Hiroko Ushikubo

We determined the complete nucleotide sequence of the human endogenous retrovirus genome HERV-K10 isolated as the sequence homologous to the Syrian hamster intracisternal A-particle (type A retrovirus) genome. HERV-K10 is 9,179 base pairs long with long terminal repeats of 968 base pairs at both ends; a sequence 290 base pairs long, however, was found to be deleted. It was concluded that a composite genome having the 290-base-pair fragment is the prototype HERV-K provirus gag (666 codons), protease (334 codons), pol (937 codons), and env (618 codons) genes. The size of the protease gene product of HERV-K is essentially the same as that of A- and D-type oncoviruses but nearly twice that of other retroviruses. A comparison of the deduced amino acid sequences encoded by the pol region showed HERV-K to be closely related to types A and D retroviruses and even more so to type B retrovirus. It was noted that the env gene product of HERV-K structurally resembles the mouse mammary tumor virus (type B retrovirus) env protein, and the possible expression of the HERV-K env gene in human breast cancer cells is discussed.

Expression of the human endogenous retrovirus genome HERV-K, homologous to mouse mammary tumor virus, was investigated in cultured human tumor cells. In several cell lines, the HERV-K genome was expressed as an 8.8-kilobase poly(A)+ RNA which appeared to be a full-size transcript of this genome. In the human breast cancer cell line T47D, stimulation of HERV-K genome expression by progesterone was observed after estradiol treatment.

Ocular pathologic angiogenesis is a causative factor for retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. In the present study, we examined the effects of rapamycin and everolimus, inhibitors of mammalian target of rapamycin (mTOR), on retinal pathologic angiogenesis in mice with oxygen-induced retinopathy (OIR), an animal model of proliferative ischemic retinopathy. Mice were exposed to 80% oxygen from postnatal day (P) 7 to P10, and were then brought into room air and subcutaneously injected with rapamycin and everolimus. The neovascular tufts, the size of the central avascular zone, and the immunoreactivity for phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTOR activity, were evaluated in flat-mounted retinas. Retinal neovascular tufts and vascular growth in the avascular zone were observed in P15 mice with OIR. In addition, intense immunoreactivity for pS6 was detected in the neovascular tufts and in endothelial cells located at the vascular-avascular border. Both rapamycin and everolimus reduced the extent of retinal neovascular tufts and pS6 immunoreactivity, but they also increased the size of the avascular zone. Thus, activation of the mTOR pathway in endothelial cells contributes to retinal pathologic angiogenesis, and mTOR inhibitors that target proliferating endothelial cells are promising candidates as anti-angiogenic agents for the treatment of vasoproliferative retinal diseases.

Cigarette smoking is a risk factor for the development of airway hyperresponsiveness and chronic obstructive pulmonary disease. Little is known concerning the effect of cigarette smoking on the contractility of airway smooth muscle. The current study was performed to determine the responsiveness of bronchial smooth muscles isolated from rats that were subacutely exposed to mainstream cigarette smoke in vivo. Male Wistar rats were exposed to diluted mainstream cigarette smoke for 2 h/d every day for 2 wk. Twenty-four hours after the last cigarette smoke exposure, a marked airway inflammation (i.e., increases in numbers of neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid and peribronchial tissues) was observed. In these subacutely cigarette smoke-exposed animals, the responsiveness of isolated intact (nonpermeabilized) bronchial smooth muscle to acetylcholine, but not to high K+ -depolarization, was significantly augmented when compared with the air-exposed control group. In alpha-toxin-permeabilized bronchial smooth muscle strips, the acetylcholine-induced Ca2+ sensitization of contraction was significantly augmented in rats exposed to cigarette smoke, although the contraction induced by Ca2+ was control level. Immunoblot analyses revealed an increased expression of RhoA protein in the bronchial smooth muscle of rats that were exposed to cigarette smoke. Taken together, these findings suggest that the augmented agonist-induced, RhoA-mediated Ca2+ sensitization may be responsible for the enhanced bronchial smooth muscle contraction induced by cigarette smoking, which has relevance to airway hyperresponsiveness in patients with chronic obstructive pulmonary disease.