In 15-month-old db/db mice, signs of diabetic retinopathy, including blood-retinal barrier breakdown, loss of pericytes, neuro-retinal apoptosis, glial reactivation, and proliferation of blood vessels, were evident. These changes in the diabetic retina were associated with increased expression of aldose reductase (AR). To further understand the role of AR in the pathogenesis of diabetic retinopathy, we generated db/db mice with an AR null mutation (AR ؊/؊ db/db). AR deficiency led to fewer retinal blood vessels with IgG leakage, suggesting that AR may contribute to blood-retinal barrier breakdown. AR deficiency also prevented diabetes-induced reduction of platelet/endothelial cell adhesion molecule-1 expression and increased expression of vascular endothelial growth factor, which may have contributed to blood-retinal barrier breakdown. In addition, long-term diabetes-induced neuro-retinal stress and apoptosis and proliferation of blood vessels were less prominent in AR ؊/؊ db/db mice. These findings indicate that AR is responsible for the early events in the pathogenesis of diabetic retinopathy, leading to a cascade of retinal lesions, including blood-retinal barrier breakdown, loss of pericytes, neuro-retinal apoptosis, glial reactivation, and neovascularization. Diabetes 54:3119 -3125, 2005

Stroke patients have increased levels of endothelin-1 (ET-1), a strong vasoconstrictor, in their plasma or cerebrospinal fluid. Previously, we showed high level of ET-1 mRNA expression in astrocytes after hypoxia/ischemia. It is unclear whether the contribution of ET-1 induction in astrocytes is protective or destructive in cerebral ischemia. Here, we generated a transgenic mouse model that overexpress ET-1 in astrocytes (GET-1) using the glial fibrillary acidic protein promoter to examine the role of astrocytic ET-1 in ischemic stroke by challenging these mice with transient middle cerebral artery occlusion (MCAO). Under normal condition, GET-1 mice showed no abnormality in brain morphology, cerebrovasculature, absolute cerebral blood flow, blood-brain barrier (BBB) integrity, and mean arterial blood pressure. Yet, GET-1 mice subjected to transient MCAO showed more severe neurologic deficits and increased infarct, which were partially normalized by administration of ABT-627 (ET A antagonist) 5 mins after MCAO. In addition, GET-1 brains exhibited more Evans blue extravasation and showed decreased endothelial occludin expression after MCAO, correlating with higher brain water content and increased cerebral edema. Aquaporin 4 expression was also more pronounced in astrocytic end-feet on blood vessels in GET-1 ipsilateral brains. Our current data suggest that astrocytic ET-1 has deleterious effects on water homeostasis, cerebral edema and BBB integrity, which contribute to more severe ischemic brain injury.

No abstract

Inappropriate coordination of oocyte nuclear and cytoplasmic maturation is thought to contribute to the poor efficiency of embryo production in vitro. With the aim of improving this coordination, the effects of milrinone, an inhibitor of type 3 phosphodiesterases, and butyrolactone-I, a selective inhibitor of cdc2 kinases, on porcine oocyte maturation were investigated. Oocytes recovered from slaughterhouse-derived ovaries of prepubertal animals were treated with the inhibitors for 24 h. At concentrations of 50 and 250 microm, milrinone reversibly inhibited meiotic progression in 57% and 71% of oocytes, respectively. The presence or absence of milrinone in the medium used to wash oocytes for 30 min did not alter the inhibitory effect of the 24 h treatment. At concentrations of 25 and 50 microm, butyrolactone-I inhibited meiotic progression in 61% and 66% of oocytes, respectively, but the effect was not fully reversible in the absence of follicle-stimulating hormone (FSH). The presence of FSH during the butyrolactone-I treatment period increased the ability of oocytes to subsequently complete meiosis at 44 h without changing the inhibitory effect at 24 h. Following in vitro fertilisation at 44 and 50 h, treatment with butyrolactone-I and milrinone, alone or in combination, did not alter embryo cleavage rate, blastocyst formation rate or blastocyst cell number. Despite the different actions of milrinone and butyrolactone-I, the present study demonstrates that these reagents inhibit meiotic progression to a similar extent in the presence of FSH while maintaining developmental competence in porcine oocytes.

Chromosomal instability (CIN) is a common characteristic in testicular germ cell tumour (TGCT). A functional mitotic checkpoint control is important for accurate chromosome segregation during mitosis. Mitotic arrest deficient 2 (MAD2) is a key component of this checkpoint and inactivation of MAD2 is correlated with checkpoint impairment. The aim of this study was to investigate the function of mitotic checkpoint control in TGCT cells and to study its association with MAD2 expression using 8 TGCT cell lines as well as 23 TGCT tissue samples. We found that in response to microtubule disruption, 6 of 8 TGCT cell lines (75%) failed to arrest in mitosis demonstrated by the decreased mitotic index and aberrant expression of mitosis regulators, indicating that mitotic checkpoint defect is a common event in TGCT cells. This loss of mitotic checkpoint control was correlated with reduced MAD2 protein expression in TGCT cell lines implicating that downregulation of MAD2 may play a critical role in an impaired mitotic checkpoint control in these cells. In addition, immunohistochemistry studies on 23 seminomas and 12 normal testis tissues demonstrated that nuclear expression of MAD2 was much lower in seminomas (p<0.0001) but cytoplasmic MAD2 expression was higher in seminomas (p=0.06) than normal samples. Our results suggest that aberrant MAD2 expression may play an essential role in a defective mitotic checkpoint in TGCT cells, which may contribute to CIN commonly observed in TGCT tumours.

No abstract

Overexpression of the helix-loop-helix protein Id-1 has been reported in over 20 types of cancer. While a number of factors have been demonstrated to regulate Id-1 gene transcription, little is known about the mechanisms responsible for its degradation. In this study, we have demonstrated that Id-1 protein stability was regulated by TNFalpha in prostate cancer cells. We found that exposure of prostate cancer cell lines, DU145 and PC-3, to TNFalpha resulted in a rapid and significant downregulation of the Id-1 protein level. The fact that neither the Id-1 promoter activity nor the Id-1 mRNA level was affected by the TNFalpha treatment suggested that the decrease in Id-1 protein was not due to the suppression of gene transcription. In addition, the half-life of the Id-1 protein was decreased in both cell lines in the presence of TNFalpha, and the addition of an ubiquitin/proteasome inhibitor (MG-132) prior to the TNFalpha treatment completely blocked the effect of the TNFalpha-induced Id-1 protein degradation. Furthermore, introduction of a Flag-tag sequence into the N-terminus region of the Id-1 protein, which has been shown to stabilize the protein, was able to protect the Id-1 protein from TNFalpha-induced degradation. These results suggest that TNFalpha downregulated Id-1 through activation of the ubiquitin/proteasome degradation pathway in prostate cancer cells. Interestingly, in both DU145 and PC-3 cells, the decrease of Id-1 protein was associated with the activation of apoptotic pathway, as evidenced by the increased expression of cleaved PARP and caspase 3. In addition, TNFalpha failed to downregulate Id-1 in a sub-line of LNCaP cells that was resistant to TNFalpha-induced apoptosis. These results further suggest that the downregulation of Id-1 may facilitate TNFalpha-induced apoptosis in prostate cancer cells. In conclusion, our findings indicate that Id-1 protein may be regulated by TNFalpha through the ubiquitin/proteasome degradation pathway and the stability of the Id-1 protein appears to correlate with the sensitivity of TNFalpha-induced apoptosis.

It is generally believed that too high or low levels of endothelin-1 (ET-1), a strong vasoconstrictor, may be detrimental to animals. Therefore, in order to understand the in vivo function of ET-1, we used a conditional transgenic approach, Cre/loxP recombination system, to generate transgenic mice that over-express ET-1 in a tissue-specific manner. In such a strategy a single transgenic mouse line, ELSE, was initially generated where a general promoter, human elongation factor 1alpha (hEF1alpha) promoter, was used to drive the expression of a loxP-flanked sequence containing the lacZ reporter gene and a STOP cassette before the ET-1 cDNA, the recombinational competency of which was confirmed in an Escherichia coli test system. In ELSE mice, expression of the reporter lacZ was limited to spermatozoa and spermatogonia as well as Sertoli, Leydig and endothelial cells in the testis, thus confirming the suitability of these mice for the generation of testes-limited ET-1 expression. To generate transgenic progeny with ET-1 over-expression in the testis (successful recombination, ELSE/ELT), ELSE mice were mated with EIIa-cre mice expressing Cre recombinase in pre-implantation mouse embryos. These ELSE/ELT mice exhibiting testis-specific ET-1 over-expression had normal reproductive function and showed no obvious alterations in gross testicular morphology. Although over-expression of ET-1 leads to reduction of testicular blood flow, young adult ELSE/ELT mice showed no obvious signs of inflammation, fibrosis or abnormal proliferation of cells in the testes of young ELSE/ELT mice by histochemical analyses.