c-Jun N-terminal kinase specifically phosphorylates p66ShcA at serine 36 in response to ultraviolet irradiation.

Mice lacking expression of the p66 isoform of the ShcA adaptor protein (p66(ShcA)) are less susceptible to oxidative stress and have an extended life span. Specifically, phosphorylation of p66(ShcA) at serine 36 is critical for the cell death response elicited by oxidative damage. We sought to identify the kinase(s) responsible for this phosphorylation. Utilizing the SH-SY5Y human neuroblastoma cell model, it is demonstrated that p66(ShcA) is phosphorylated on serine/threonine residues in response to UV irradiation. Both c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases are activated by UV irradiation, and we show that both are capable of phosphorylating serine 36 of p66(ShcA) in vitro. However, treatment of cells with a multiple lineage kinase inhibitor, CEP-1347, that blocks UV-induced JNK activation, but not p38, phosphatidylinositol 3-kinase, or MEK1 inhibitors, prevented p66(ShcA) phosphorylation in SH-SY5Y cells. Consistent with this finding, transfected activated JNK1, but not the kinase-dead JNK1, leads to phosphorylation of serine 36 of p66(ShcA) in Chinese hamster ovary cells. In conclusion, JNKs are the kinases that phosphorylate serine 36 of p66(ShcA) in response to UV irradiation in SH-SY5Y cells, and blocking p66(ShcA) phosphorylation by intervening in the JNK pathway may prevent cellular damage due to light-induced oxidative stress.

Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family.

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.

beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation.

beta-Amyloid (A beta) has been strongly implicated in the pathophysiology of Alzheimer's disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c-Jun N-terminal kinases (JNKs) and of their substrate, c-Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated A beta. The activities of JNK family members increased in neuronal PC12 cells within 2 h of A beta treatment and reached 3--4-fold elevation by 6 h. To test the role of these changes in death caused by A beta, we examined the effects of CEP-1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP-1347 (100--300 nM) in the culture medium effectively blocked the increases in cellular JNK activity caused by A beta and, at similar concentrations, protected both PC12 cells and sympathetic neurons from A beta-evoked-death. Effective protection required addition of CEP-1347 within 2 h of A beta treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant-negative c-Jun construct also conferred protection from A beta-evoked death, supporting a model in which JNK activation contributes to death via activation of c-Jun. Finally, CEP-1347 blocked A beta-stimulated activation of caspase-2 and -3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by A beta and hence identify it as a potential therapeutic target in AD.

A radioactive binding assay for inhibitors of trkA kinase.

The high-affinity receptor for nerve growth factor (NGF), trkA, is a receptor-linked tyrosine kinase. The binding of NGF to trkA, depending on the context of its environment, can cause beneficial or deleterious responses in the target cells. For example, the activation of trkA in sympathetic and sensory neurons causes the subsequent survival and differentiation of these cells. On the other hand, the activation of trkA by NGF in other cells has been implicated in several pathologies including inflammation-induced hyperalgesia and several cancers. A radioactive binding assay to evaluate inhibitors of the kinase domain of trkA has been developed and validated. The assay monitors the specific binding of an inhibitor of trkA kinase activity, the indolocarbazole K-252a, to the trkA receptor. [3H]K-252a binds with high affinity to one site on the cytoplasmic kinase domain of the trkA receptor. Binding is saturable and reversible with a dissociation constant (Kd) of 1.5 nM. The binding assay has been used in competition binding experiments to determine the inhibition constants for other indolocarbazole compounds. The IC50 values for compounds obtained in the binding assay correlate very well with the IC50 values obtained in an enzyme-linked immunosorbent assay for trkA tyrosine kinase activity.

Membranes of human neuroblastoma SH-SY5Y cells contain specific binding sites for [3H]K-252A.

K-252a and the structurally similar compound staurosporine promote neurotrophic responses in several cell lines (PC12, SH-SY5Y human neuroblastoma) and in cultures of primary neurons. The molecular mechanisms involved in the induction of these neurotrophic activities are unknown. It is demonstrated in this report that [3H]K-252a binds to SH-SY5Y membranes and that the binding is specific and saturable with a Kd of 2.7 nM and a Bmax of 100,000 sites per cell. The association of [3H]K-252a with its binding site is rapid and reversible, and the binding was inhibited by unlabeled K-252a and by staurosporine. Binding of [3H]K-252a was not inhibited by the potent protein kinase C (PKC) inhibitor GF109203X. Down regulation of PKC by treating SH-SY5Y cells with a phorbol ester did not cause a reduction in the specific binding of [3H]K-252a to membranes, suggesting that the binding is not to PKC. Treatment of the SH-SY5Y membranes with trypsin and by boiling destroyed all specific binding of [3H]K-252a. These results suggest that the [3H]K-252a binds to a specific protein site that is associated with membranes of SH-SY5Y cells.

A rapid fluorometric assay to measure neuronal survival in vitro.

We report the development and characterization of a rapid fluorometric microassay suitable for quantifying neuronal cell survival. The method can be used in two formats: (1) a time course analysis of survival response or (2) as a simple endpoint assay for the assessment of neuronal survival promoted by a variety of reagents. The assay uses calcein AM, a non-fluorescent, electrically neutral, non-polar analogue of fluorescein diacetate, which passively crosses cell membranes and is cleaved to a fluorescent derivative by non-specific intracellular esterases. Once cleaved in viable cells, the resultant fluorescent salts are retained by intact cell membranes. The relative number of viable cells under various conditions can be quantified by measuring the emitted fluorescence. Described herein are the conditions that allow the determination of low viable neuronal cell numbers (10(2)-10(3) cells/cm2).

Australian trial of topical minoxidil and placebo in early male pattern baldness.

One hundred and sixty nine men with early male pattern baldness (androgenic alopecia) were treated in a random, double-blind fashion with either 2% minoxidil solution or placebo vehicle for 24 weeks, one ml applied twice daily. After 24 weeks all patients received the active solution until week 48. After 24 weeks the minoxidil treated patients had increased their non-vellus hair counts significantly more than the placebo treated group; means were 37.6 and 8.8 hairs per reference area, 95% C.I. for difference = 10.85 to 60.75. The rate of non-vellus hair regrowth was also greater among minoxidil treated patients than placebo treated patients. Nine (12.5%) evaluable minoxidil treated patients compared with 2 (2.7%) evaluable placebo treated patients reported moderate or dense hair regrowth at week 24. Minimal regrowth was reported by 18 (25%) active group and 15 (20%) placebo group patients. The investigators considered that 3 (2%) of the minoxidil group and none of the placebo group had moderate hair regrowth and that none had dense regrowth. After 48 weeks treatment 28 (23%) patients considered that they had moderate hair regrowth and the investigators considered that 14 (12%) patients had moderate regrowth. None had dense growth. No serious adverse reactions or deaths were reported. Minoxidil solution appeared to be an efficacious and safe treatment for early androgenic alopecia.

Generalised eruptive syringoma--a papular dermatosis.

Generalized eruptive syringoma is a rare condition characterised by multiple papules over the anterior trunk, sometimes extending on to the flexural aspects of the arms and thighs. A 22 year old Ethiopian woman with this disorder is presented.

Acquired zinc deficiency in a breast-fed premature infant.

Zinc deficiency that was diagnosed at 14 weeks of age developed in a breast-fed premature infant. There was a rapid response to zinc supplements (20 mg/day) and therapy was stopped after three weeks without recurrent disease. The maternal breast milk had a low level of zinc and this could not be corrected by oral zinc supplements.

The distribution of uterine glycogen during early pregnancy in the young and senescent golden hamster.

Glycogen distribution in the uterus of young and old hamsters was examined histochemically during the implantation of the embryo. Decidual cells and glycogen first appeared at 96 hours (post-ovulation) in the young animal. Decidualization did not occur in the senescent animal until 108 hours of development and the appearance of glycogen in the antimesometrial stroma was further delayed until 120 hours of development in the aged female. By 132 hours glycogen synthesis in the antimesometrial decidua was similar in both groups. Mesometrial glycogen synthesis in the aged animal continued to lag behind that of the young animal throughout the period investigated. The role of the delayed decidual cell response and the appearance of glycogen as possible causal factors in the observed decreased reproductive output in senescent animals is discussed.