UVEAL MELANOMA IN A 15-YEAR-OLD GIRL. CASE REPORT.

Uveal melanoma is the most common intraocular tumour in adults, it is a form of cancer that affects mostly older adults, as the average age at detection of this tumour is 60 years, but it can occur in any age group with no significant gender difference. However, uveal melanoma is very rare in children compared to the adult population, accounting for 1 % of all cases. In pediatric patients, malignant uveal melanoma is more frequently manifested during puberty, leading to speculation of an association between uveal melanoma and growth hormone levels. Prognostic factors for uveal melanoma include tumour histology, chromosomal abnormalities, tumour size, extrascleral spread and tumour location. Risk factors for uveal melanoma include melanocytosis, neurofibromatosis type 1 and dysplastic naevus syndrome. Some studies point to a significantly lower risk of developing metastases in younger patients, but the prognosis of uveal melanoma in children is not yet fully known. Clinical signs and treatment options for malignant uveal melanoma in children are still under discussion. Differential diagnosis of uveal lesions in children can sometimes be very difficult, as evidenced by following case report in which authors describe a case of choroidal melanoma in a 15-year-old girl.

Primary orbital teratoma - case study.

Teratomas are tumours deriving from embryonal stem cells. They consist of elements of all three germinal layers. Orbital localization of this type of tumour is very rare. Symptoms develop in the first months of life, rarely later, as cystoid components of the tumour grow in size. Surgical treatment is extremely difficult, eye is spared in individual cases. In our case study, we present baby girl with a diagnosis of primary intraorbital teratoma, who underwent partial resection of the tumour with not just the eye globe spared, but also with visual functions present after the surgery.

[Identification of a Family with SUFU Germline Deletion Based on a Case of Desmoplastic Medulloblastoma in an Infant]. / Identifikace rodiny s nosicstvím germinální delece genu SUFU na podklade dia-gnózy desmoplastického meduloblastomu u batolete.

BACKGROUND: Medulloblastoma, an embryonal neuroectodermal tumor of the cerebellum, is the most common malignant brain tumor in children. There are approximately 15 cases diagnosed in the Czech Republic each year. The recent World Health Organization classification recognizes several histopathological subtypes of medulloblastoma: classical, desmoplastic/ nodular with its extensive-nodularity variant, and anaplastic/ large-cell variant. Further molecular analysis identified four basic subgroups of medulloblastoma: WNT, SHH, Group 3, and Group 4. The subgroup of SHH meduloblastoma is associated with somatic mutations of SHH, PTCH1, SUFU, SMO and TP53, while the most common mutations found in infants up to three years of age were PTCH1 and SUFU. The majority of medulloblastomas are sporadic diseases, whereas only about 5- 10% of all cases occur in connection with hereditary genetic syndromes. CASE: We present a case of a 21-months old girl diagnosed with a localized posterior fossa tumor. The histopathological examination revealed a desmoplastic/ nodular medulloblastoma. The treatment comprised a radical exstirpation of the tumor followed by adjuvant chemotherapy. With the use of array-CGH, a partial biallelic deletion of the SUFU gene (locus 10q24.32) was detected in the tumor DNA, whereas a monoallelic deletion was found in the peripheral lymphocyte DNA of the patient. These findings were confirmed by an independent qPCR method. Monoallelic germline deletion of SUFU was also identified in the patients mother, who was a healthy carrier. Pedigree of the family suggested a transition of the germline deletion of SUFU, since another brain tumors (including one case diagnosed before the age of three years) were identified in previous generations. CONCLUSION: Germline mutations in SUFU gene are believed to predispose to infant desmoplastic/ nodular medulloblastomas, basal cell carcinomas and meningiomas. The susceptibility gene shows autosomal dominant inheritance with an incomplete penetrance. There is no evidence-based surveillance strategy suggested for the carriers of germline SUFU mutations/ deletions so far. Our recommendation is based both on a family history of our patient and similar cases described in the literature. Since the germinal mutations in SUFU are responsible for up to 50% of all desmoplastic medulloblastomas in children under three years of age, genetic testing of SUFU should be encouraged in this population of patients.

[Autosomal dominant polycystic kidney disease in the fetus with seemingly negative family history. A case report]. / Autosomálne dominantní polycystóza ledvin u plodu se zdánlive negativní rodinnou anamnézou--kazuistika.

An unusual case of fetal polycystic kidney disease is reported. Oligohydramnios and enlarged hyperechogenic kidneys were found at 21 weeks. The pregnancy was terminated and fetal autopsy performed. The histopathological pattern of fetal kidneys was consistent with glomerulocystic disease and this raised suspicion of autosomal dominant polycystic kidney disease (ADPKD). Initially, the family history seemed to be negative for ADPKD. The mother's diagnosis was established only after the abortion of the affected fetus. She had no symptoms of renal disease. Multigenerational involvement was revealed on the mother@s side. Mechanisms leading to prenatal ADPKD and prognosis of the pediatric patients are discussed.

[Spontaneus abortion caused by Listeria monocytogenes--report of three cases]. / Listeria monocytogenes jako prícina spontánního abortu--popis trí prípadu.

This paper describes three cases of spontaneous abortion caused by Listeria monocytogenes which were diagnosed during the outbreak of listeriosis in late 2006. Both clinical and morphological features were highly consistent. The abortion between 19 and 20 weeks of gestation was preceded by high maternal fever. The fetal disease was miliary widespread with involvement of adrenals, liver and lungs in particular. There was always present a pustular rash. Histological examination of the placentas revealed acute chorioamnionitis with massive growth of gram-positive rods in the amnion in all cases. Acute villitis and intervillous microabscesses were additionally noted in one case. The diagnosis in all reported cases was not established until fetal autopsy. The final diagnosis was confirmed by microbiological examination using samples submitted during the autopsy examination.

Brain angiotensin II, an important stress hormone: regulatory sites and therapeutic opportunities.

The presence of a brain Angiotensin II (Ang II) system, separated from and physiologically integrated with the peripheral, circulating renin-angiotensin system, is firmly established. Ang II is made in the brain and activates specific brain AT(1) receptors to regulate thirst and fluid metabolism. Some AT(1) receptors are located outside the blood-brain barrier and are sensitive to brain and circulating Ang II. Other AT(1) receptors, located inside the blood-brain barrier, respond to stimulation by Ang II of brain origin. AT(1) receptors in the subfornical organ, the hypothalamic paraventricular nucleus (PVN), and the median eminence are involved in the regulation of the stress response. In particular, AT(1) receptors in the PVN are under glucocorticoid control and regulate corticotrophin-releasing hormone (CRH) formation and release. In the PVN, restraint elicits a fast increase in AT(1) receptor mRNA expression. The expression of paraventricular AT(1) receptors is increased during repeated restraint and after 24 h of isolation stress, and their stimulation is essential for the hypothalamic-pituitary-adrenal axis activation, the hallmark of the stress response. Peripheral administration of an AT(1) receptor antagonist blocks peripheral and brain AT(1) receptors, prevents the sympathoadrenal and hormonal response to isolation stress, and prevents the gastric stress ulcers that are a characteristic consequence of cold-restraint stress. This evidence indicates that pharmacologic inhibition of the peripheral and brain Ang II system by AT(1) receptor blockade has a place in the prevention and treatment of stress-related disorders.

Angiotensin II AT1 and AT2 receptor types regulate basal and stress-induced adrenomedullary catecholamine production through transcriptional regulation of tyrosine hydroxylase.

The sympathoadrenal response to stress includes a profound increase in adrenomedullary catecholamine synthesis driven by stimulation of tyrosine hydroxylase (TH) transcription. We studied the role of Angiotensin II type 1 and 2 (AT(1) and AT(2)) receptors during isolation stress, and under basal conditions. Pretreatment of rats with the AT(1) receptor antagonist candesartan for 14 days prior to isolation completely prevented the stress-induced stimulation of catecholamine synthesis, decreasing tyrosine hydroxylase transcription by preventing the expression of the transcriptional factor, Fos-related antigen 2 (Fra-2). In addition, AT(1) receptor antagonism prevented the stress-induced increase in adrenomedullary AT(2) receptor binding and protein. Treatment of non-stressed, grouped animals under basal conditions with the AT(1) receptor or with PD 123319, an AT(2) receptor antagonist, decreased the adrenomedullary norepinephrine (NE) content and TH transcription. While AT(1) receptor antagonism decreased the levels of Fra-2 and the phosphorylated forms of cAMP responsive element binding protein (pCREB) and EKR2 (p-ERK2, phosphor-p42 MAP kinase), the AT(2) antagonist decreased Fra-2 with no change in the phosphorylation of CREB or EKR2. Our results demonstrate that both adrenomedullary AT(1) and AT(2) receptor types maintain and promote the adrenomedullary catecholamine synthesis and the transcriptional regulation of TH. Instead of opposing effects, however, our results indicate a complex synergistic regulation between the AT(1) and AT(2) receptor types.

Angiotensin II AT1 receptor blockade prevents gastric ulcers during cold-restraint stress.

Cold-restraint stress reduces gastric blood flow and produces acute gastric ulcers. We studied the role of Angiotensin II (Ang II) on gastric blood flow and gastric ulceration during stress. Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated for 14 days with the AT(1) receptor antagonist candesartan before cold-restraint stress. AT(1) blockade increased gastric blood flow 40% to 50%; prevented gastric ulcer formation by 70% to 80%; reduced the increase in adrenomedullary epinephrine and TH mRNA without preventing the stress-induced increase in adrenal corticosterone; decreased the stress-induced expression of tumor necrosis factor alpha (TNF-alpha) and adhesion protein ICAM-1 in arterial endothelium, and neutrophil infiltration in the gastric mucosa; and decreased PGE(2) content. AT(1) receptor blockers prevent stress-induced ulcerations by a combination of gastric blood flow protection, decreased sympathoadrenal activation, anti-inflammatory effects with reduction in TNF-alpha, and ICAM-1 expression, leading to reduced neutrophil infiltration while maintaining the protective glucocorticoid effects and PGE(2) release. Ang II has a crucial role, through stimulation of AT(1) receptors, in the production and progression of stress-induced gastric injury, and AT(1) receptor antagonists could be of therapeutic benefit.

Angiotensin II AT1 receptor antagonism downregulates stress-related gene expression in brain microvessels from spontaneously hypertensive and normotensive rats.

We studied the effect of treatment with the Angiotensin II AT(1) receptor antagonist candesartan (0.3 mg/kg/day via osmotic minipumps for 4 weeks compared with administration of vehicle) in brain microvessels in adult spontaneously hypertensive rats (SHR) that were vulnerable to stroke and normotensive control rats (WKY). At the dose administered, candesartan normalized blood pressure in SHR without significantly affecting blood pressure in WKY rats. We performed the gene expression analysis in rat brain microvessels using the Affymetrix Gene Chip Expression Analysis Technique. From a total of 8,799 probe array sets analyzed, we found abundant abnormalities in gene expression in SHR. Because stress has been suggested as a precipitant factor in brain ischemia and treatment with AT(1) receptor antagonist candesartan prevents the hormonal and sympathoadrenal reaction to isolation stress and protects from stress-induced gastric ulcers, we focused on the expression of stress-related genes. We found a higher number of probe array sets modified by candesartan treatment in normotensive WKY rats than in hypertensive SHR. AT(1) receptor blockade decreased the transcription levels of the stress-related tyrosine kinase receptor, stathmin, and fibroblast growth receptor genes in WKY and SHR rats. Our results indicate that Angiotensin II and its AT(1) receptors can influence gene expression independently of the effects on blood pressure. In addition, AT(1) receptor regulation of stress-related genes in brain microvessels may explain the proposed association between stress and ischemic disorders of the brain.

Thermal destabilization of ovarian LH/hCG receptors by negatively charged lipids.

The stabilizing effect of BSA on the rat ovarian LH/hCG receptor was analyzed by thermal perturbation technique. Thermal destabilization of the receptor with arachidonic acid along with digestion of membrane with phospholipase A2 and reversal of these effects when BSA was used as fatty acids scavenger, may indicate that free fatty acids are responsible for instability of the LH/hCG receptor. This destabilizing effect may be caused by the presence of a net negative surface charge provided by fatty acids. This presumption was corroborated by the reconstitution of delipidated LH/hCG receptor into proteoliposomes. Delipidated receptor lost to a great extent its binding activity and thermal stability. The receptor was fully reactivated by the reconstitution into proteoliposomes with neutral phosphatidylcholine but not with negatively charged phosphatidylserine and phosphatidylglycerol. Thermal inactivation of the LH/hCG receptor by delipidation was entirely inverted by treatment with phosphatidylcholine but the presence of negatively charged phospholipids did not change the heat inactivation profile of hCG-binding sites.

Luteinization factor-stimulated steroidogenesis in porcine granulosa cells.

Luteinization stimulator (LS), an intrafollicular compound of preovulatory (5-8 mm) follicles, increased both the basal and gonadotropins-stimulated production of progesterone by immature (1-3 mm) granulosa cells. The mechanism by which LS enhance steroidogenesis was investigated by studying the modulation of progesterone biosynthesis from exogenous cholesterol and pregnenolone in cultured porcine granulosa cells in serum-free medium. Progesterone production by cultured granulosa cells was stimulated by FSH, while treatment with 22-OH-cholesterol further enhanced the gonadotropin action. The activity of LS was found in cell conditioned media obtained after 3-day cultivation of preovulatory granulosa cells. Conversion of 22-OH-cholesterol into progesterone by granulosa cells isolated from small follicles was significantly stimulated in the presence LS in culture media. Also, progesterone production by granulosa cells in the presence of pregnenolone was increased considerably. Concomitant treatment with LS led to a further augmentation in progesterone synthesis. Endogenous formation of pregnenolone was inhibited by aminoglutethimide. Thus, LS enhancement of progesterone production in cultured porcine granulosa cells is associated with an increase in the activity of cytochrome P450 cholesterol side-chain cleavage and 3beta hydroxysteroid dehydrogenase enzymes.

Effect of intraovarian factors on porcine follicular cells: cumulus expansion, granulosa and cumulus cell progesterone production.

The role of granulosa cell conditioned media (CM) containing luteinization stimulator (LS), and the role of EGF in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral follicles was investigated. The CM were prepared by incubation of granulosa cells isolated from large antral follicles. After 24h incubation, more than 61 or 64% of OCC expanded to the +3 and +4 stage in the presence of CM (50%) or EGF (10ng/ml), respectively. The stimulatory effect of LS and EGF on the cumulus expansion was accompanied by the enhanced hyaluronic acid synthesis. Complete suppression of cumulus expansion stimulated by LS and EGF was observed in the presence of 10 micromol/l genistein (tyrosine kinase inhibitor), in the presence of 10mmol/l LiCl (the inhibitor of inositol 1,4,5-trisphosphate metabolism), and 100 micromol/l gallopamil, verapamil and norverapamil (calcium channel blockers). Stimulatory effect of EGF on the cumulus expansion of OCC isolated from large follicles was accompanied by the increased cumulus cell progesterone production. However, EGF did not affect the progesterone production by OCC isolated from small follicles. To determine whether EGF could modulate the granulosa cell steroidogenesis also, the effect of EGF on granulosa cells isolated from large (LGC) and small (SGC) follicles was compared. EGF (10ng/ml) failed to affect the progesterone synthesis during 72h culture of SGC but significantly enhanced the LGC progesterone production. Our results indicate that luteinization factor stimulates the cumulus expansion and hyaluronic acid synthesis by the OCC isolated from large antral follicles. The mechanism of LS- and EGF-induced cumulus expansion may involve tyrosine kinase activation and calcium mobilization. In addition, these results indicate the different response of porcine cumulus and granulosa cells originating from small and large follicles on the stimulatory effect of EGF.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Gossypol inhibits follicle-stimulating hormone- and epidermal growth factor-stimulated expansion of oocyte-cumulus complexes from porcine preovulatory follicles.

The role of gossypol in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral porcine follicles was investigated. Marked suppression of cumulus expansion stimulated with follicle-stimulating hormone (FSH) and epidermal growth factor (EGF) was observed in the presence of different concentrations of gossypol. Comparable inhibitory effects were obtained in the presence of NO donor, S-nitroso-N-acetylpenicillamine or sodium nitroprusside, suggesting that the inhibitory effect of gossypol may be mediated via NO generation. The inhibitory effect of gossypol on cumulus expansion of OCC was accompanied by inhibition of progesterone secretion of OCC and the decrease of [125I]EGF binding to granulosa cells.

Involvement of membrane surface charge in thermal stability of the rat ovarian LH/hCG receptor.

Analysis of fluorescence of membrane-bound 8-anilino-1-naphthalene sulfonate and monodansylcadaverine probes revealed that a negative membrane surface charge derived from free fatty acids (FFA) resulted in destabilization of structure-functional properties of the rat ovarian LH/hCG receptor. Removal of FFA from rat luteal and porcine ovarian granulosa cells by BSA increased gonadotropin responsiveness of cells in cAMP formation.

Hormonal modulation of structural alteration of rat ovarian luteinizing/human chorionic gonadotropin receptors.

The structure-stabilizing effect of homologous and heterogeneous desensitization and albumin on rat ovarian LH/hCG receptors was analyzed by thermal perturbation technique. HCG-induced down-regulation shifted the heat inactivation profile of hCG-binding sites to a temperature lower by about 7 degrees C (T50 values). In heterogeneous desensitization, which also involves uncoupling of receptors from adenylyl cyclase system, only follicle stimulating hormone (FSH) changed the stability of ovarian LH/hCG receptors. Stimulation of other hormonal receptors, which belong to the family of membrane spanning G protein-linked receptors, i.e. beta-adrenergic, glucagon, serotonin and prostaglandin E (PGE) had no effect on the stability of the LH/hCG receptor. Reduction of the stability of the LH/hCG receptor by about 3 degrees C after PGF2alpha injection to luteinized rats may be connected with specific process of luteolysis. On the other hand, albumin had a stabilizing effect on the receptor. The receptor destabilizing action of oleic acid incorporated into ovarian membranes along with calcium stimulation of endogenous phospholipase A (PLA) activity and reversal of these effects when BSA was used as fatty acid scavenger, may indicate that free fatty acids are responsible for the thermal instability of hCG-binding sites. Fluorescence quenching studies indicated that extraction of free fatty acids by albumin elevated the accessibility of fluorophores for acrylamide, and suggest that modificated lipid-protein interactions may affect the stability of the LH/hCG receptor structure.

Inhibitory effect of gossypol on basal and luteinization factor-stimulated progesterone synthesis in porcine granulosa cells.

Gossypol, a polyphenolic aldehyde, inhibits steroidogenesis and the reproductive system in both sexes. The present study was undertaken to investigate whether gossypol may affect progesterone biosynthesis in cultured porcine granulosa cells isolated from small (1-2 mm) follicles (SGC). SGC were cultured with gossypol, NO donor S-nitroso-N-acetylpenicillamine (S-NAP) or the specific NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), in the presence or absence of follicular fluid isolated from large (5-8 mm) follicles (LFF) or conditioned media (CM) of granulosa cells isolated from large follicles (LGC). Gossypol enhanced the nitrite content in culture media of SGC and inhibited basal progesterone secretion by SGC. S-NAP (10(-3) M) inhibited progesterone secretion and enhanced the formation of cGMP by SGC. L-NAME had no effect on progesterone accumulation by SGC. The stimulatory effect of LFF or CM media on progesterone production by SGC in culture was also inhibited by S-NAP (10(-3)) and gossypol (10(-4) M). Moreover, gossypol inhibited forskolin-stimulated progesterone secretion, as well as substrate-enhanced conversion of 22-OH-cholesterol and pregnenolone to progesterone. These results suggest that the inhibitory effect of gossypol on progesterone secretion in culture of SGC may be mediated via NO generation.

Structure-stabilizing effect of albumin on rat ovarian LH/hCG receptors.

The stabilizing effect of albumin on structure-functional alteration of LH/hCG receptors was analyzed by thermal perturbation technique. On exposing the membranes to bovine serum albumin (BSA) the heat inactivation profile of hCG-binding sites was shifted to a temperature higher by about 5 degreesC (T50 values). The receptor destabilizing action of arachidonic and oleic acids incorporated into ovarian membranes and reversal of this effect when BSA was used as fatty acid scavenger, may indicate that free fatty acids are responsible for the thermal instability of hCG-binding sites. This presumption was corroborated by digestion of membranes with phospholipase A2 (PLA2). This enzyme exerted effects on the thermal stability of the receptor protein resembling those observed upon insertion of fatty acids. The membrane fluidization induced by arachidonic acid can be reversed by BSA. However, alterations of lipid fluidity in membranes were not found to be a necessary prerequisite for stabilization of the LH/hCG receptor structure. Fluorescence quenching studies indicated that incorporation of oleic acid or digestion of membrane phospholipids with PLA2 elevated the accessibility of fluorophores for acrylamide. BSA scavenging of free fatty acids approached the quenching rate of control membranes. Analysis of fluorescence of membranes bound to monodansylcadaverine probe revealed that the negative surface charge derived from free fatty acids resulted in destabilization of the receptor protein. The effects of free fatty acids on membranes suggest that altered lipid-protein interactions may directly affect the stability of the LH/hCG receptor structure.

Modulation of LH/hCG receptors and physical state of ovarian membranes in rat pseudopregnancy.

Previous investigations have demonstrated that increased ovarian function during pseudopregnancy in the rat may be associated with alterations of the physical state of membranes. Changes in rigidity of membrane lipids were observed during the formation as well as regression of corpora lutea. The effects of cyclooxygenase inhibitors (indomethacin and acetylsalicylic acid (ASA)) and of selected steroids (estradiol, testosterone and dihydrotestosterone) on the functional state of luteinized ovaries were studied. The compounds were administered to the animals in silastic capsules on different days after hCG injection. ASA and indomethacin administration on days 10 and 11 after hCG injection resulted in an increase in the LH/hCG receptor binding activity and rigidity of ovarian membrane lipids, as determined by fluorescence polarization of 1,6-diphenyl-1,3,5 hexatriene (DPH) probe. This effect was apparent within 7 days after indomethacin and ASA treatment. Both estradiol and testosterone significantly increased the ovarian LH/hCG binding activity, however estradiol did not affect the membrane lipid rigidity. Unlike testosterone, the administration of dihydrotestosterone induced a decrease in membrane lipid rigidity and reduced the accessibility of the LH/hCG receptor. Inhibitors of prostaglandin F2alpha (PGF2alpha) synthesis, as the endogenous mediator of luteolysis, were shown to delay the regression of the corpora lutea and to prolong the luteal activity in pseudopregnant rats.

Effect of local anesthetics on accessibility and thermal stability of the rat ovarian LH/hCG receptor.

Perturbation of rat ovarian membranes induced by local anesthetics altered the accessibility and thermal stability of the LH/hCG receptor. Incubation of ovarian membranes with tetracaine and benzyl alcohol resulted in a dose-dependent loss of binding activity of the LH/hCG receptor. Possible structure-functional properties of the receptor in membranes treated with local anesthetics were analyzed by the thermal perturbation technique. The heat inactivation profile of the LH/hCG binding sites in 10 mM tetracaine and 20 mM benzyl alcohol treated membranes was shifted to lower temperatures of about 15 degrees C and 4 degrees C (T50 values), respectively. The thermal stability of the receptor decreased with an increasing concentration of the local anesthetics. Thermal destabilization of the LH/hCG receptor induced by the action of tetracaine was higher at pH 9.5 than at pH 5.5. Treatment of ovarian membranes with 5 mM tetracaine modified and with 20 mM benzyl alcohol failed to change the quenching of protein fluorescence, characteristic for control membranes. Incubation of ovarian membranes with tetracaine and benzyl alcohol increased the membrane lipid fluidity, as determined by fluorescence polarization of the 1,6-diphenyl-1,3,5-hexatriene probe (DPH).