Expression and localization of mutant p16 proteins in melanocytic lesions from familial melanoma patients

Little is known about the correlation between the loss of p16 expression and tumor progression in familial melanoma; no systematic study has been conducted on p16 expression in melanocytic tumors from patients carrying germline CDKN2A mutations. We analyzed 98 early primary lesions from familial patients, previously tested for germline CDKN2A status, by quantitative immunohistochemistry using 3 p16 antibodies. We found that p16 expression was inversely correlated with tumor progression and was significantly lower in melanomas, including in situ lesions, than in nevi. Of other features analyzed, tumor thickness showed the most significant correlation with p16 levels. Lesions from mutation-negative patients displayed combined nuclear and cytoplasmic staining. However, some mutation-positive lesions (ie, G101W, 113insR, M53I, R24P, and 33ins24), including benign nevi, showed nuclear mislocalization, confirming previous studies suggesting that subcellular distribution indicates functional impairment of p16.     

Protective effects of PJ34, a novel, potent inhibitor of poly(ADP-ribose) polymerase (PARP) in in vitro and in vivo models of stroke

Focal cerebral ischemia activates the nuclear protein poly(ADP-ribose) polymerase (PARP) by single DNA strand breaks which leads to energy depletion and cell necrosis. Deletion or inhibition of PARP protects against ischemic brain injury. Here we examined the neuroprotective effect of PJ34, a novel potent inhibitor of PARP in vitro and in vivo. Serum-free primary neuronal cultures derived from rat cortex (E15-17) and kept in culture for 10 days were exposed to oxygen glucose deprivation (OGD) in vitro. Neuronal injury was quantified by LDH release after 24 h. Pretreatment with 30-1000 nM PJ34 significantly protected from OGD-induced cell injury in a dose-dependent manner. For in vivo experiments SV/129 mice were treated with PJ34 (50 microg) by intraperitoneal injection 2 h before 1 h middle cerebral artery occlusion (MCAo) and again 6 h later. Twenty-three h after reperfusion ischemic injury was significantly decreased compared to vehicle-treated controls (infarct volume reduction of 40%, p<0.05). Similarly, in a rat model of MCAo (2 h occlusion followed by up to 22 h reperfusion), PJ34 administration (10 mg/kg i.v.) significantly reduced infarct size, and the effect of the drug was maintained even if it was given as late as 10 min prior to reperfusion time. PJ34 significantly protected in a 4 h, but not in a 24 h permanent occlusion model. In conclusion, PJ34, a novel, potent inhibitor of PARP exerts massive neuroprotective agents, with a significant therapeutic window of opportunity. The present work strengthens the concept that pharmacological PARP inhibition may be a suitable approach for the treatment of acute stroke in man.     

Depletion of calcitonin gene-related peptide from the caudal trigeminal nucleus of the rat after electrical stimulation of the Gasserian ganglion

Electrical stimulation of the Gasserian ganglion resulted in partial depletion of calcitonin gene-related peptide (CGRP) from ipsilateral central terminals of pseudounipolar primary sensory ganglion cells. Affected terminals exhibit decreased CGRP immunoreactivity as shown by cytophotometric densitomery of the caudal trigeminal nucleus. The decrease in CGRP immunoreactivity is statistically significant only in the medial one-third of the caudal trigeminal nucleus. Since earlier studies have shown that electrical stimulation of the Gasserian ganglion induces first accumulation then depletion of CGRP from perivascular sensory terminals in the dura mater, the present experiments suggest that CGRP is depleted also from central terminals of primary sensory trigeminal neurons, which might be of importance in the pathogenesis of migraine headache. Received: 18 December 1996 / Accepted: 26 June 1997     

Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases

Tay-Sachs and Sandhoff diseases are autosomal recessive neurodegenerative diseases resulting from the inability to catabolize GM2 ganglioside by beta-hexosaminidase A (Hex A) due to mutations of the alpha subunit (Tay-Sachs disease) or beta subunit (Sandhoff disease) of Hex A. Hex B (beta beta homodimer) is also defective in Sandhoff disease. We previously developed mouse models of both diseases and showed that Hexa-/- (Tay-Sachs) mice remain asymptomatic to at least 1 year of age while Hexb-/- (Sandhoff) mice succumb to a profound neurodegenerative disease by 4-6 months of age. Here we find that neuron death in Hexb-/- mice is associated with apoptosis occurring throughout the CNS, while Hexa-/- mice were minimally involved at the same age. Studies of autopsy samples of brain and spinal cord from human Tay-Sachs and Sandhoff diseases revealed apoptosis in both instances, in keeping with the severe expression of both diseases. We suggest that neuron death is caused by unscheduled apoptosis, implicating accumulated GM2 ganglioside or a derivative in triggering of the apoptotic cascade.      

The role of recombination signal sequences in the preferential joining by deletion in DH-JH recombination and in the ordered rearrangement of the IgH locus

The bias favoring deletion over inversion in DH-JH rearrangement has been known for years, but the underlying mechanism has yet to be fully defined. It has been suggested that the ratio of deletion/inversion is determined by the combined effect of two factors: (i) the relative strengths of 5' and 3' recombination signal sequences (RSS) of a DH segment, and (ii) the efficiency with which the deletional product (one joint) forms relative to the inversional product (two joints). In this study, we analyzed for the first time the effect of factor 1 alone on the biased 3' RSS utilization in DH-JH joining by using deletional plasmids in an extrachromosomal substrate V(D)J recombination assay. It was found that the 3' RSS and associated coding end (12 bp) mediate recombination more efficiently than the 5' RSS/coding end DH-JH plasmids. These results demonstrate that the effect of the RSS/coding end alone can account, at least partially, for the predominant deletion in DH-JH recombination. The potential effect of the relative strength of RSS and associated coding end on the ordered rearrangement of DH-JH followed by VH to DH-JH was also assessed. When recombination frequencies of D-->J (3' DH to J3) were compared with frequencies of V-- >D (VHPJ14 to 3' DH or VHOX2 to 3' DH), it was found that V-->D joining was, if anything, more efficient than D-->J joining. Therefore, if all three segments were accessible, RSS/coding end effects would not contribute to the ordered rearrangement of the IgH locus.      

Mechanisms of enhanced prevalence of asthma and atopy in developed countries

Atopy — a T helper 2 cell driven hypersensitivity to innocuous antigens (allergens) which causes most cases of asthma — is of complex genetic and environmental origins. There is compelling epidemiological evidence for a rise in atopic disease in ‘westernised’ communities. The changing pattern of microbial exposure in early childhood is suggested to be the principal candidate mechanism for this rise.     

Issues to debate on the Women's Health Initiative: estrogen: an instrument or the conductor of the orchestra?

Although it is well known that cyclic production of sex hormones is essential to establish reproductive function and female characteristics, distant impacts of the activity of the female endocrine system result from a concert of delicate mechanisms. Estrogen is rather an instrument than a conductor in this physiological orchestra of the female. Thus, controversies in the explanation of results from studies on hormone replacement therapy (HRT) and cardiovascular disease (CVD) prevention might be eliminated, if we analyse not only the role of estrogen but a broader spectrum of factors leading to CVD. Authors would like to hypothesize that haemorheological changes in women around menopause, such as increased blood and plasma viscosity, haematocrit and fibrinogen, are largely responsible for the increased mortality in the post-menopausal life period. We believe that a cyclic withdrawal bleeding establishes a more favourable haemorheological condition, thus, sequentially administered estrogen might be protective in post-menopausal women. Nevertheless, other factors, that decrease blood viscosity, such as daily exercise, intake of ample amount of fluids as well as ideal nutrition, are equally important. We are confident that sequential HRT, as well as healthy life style and risk prevention programmes have their proper place in the management of this issue.     

Dietary micronutrients/antioxidants and their relationship with bronchial asthma severity

BACKGROUND: Because little is known about micronutrient/antioxidant intake and asthma severity, we investigated dietary intake and plasma/serum levels of micronutrients/antioxidants in a group of asthma patients with various degrees of severity, and compared the results with healthy subjects. METHODS: A case control study was carried out on 118 asthma patients and 121 healthy subjects. The severity of the disease was classified by division of patients into four groups. Normal dietary micronutrient/antioxidant intake was estimated from a food frequency questionnaire. Plasma/serum levels of vitamins C, E, and A, selenium, magnesium, zinc, and platelet glutathione peroxidase (GSH-Px) activity were also determined. RESULTS: No differences in daily micronutrient/antioxidant intake were seen between patients and healthy subjects. The severity of the disease showed no significant relationship with micronutrient/antioxidant intake. There were no differences in plasma/serum levels in any of the micronutrients/antioxidants between healthy subjects and asthmatics. Nor were any differences found between asthma groups in severity in the biochemical measures, except in platelet GSH-Px activity, which was significantly lower in the most severe groups. CONCLUSIONS: In this study, we found no evidence of any association between micronutrient/antioxidant intake or plasma/serum levels of micronutrients/antioxidants and asthma. Reduction of platelet GSH-Px activity in the most severe patients suggests that these patients have a diminished capacity to restore part of the antioxidant defences.