. Molecular profiling included ARID1A, RNF43, TP53, PIK3CA, KRAS, TGFBR2, and human epidermal growth factor 2 (HER2). ICIs-treated patients were classified as achieving clinical benefit (complete response, partial response, or durable stable disease ≥16 weeks) or no clinical benefit using iRECIST v1.1 and clinical assessment. Overall survival (OS) was estimated by using the Kaplan-Meier method. RESULTS: Thirty-four patients were identified (median age, 66 years; 53% male), including 21 with stage IV disease. Tumors were predominantly poorly differentiated (65%) and HER2-negative (94%), and 18% of patients had Lynch syndrome. Among 22 ICI-treated patients, 55% achieved clinical benefit, which was strongly associated with prolonged OS (P < .01). Most responses occurred at the first radiographic assessment (approximately 12 weeks). Elevated tumor mutational burden (TMB; ≥20 mutations/Mb) was present in 56% of patients but was not associated with clinical benefit (P = .40), and no individual genomic alteration significantly correlated with treatment outcome. Exploratory analyses suggested longer OS among patients with liver versus peritoneal metastases. Treatment was well tolerated, with predominantly low-grade immune-related adverse events. Baseline Eastern Cooperative Oncology Group performance status (0-1 v ≥ 2) was associated with clinical benefit (P = .049). CONCLUSION: Approximately half of the patients with MSI-H/dMMR gastric/GEJ adenocarcinoma cancers derived durable clinical benefit from ICIs, and treatment response was strongly associated with survival. Conventional genomic features, including TMB, did not predict clinical benefit, highlighting the need for additional biomarkers.

Humans↗

PubMed1984

Mechanisms subserving calcium's modulation of luteinizing hormone action in isolated swine granulosa cells.

We studied the mechanism(s) by which calcium ions modulate progesterone biosynthesis by isolated swine granulosa cells incubated in chemically defined medium in vitro. In selectively calcium-deficient incubations, the capacity of 8-bromo-cAMP to stimulate pregnenolone synthesis from endogenous sterol substrate was significantly impeded. This effect of calcium ions was specific, because calcium ions did not influence basal pregnenolone production or alter progesterone production in response to exogenously supplied cholesterol substrate. Moreover, calcium ions did not modify other biosynthetic processes in granulosa cells, such as de novo synthesis of cholesterol from [14C]acetate or the aromatization of testosterone to 17 beta-estradiol. The possible role of calmodulin in mediating calcium's actions in pig granulosa cells was tested by measuring the calmodulin content of these cells and assessing the functional responses to classical calmodulin antagonists. By immunoassay, swine granulosa cells contained high concentrations of calmodulin, viz. 4.21-4.88 micrograms calmodulin/mg protein. Moreover, calmodulin antagonists inhibited LH-stimulated progesterone production with the following rank order of potencies [estimated by half-maximally inhibitory concentrations (ID50)]: penfluridol (1 microM), trifluoroperazine (9 microM), chlorpromazine (95 microM), and trifluoperazine sulfoxide (greater than 300 microM). In addition, the nonphenothiazine calmodulin antagonist W7 inhibited stimulated progesterone production with an ID50 of 16.7 microM. W5 was less active. None of these antagonists significantly suppressed LH-stimulated cAMP generation at the low concentrations capable of inhibiting progesterone production. The effects of calcium ions seemed to depend upon the availability of intracellular pools of calcium, because TMB-8, an inhibitor of intracellular calcium mobilization, effectively suppressed LH-stimulated progesterone production (ID50, 18 microM). However, even 100 microM TMB-8 failed to alter basal progesterone production or suppress LH-stimulated cAMP generation in these cells. In summary, the present studies indicate that calcium ions significantly modulate LH's stimulation of pregnenolone biosynthesis from endogenous cholesterol substrate in swine ovarian cells. Calcium does not influence basal pregnenolone production, estrogen synthesis from androgen substrate, de novo biosynthesis of cholesterol from [14C]acetate, or progesterone production from exogenously supplied sterol substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1987

Lipoxygenase inhibitors reduce insulin secretion without impairing calcium mobilization.

Inhibitors of pancreatic islet lipoxygenase (LPX) impair nutrient-induced insulin (I) release. To define the mechanism of action of these inhibitors, studies were carried out at subthreshold glucose concentrations (0-1.7 mM) in order to minimize any effects of LPX blockade on the potentiating effect of extracellular fuels. Barium chloride (Ba2+; 2 mM) increased 45Ca2+ release from prelabeled islets in Ca2+-free medium and, thus, is a model for the mobilization of intracellular Ca2+ stores. Inhibition of LPX (using nordihydroguaiaretic acid, BW755c [3-amino-1-(trifluomethyl-phenyl)2-pyrazoline] or butylated hydroxytoluene) did not have any consistent effect on the influx of Ba2+ (as assessed by 133Ba uptake) or on the consequent release of cellular Ca2+ stores; however, each LPX inhibitor vitiated Ba2+-induced I release. The LPX inhibitors were not merely acting as nonspecific antioxidants, since two inhibitors which do not act by scavenging hydroperoxides (5,8,11,14-eicosatetraynoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid) also impeded the effect of Ba2+ on I secretion; furthermore, a series of hydroxyl radical scavengers, reducing agents, or agents that block nonenzymatic and/or NADPH-activated lipid peroxidation did not inhibit I secretion. LPX inhibitors also blocked the residual I response to 16.7 mM glucose in Ca2+-free medium. Additionally, they reduced secretion induced by 46 mM K+ or 1 mM isobutylmethylxanthine (provided in the presence of extracellular Ca2+), without inhibiting K+- or isobutylmethylxanthine-induced Ca2+ fluxes. Stimuli sensitive to LPX blockade were also antagonized by antimycin A (an inhibitor of energy flux) or TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride; which appeared to deplete critical intracellular Ca2+ stores]. In contrast, the effects of exogenous phospholipase C (and several other Ca2+-dependent membrane-active agonists) were resistant to the LPX inhibitors, TMB-8, and antimycin A; thus, LPX inhibitors are not nonspecific global poisons of all Ca2+-dependent exocytotic hormone release. We conclude that LPX (or a very similar enzyme) may modulate the effects (or redistribution) of an ATP-dependent trigger pool of Ca2+ at a site distal to and independent of its mobilization by primary islet agonists. LPX inhibitors also blocked secretion induced by 12-O-tetradecanoyl-phorbol-13-acetate; this effect may reflect an effect of LPX on the activation of protein kinase C or a modulation of its synergism with the same trigger Ca2+ pool(s).

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1988

Regulation of relaxin release from monodispersed porcine luteal cells: effect of calcium ionophore A23187 and calcium channel blockers.

The role of calcium ion mobilization in modulation of secretion of the ovarian protein hormone relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells. The rate of development of plaques in time-course experiments was used in this study as an index of the rate of relaxin release. Exposure of luteal cell-containing monolayers to the calcium-mobilizing agent A23187 (40 nM to 5 microM) resulted in a dose-related increase in the rate of relaxin-plaque formation. This effect [and the influence of a stimulatory secretagogue, prostaglandin E2 (PGE2; 1 microM)] was suppressed by coculture with Co2+ (5 mM), a calcium channel blocker. These results are consistent with the view that calcium ion redistribution within porcine luteal cells forms a pathway that subserves, at least in part, the rates of basal and stimulated relaxin release in vitro. However, A23187 was equally effective in enhancing the rate of plaque formation when the monolayers were bathed in a low calcium medium (mean +/- SEM, 6.61 +/- 0.92 microM Ca2+), rather than a calcium-replete medium (1.56 +/- 0.09 mM Ca2+). Likewise, neither basal nor PGE2-stimulated (1 microM) relaxin secretion was abrogated by culture of monolayers in low calcium medium. These data suggest that the stimulatory effect of A23187 (and perhaps PGE2) arose predominantly through redistribution of calcium stored within intracellular sites in luteal cells, rather than entrance of calcium into the cell from the extracellular medium. Yet, incompatible with this interpretation, we observed that TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride; a putative inhibitor of intracellular calcium redistribution] enhanced rather than blocked A23187- or PGE2-stimulated relaxin release. This result is consistent with the possibility that TMB-8 mobilized (rather than blocked) calcium in this cell system or that it acted via calcium-independent mechanisms. We conclude that calcium mobilization has the potential to act as a molecular pathway that transduces secretion of an ovarian peptide hormone, relaxin. However, the exact nature and physiological role(s) of the Ca2+ pathway in the control of relaxin secretion and the interrelationships of this mechanism with other intracellular messengers that may also modulate ovarian peptide secretion remain to be more clearly defined.

Animals↗

PubMed1989

Intracellular Ca2+ mobilization by thyrotropin, carbachol, and adenosine triphosphate in dog thyroid cells.

The effect of TSH, carbachol (CC), and ATP on intracellular calcium concentration ([Ca2+]i) in primary cultures of dog thyroid cells was examined using the fluorescent Ca2+ indicator fura-2. TSH caused an increase in [Ca2+]i at 37 C, but not 22 C, while it increased cAMP formation in these cells at both 22 and 37 C. CC and ATP increased [Ca2+]i at both 22 and 37 C. The CC-induced increase in [Ca2+]i was under muscarinic receptor control, and it was biphasic, with an initial spike followed by a sustained increase at a lower level. TSH and ATP were weaker agonists compared to CC, since maximal doses of TSH (100-500 mU/ml) and ATP (100-500 microM) increased [Ca2+]i by 40-70% over basal levels, compared to a 2- to 4-fold increase in [Ca2+] induced by maximal doses of CC (10-50 microM). The TSH-induced increase in [Ca2+]i was transient, returning to basal levels within 1-2 min after application of the agonist. All three agents were able to transiently increase [Ca2+]i to be internal stores. In the presence of the inorganic Ca2+ channel blockers La3+, Ni2+, and Co2+, the peak [Ca2+]i change was little affected, while the persistent response to CC and ATP was blocked, indicating dependence of this phase on influx of Ca2+. Paradoxically, these channel blockers abolished the effect of TSH on [Ca2+]i. TSH stimulation of cAMP formation was also inhibited 80-90% by these blockers, but not in Ca2+-free/EGTA buffer. These results suggest that the Ca2+ channel blockers may have actions in addition to inhibition of Ca2+ entry in these cells. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl] specifically blocked both the initial and sustained increase induced by CC, while having no effect on ATP or TSH-induced [Ca2+]i, suggesting that TMB-8 may not be a general antagonist of Ca2+ mobilization. Activators of protein kinase-C, such as phorbol esters or an analog of diacylglycerol, inhibited the [Ca2+]i rise induced by all the three agonists used, indicating a regulatory role of protein kinase-C activation on [Ca2+]i in these cells. In FRTL-5 cells, [Ca2+]i was also increased by TSH and ATP, but not by CC. ATP, however, was a more effective agonist than in dog thyroid cells, while TSH increased [Ca2+]i by a similar magnitude in both cell types. The results of the present study demonstrate that TSH, albeit of lesser potency than CC, increases [Ca2+]i by causing intracellular Ca2+ mobilization in cultured dog thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1994

Neuropeptide Y stimulates luteinizing hormone-releasing hormone release from superfused hypothalamic GT1-7 cells.

The effects of neuropeptide Y (NPY) on LHRH release from an immortalized cell line were investigated using a flow-through cell culture superfusion system. Immortalized hypothalamic GT1-7 cells were cultured for 72 h and superfused for a total of 180 min. In initial experiments, discrete 5-min pulses of NPY (10(-12)-10(-5) M) were administered to the cells. A clear dose-dependent stimulatory effect on NPY on LHRH release from the cells was observed with a calculated 50% effectiveness concentration of 33 nM. The stimulatory effects of brief NPY exposure were rapid and robust, e.g. reaching and maintaining levels of 173% over baseline for 20 min at the 10(-7) dose. The lowest dose of NPY that showed a significant effect was 10(-10) M; maximal responses were observed at 10(-6) M and reached a plateau thereafter. Control pulses of Dulbecco's modified Eagle's medium (DMEM) and 10(-6) M substance P or arg-vasopressin were also presented to the cells to serve as controls for our pulse protocol, and these challenges produced no significant LHRH responses. The NPY receptor antagonists, PYX1 and PYX2, at 10(-8) M, completely blocked the observed NPY responses in these cells. To assess the NPY receptor subtypes that mediate the NPY effects pharmacologically, GT1-7 cells were challenged with a Y1 receptor agonist, (Leu31Pro34)NPY, a Y2 receptor agonist, NPY(13-36), or peptide YY, at doses 10(-12)-10(-5) M. All four peptides stimulated LHRH release from GT1-7 cells with a rank-ordered potency of NPY = peptide YY > Y1 agonist = Y2 agonist. To examine possible signal transduction mechanism(s) involved in mediating this effect, pertussis toxin, RpcAMPs (cyclic adenosine-3'5'-monophosphothioate Rp diastereomer), Ca(2+)-free DMEM and TMB-8 (3, 4, 5-trimethoxybenzoic acid 8-(diethylamino) octylester) were used to treat the cells before and during superfusion with NPY. Treatment with pertussis toxin, RpcAMPs, and Ca(2+)-free DMEM did not significantly alter NPY-stimulated LHRH release responses to 10(-7) M NPY. However, the addition of 100 microM and 250 microM TMB-8 to Ca(2+)-free DMEM almost completely blocked this NPY effect, as did 10 microM ryanodine. Finally, the locus of action for this NPY effect was examined using tetrodotoxin to reduce action potential propagation in the GT1-7 cells. Tetrodotoxin treatment blocked the LHRH response to NPY by more than 50%.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed2002

An IP3-activated Ca2+ channel regulates fungal tip growth.

Hyphal extension in fungi requires a tip-high Ca(2+) gradient, which is generated and maintained internally by inositol (1,4,5)-trisphosphate (IP(3))-induced Ca(2+) release from tip-localized vesicles and subapical Ca(2+) sequestration. Using the planar bilayer method we demonstrated the presence of two types of IP(3)-activated Ca(2+) channels in Neurospora crassa membranes with different conductances: one low (13 picosiemens), the other high (77 picosiemens). On sucrose density gradients the low conductance channel co-localized with endoplasmic reticulum and plasma membrane, and the high conductance channel co-localized with vacuolar membranes. We correlated the effect of inhibitors on channel activity with their effect on hyphal growth and Ca(2+) gradients. The inhibitor of IP(3)-induced Ca(2+) release, 2-aminoethoxidiphenylborate (2-APB), inhibits both channels, while heparin, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8) and dantrolene inhibit only the large conductance channel. Because 2-APB inhibits hyphal growth and dissipates the tip-high cytosolic [Ca(2+)] gradient, whereas heparin microinjection, TMB-8 and dantrolene treatments do not affect growth, we suggest that the small conductance channel generates the obligatory tip-high Ca(2+) gradient during hyphal growth. Since IP(3) production must be catalyzed by tip-localized phospholipase C, we show that a number of phospholipase C inhibitors [neomycin, 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]- 1H-pyrrole-2,5-dione (U-73122) (but not the inactive pyrrolidine U-73343), 3-nitrocoumarin] inhibit hyphal growth and affect, similarly to 2-APB, the location of vesicular Ca(2+) imaged by chlortetracycline staining.

Calcium Channels↗

PubMed1987

Effects of calcium antagonists on cyclic AMP phosphodiesterase induction in Dictyostelium discoideum.

Previous studies have suggested that cyclic GMP and/or Ca2+ might function as second messengers in the induction by exogenous cyclic AMP of the cyclic AMP phosphodiesterase (PD) in Dictyostelium discoideum. To assess further the role of Ca2+ in PD induction we examined the effect on this process of a number of putative Ca2+-channel blockers. At relatively low micromolar concentrations, TMB-8, nicardipine, nifedipine, diltiazem and verapamil all altered cell shape and inhibited PD induction in a similar dose-dependent fashion. Concentrations of these drugs that abolished PD induction had no effect on cell viability; however, higher concentrations reduced viability and caused cell lysis. All effects of these compounds on the cells were antagonized at least partially by 5-10 mM-Ca2+. Other cations tested were considerably less effective. Like the organic inhibitors, La3+ also altered cell shape, inhibited PD induction and reduced cell viability at elevated concentrations, but its effect on the cells appeared to be more complex. Inhibition of PD induction by the organic antagonists could not be attributed solely to an impaired uptake of extracellular Ca2+, a reduction of ATP pools in the cells or a direct effect on calmodulin. Concentrations of TMB-8 that inhibited PD induction had little effect on the cyclic GMP response. Therefore, this compound did not inhibit PD induction indirectly by blocking cyclic GMP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1988

Regulation of cell shape in Euglena gracilis. V. Time-dependent responses to Ca2+ agonists and antagonists.

The daily changes in cellular shape observed in growth-synchronized cultures of Euglena gracilis Klebs strain Z, were altered by exposure to Ca2+ channel agonists and antagonists. The response of the cells to these pharmacological agents depended, in part, on the time in the growth cycle that the cells were exposed. The Ca2+ channel blockers verapamil and nifedipine and the intracellular Ca2+ antagonist TMB-8 all caused cell rounding when elongated cells from the middle of the light cycle were treated. These results were the same as with other methods used to deprive cells of extracellular Ca2+, such as exposure to EGTA or resuspension in Ca2+-free medium. The cell response in mid light cycle to the channel blockers was reversible by simultaneous exposure to CaCl2, and the nifedipine response was also reversed by simultaneous exposure to the structurally related Ca2+ agonist BAY-K 8644. Exposure of cells in the first hour of the light cycle to verapamil, nifedipine or TMB-8 caused an unexpected result. Instead of preventing the round cells from elongating in the first portion of the light cycle, as do LaCl3, EGTA or resuspension in Ca2+-free medium, a greater than expected percentage of elongated cells was found in the treated population. This represents the first instance in which the biological clock control over the rate and extent of cell elongation was accelerated. The calcium agonist CGP-28392 did not have an effect on cell elongation in the early portion of the light cycle but caused cell rounding in the middle of the light cycle. The calcium agonist BAY-K 8644 did not cause any shape changes alone, but was capable of reversing the effects of nifedipine in the middle of the light cycle.

Animals↗

PubMed1998

Cry1Ac, a bacillus thuringiensis toxin, triggers extracellular Ca2+ influx and Ca2+ release from intracellular stores in Cf1 cells

Intracellular Ca2+ concentration was measured in single Cf1 cells (Choristoneura fumiferana, spruce budworm) loaded with Fura-2, a Ca2+-sensitive fluorescent probe. Cf1 cells displayed Ca2+ surges in response to Cry1Ac and Cry1C proteins, two Cf1-toxic Bacillus thuringiensis products, but not to Cry1Aa and Cry3A, which are not toxic to Cf1 cells. In the presence of extracellular Ca2+, the toxin-induced Ca2+ response was insensitive to methoxyverapamil, a voltage-dependent Ca2+ channel blocker, but was abolished by lanthanum, a general inhibitor of Ca2+ transport. In the absence of external Ca2+, Cry1Ac induced a small intracellular Ca2+ transient which was inhibited by TMB-8, a blocker of Ca2+ release from inositol-1,4,5-trisphosphate-sensitive pools. Under these conditions, thapsigargin, which inhibits intracellular Ca2+-ATPases, elicited a Ca2+ surge when applied alone. However, subsequent addition of Cry1Ac failed to induce a Ca2+ signal, indicating a depletion of intracellular Ca2+ pools. In Cf1 cells, therefore, bioactive B. thuringiensis toxins triggered intracellular Ca2+ surges which were mainly due to the influx of extracellular Ca2+ through toxin-made pores, as confirmed by planar lipid bilayer experiments. Furthermore, TMB-8- and thapsigargin-sensitive Ca2+ stores contributed to the Cry1Ac-induced Ca2+ signal.

Journal Article↗

PubMed1989

Sensitivity of steroid enzyme immunoassays. Comparison of four label enzymes in an assay system using a monoclonal anti-steroid antibody.

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), beta-galactosidase (beta-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways, using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl beta-D-galactopyranoside and 4-methylumbelliferyl beta-D-galactopyranoside were used for beta-GAL, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (Ka = 2 x 10(10) M-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed2001

[Formation mechanism of 4,4-methylenebis(N,N-dimethylaniline) by the anodic oxidation of N,N-dimethylaniline].

Preparative constant-current electrolysis of N,N-dimethylaniline(100 mM, DMA) was carried out in dry acetonitrile containing Et4NClO4 to give N,N,N',N'-tetra-methylbenzidine(TMB) and 4,4'-methylenebis(N,N-dimethylaniline) (MDMA). In the presence of HCl, DMA was quantitatively oxidized to TMB. Formation mechanism of MDMA was discussed using the results of semi-empirical calculations that were PM3 and AM1. The proposed mechanism by Hand and coworker was ruled out and an alternative one is suggested as follows. DMA undergoes one-electron oxidation at the anode and gives the corresponding N,N-dimethylaniline cation (A). Aydroxy ion, instead of DMA, abstracts a proton from A and gives H2O and methyl(phenyl)aminomethyl radical (B). B reacts with DMA to give a 1-dimethylamino-4-(N-methyl-N-phenylamino)methyl-2,5-cyclohexadienyl radical (I). The radical I decomposes to N-methylaniline and a 1-dimethylamino-4-methylene-2,5-cyclohexadienyl radical (J). The radical J reacts with DMA to give a 1-dimethylamino-4-(4-dimethylaminobenzyl)-2,5-cyclohexadienyl radical (K), which is oxidized to MDMA by the anode.

Aniline Compounds↗

PubMed1995

Irsogladine inhibits ionomycin-induced decrease in intercellular communication in cultured rabbit gastric epithelial cells.

Effects of irsogladine on ionomycin-induced decrease in intercellular communication and increase in intracellular concentration of Ca2+ ([Ca2+]i) were investigated in cultured rabbit gastric epithelial cells. Ionomycin (10(-7)-10(-6) M) transiently and concentration-dependently inhibited intercellular communication concomitantly with the elevation of [Ca2+]i in the presence and absence of extracellular Ca2+. Irsogladine (10(-5) M), which has been shown to facilitate intercellular communication, suppressed the ionomycin-induced elevation of [Ca2+]i and decrease in intercellular communication. The suppression of the ionomycin effects by irsogladine was independent of extracellular Ca2+. TMB-8 [8-(diethylamino)octyl-3,4,5-trimethoxy-benzoate hydrochloride] (10(-6) M) also suppressed the ionomycin-induced elevation of [Ca2+]i and decrease in intercellular communication. These results indicate that the ionomycin-induced decrease in intercellular communication may be due to Ca2+ mobilization from intracellular stores. Inhibitory effects of irsogladine and TMB-8 on the ionomycin-induced decrease in intercellular communication may be produced by suppressing Ca2+ mobilization.

Animals↗

PubMed2024

A comprehensive analysis of ribonucleotide reductase subunit M2 for carcinogenesis in pan-cancer.

BACKGROUND: Although there is evidence that ribonucleotide reductase subunit M2 (RRM2) is associated with numerous cancers, pan-cancer analysis has seldom been conducted. This study aimed to explore the potential carcinogenesis of RRM2 in pan-cancer using datasets from The Cancer Genome Atlas (TCGA). METHODS: Data from the UCSC Xena database were analyzed to investigate the differential expression of RRM2 across multiple cancer types. Clinical data such as age, race, sex, tumor stage, and status were acquired to analyze the influence of RRM2 on the clinical characteristics of the patients. The role of RRM2 in the onset and progression of multiple cancers has been examined in terms of genetic changes at the molecular level, including tumor mutational burden (TMB), microsatellite instability (MSI), biological pathway changes, and the immune microenvironment. RESULTS: RRM2 was highly expressed in most cancers, and there was an obvious correlation between RRM2 expression and patient prognosis. RRM2 expression is associated with the infiltration of diverse immune and endothelial cells, immune checkpoints, tumor mutational burden (TMB), and microsatellite instability (MSI). Moreover, the cell cycle is involved in the functional mechanisms of RRM2. CONCLUSIONS: Our pan-cancer study provides a comprehensive understanding of the carcinogenesis of RRM2 in various tumors.

Humans↗

PubMed1993

Potential role for arachidonic acid and eicosanoids in modulating progesterone secretion by ovine chorionic cells.

The present study was conducted to investigate whether arachidonic acid and its metabolites can modulate progesterone (P4) secretion in ovine chorionic cells. At concentrations of 7.5 mumol/l and 12.5 mumol/l, arachidonic acid caused an increase of basal P4 secretion (about 1.8-fold (p < 0.01) and 2.5-fold (p < 0.001), respectively, over control). Such a stimulatory effect was suppressed when the concentration of arachidonic acid attained 25 mumol/l, and at 50 mumol/l the fatty acid led to a decline of basal P4 synthesis (about 35%, p < 0.01). Phospholipase A2 (PLA2) and melittin had a similar dual effect to that observed when arachidonic acid was added exogenously. In contrast, eicosatrienoic acid (a closely related fatty acid) did not stimulate P4 secretion but inhibited it at a concentration of 50 mumol/l (about 40% inhibition, p < 0.01). The possible involvement of calcium on the effects of arachidonic acid was explored. Interestingly, 3 mmol/l ethylene glycol bis(beta-aminoethyl ether)-N,N,N,N'-tetraacetic acid (EGTA) and 10 mumol/l 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) further enhanced the steroidogenic effect of 12.5 mumol/l arachidonic acid (p < 0.05 and p < 0.01 vs the corresponding value in the absence of EGTA or TMB-8, respectively). In contrast, these agents failed to modify P4 secretion observed in the presence of 50 mumol/l arachidonic acid. We also tested the effect of inhibition of arachidonic acid metabolism via cyclooxygenase and lipoxygenase pathways. Indomethacin (10 mumol/l) failed to block the effects of arachidonic acid, but nordihydroguaiaretic acid (10 mumol/l) prevented the stimulatory action of this fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗

PubMed2026

Associations of TILs and genomic alterations in HER2+ early breast cancer.

This study investigated the associations between tumor-infiltrating lymphocytes (TILs), genomic features, and prognosis in HER2+ early breast cancer (EBC) patients receiving adjuvant trastuzumab. We retrospectively analyzed 864 HER2+ EBC patients from Shanghai Ruijin Hospital (2009-2017). The optimal threshold of TILs for predicting disease-free survival (DFS) and overall survival (OS) was explored. Whole-exome sequencing (WES) on 261 tumors assessed the mutational profiles, tumor mutational burden (TMB), and copy number alteration (CNA). Associations between these genomic features, TIL levels, and prognosis were further evaluated. TILs showed a right-skewed distribution (median: 15%, IQR: 1-30%), and higher TIL levels were significantly associated with hormone receptor negativity and high histologic grade (P < 0.001). A 15% TIL threshold optimally predicted prognosis, with low-TIL (≤15%, 63.0%) patients showing inferior DFS (HR: 1.63, P = 0.009) and OS (HR: 2.12, P = 0.037). WES identified frequent mutations in TP53 (62.8%), PIK3CA (34.5%), and BRCA2 (9.6%). A higher TIL density was observed in TP53-wild-type, low-TMB or low-CNA tumors (P < 0.05). PIK3CA mutations conferred a significant DFS advantage. Integrating TIL level with PIK3CA or BRCA2 mutational status yielded distinct DFS trajectories (log-rank P = 0.023 and 0.040, respectively); patients with both high TIL levels and either PIK3CA or BRCA2 mutations had the most favorable outcomes. Stromal TILs at a 15% cutoff provide robust prognostic information in trastuzumab-treated HER2+ EBC. Integrating TIL levels with PIK3CA or BRCA2 mutational status enables refined risk stratification, offering a practical framework for personalized treatment decisions.

Humans↗

PubMed1987

Prostaglandin production by the guinea-pig endometrium: is calcium necessary?

The output of prostaglandin (PG) F2 alpha from guinea-pig endometrium obtained on day 15 of the oestrous cycle and maintained in tissue culture was significantly (P less than 0.05) reduced by the use of Ca2+-depleted medium, EGTA (a Ca2+ chelator), 8-(N,N-diethyl-amino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8; an intracellular Ca2+ antagonist), trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7; both calmodulin antagonists). Nifedipine inhibited PGF2 alpha output at a concentration (100 mumol/l) much greater than that usually required to block Ca2+ channels. Verapamil had a small but significant (P less than 0.05) inhibitory effect on PGF2 alpha output at 10-100 mumol/l. The outputs of PGE2 and, to a lesser extent, 6-keto-PGF1 alpha (the hydrated product of PGI2) were also reduced by using Ca2+-depleted medium. EGTA reduced the outputs of PGE2 and 6-keto-PGF1 alpha on day 1 of culture, but stimulated 6-keto-PGF1 alpha output on day 3 of culture. The outputs of PGE2 and 6-keto-PGF1 alpha were increased by TMB-8 (100 mumol/l) on day 3 of culture and by TFP and, to a smaller extent, by W-7 on all 3 days of culture. Nifedipine (100 mumol/l by not 1 or 10 mumol/l) reduced the outputs of PGE2 and 6-keto-PGF1 alpha on all 3 days of culture, whereas verapamil (100 mumol/l but not 1 or 10 mumol/l) increased the outputs of these two prostaglandins on days 2 and 3 of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗