(Pro)renin receptor is associated with angiogenic activity in proliferative diabetic retinopathy.

AIMS/HYPOTHESIS: The renin-angiotensin system (RAS) potentially has a role in the development of end-organ damage, and tissue RAS activation has been suggested as a risk factor for diabetic retinopathy. We have recently shown significant involvement of (pro)renin receptor ([P]RR) in retinal inflammation in a rodent model of early diabetes. In this study we aim to elucidate the (P)RR-associated pathogenesis of fibrovascular proliferation, a late-stage angiogenic complication in human diabetic retinopathy. METHODS: Vitreous fluids from 23 eyes of patients with proliferative diabetic retinopathy (PDR) and 16 eyes of controls with non-diabetic, idiopathic macular diseases (macular hole and epiretinal membrane) were collected. Protein levels of soluble (P)RR were measured by ELISA, and immunofluorescence was performed to assess the localisation of (P)RR and related molecules in fibrovascular tissues from PDR eyes. RESULTS: (P)RR immunoreactivity was detected in neovascular endothelial cells, colocalised with prorenin, phosphorylated extracellular signal-regulated kinase (ERK) and vascular endothelial growth factor (VEGF). Prorenin application to human retinal microvascular endothelial cells significantly upregulated mRNA expression of VEGF, especially the VEGF165 isoform, which was abolished by (P)RR or ERK signalling blockade. Proteases known to cleave (P)RR, including furin, were positive in endothelial cells in fibrovascular tissues. Protein levels of soluble (P)RR in vitreous fluids were higher in PDR eyes than in non-diabetic control eyes, and correlated significantly with vitreous prorenin and VEGF levels and the vascular density of fibrovascular tissues. CONCLUSIONS/INTERPRETATION: Our data using human samples provide the first evidence that (P)RR is associated with angiogenic activity in PDR.

Delta15N of soil N and plants in a N-saturated, subtropical forest of southern China.

We investigated the delta(15)N profile of N (extractable NH(4)(+), NO(3)(-), and organic N (EON)) in the soil of a N-saturated subtropical forest. The order of delta(15)N in the soil was EON > NH(4)(+) > NO(3)(-). Although the delta(15)N of EON had been expected to be similar to that of bulk soil N, it was higher than that of bulk soil N by 5 per thousand. The difference in delta(15)N between bulk soil N and EON (Delta(15)N(bulk-EON)) was correlated significantly with the soil C/N ratio. This correlation implies that carbon availability, which determines the balance between N assimilation and dissimilation of soil microbes, is responsible for the high delta(15)N of EON, as in the case of soil microbial biomass delta(15)N. A thorough delta(15)N survey of available N (NH(4)(+), NO(3)(-), and EON) in the soil profiles from the organic layer to 100 cm depth revealed that the delta(15)N of the available N forms did not fully overlap with the delta(15)N of plants. This mismatch in delta(15)N between that of available N and that of plants reflects apparent isotopic fractionation during N uptake by plants, emphasizing the high N availability in this N-saturated forest.

Expression of glutamine synthetase and cell proliferation in human idiopathic epiretinal membrane.

BACKGROUND/AIM: The mechanisms of the cellular origin and cell proliferation in the idiopathic epiretinal membrane (ERM) are unsolved. The aim of this study was to examine the expression of cell cycle related molecules and glutamine synthetase (GS), which is expressed in Müller cells and their processes, in ERM tissues. METHODS: The ERMs were surgically removed using pars plana vitrectomy. Formalin fixed, paraffin embedded ERM tissues were analysed by immunohistochemistry with anti-cyclin D1, p27 (KIP1), proliferating cell nuclear antigen (PCNA), and GS antibodies. RESULTS: The histopathological findings showed that all the ERMs consisted of oval or spindle mononuclear cells with thin collagen-like tissues. Immunoreactivity for GS was detected in collagen-like tissues of ERM, presenting a continuous, isodense pattern. GS immunopositive cells in all cases expressed PCNA in their nuclei. Nuclear immunoreactivity for cyclin D1 was noted in the ERM constituent cells, whereas p27 (KIP1) positive nuclei were not detected. CONCLUSION: Cyclin D1 and PCNA were expressed in the idiopathic ERM, which was mainly derived from Müller cells and extensions of their processes.

Elevations of AGE and vascular endothelial growth factor with decreased total antioxidant status in the vitreous fluid of diabetic patients with retinopathy.

BACKGROUND/AIMS: Advanced glycation end product (AGE) induces vascular endothelial growth factor (VEGF) expression in cell culture and animal models, being considered to be involved in development of diabetic retinopathy; oxidative stress also has a part in diabetic retinopathy. However, the interrelations between AGE, VEGF, and oxidative stress remain to be elucidated. In this study, vitreous AGE, VEGF, and total antioxidant levels in were determined in diabetic patients with retinopathy, and the relations among them investigated. METHODS: ELISA (enzyme linked immunosorbent assay) was used to determine the vitreous levels of AGE and VEGF in 41 patients with diabetic retinopathy and 28 non-diabetic control subjects. Total antioxidant levels in vitreous of 20 diabetic patients and 18 controls were also analysed by ELISA. RESULTS: The vitreous levels of AGE and VEGF were significantly higher in diabetic patients than in control subjects (p<0.01 for both). There was a significant correlation between the vitreous AGE and VEGF levels (p<0.001). Total antioxidant status was decreased in vitreous in patients with diabetes compared with the controls (p<0.01). Furthermore, both AGE and VEGF levels were inversely correlated with the total antioxidant status (p<0.01 and p<0.05, respectively). CONCLUSION: This study suggests that AGE and decreased total antioxidant status may contribute to the pathogenesis of diabetic retinopathy via induction of VEGF.

Dhh1 regulates the G1/S-checkpoint following DNA damage or BRCA1 expression in yeast.

BACKGROUND: Heterologous expression of the tumor suppressor BRCA1 in the yeast Saccharomyces cerevisiae is lethal. To identify potential new BRCA1-interacting gene targets, we characterized highly conserved ionizing radiation (IR) sensitive gene deletions that suppress BRCA1-induced lethality in yeast. MATERIALS AND METHODS: Previously, we exposed an isogenic collection of yeast strains individually deleted for 4746 nonessential genes to IR and identified 199 radiation sensitive deletion strains. A subset (n = 130) of these were screened for those that suppressed the G1 arrest and lethality observed following galactose-induced expression from a GAL::BRCA1 plasmid in wild type yeast. RESULTS: We found that deletions of two core components of the highly conserved CCR4-NOT transcription complex (CCR4 or DHH1) rescued BRCA1-induced G1 arrest and lethality in yeast. This was not because of down regulation of the GAL promoter since both deletion strains produce large amounts of BRCA1 that is rapidly degraded. In addition, heterologous expression of BRCA1 results in increased transcription of the DNA damage-inducible reporter construct DIN::LacZ. Reduced viability following IR and nitrogen starvation was observed among strains deleted for CCR4 or DHH1 because of a defect in G1 to S phase checkpoint transition. Lethality following nitrogen starvation and IR was partially rescued in dhh1Delta strains by expressing the human ortholog of DHH1 (DDX6) which has been identified as a breakpoint oncogene.T CONCLUSIONS: hese results suggest that BRCA1 may promote genomic stability in human cells by interacting with the highly conserved ortholog of DHH1 (DDX6) to properly activate G1/S checkpoint arrest following DNA damage.

Morphological and functional analyses of adult onset vitelliform macular dystrophy.

AIM: To evaluate the morphology and visual function of the macula in eyes with adult onset vitelliform macular dystrophy (AVMD). METHODS: 12 eyes of six patients with AVMD were examined by ophthalmoscopy, scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), and multifocal electroretinography (mfERGs). The macular lesions were bilateral in all patients and varied from the typical vitelliform (five eyes), faded vitelliform changes with retinal pigment epithelium (RPE) atrophy (five eyes), and a normal fovea associated with small flecks around the macula (two eyes). RESULTS: SLO demonstrated small abnormal bright spots in the deep retina throughout the posterior retina in all cases. OCT showed a highly reflective fusiform thickened layer at the level of the RPE and choriocapillaris in patients with a submacular yellow vitelliform lesion. A well circumscribed, optically clear space was observed beneath the retinal layer in the macular lesions with RPE atrophy. The mfERGs were significantly reduced not only in the macular area but also in the outermost ring (20-30 degrees ) of the mfERGs. CONCLUSIONS: The submacular materials that accumulate within the RPE or subepithelial layers reported in previous histopathological studies of vitelliform lesions can be detected by OCT. In the macular lesions with RPE atrophy, the material may have disappeared leaving a subretinal or subepithelial optical clear space. These SLO and mfERG observations suggest that the morphological and functional abnormalities may not be localised just in the macular area but may be present throughout the posterior pole in eyes with AVMD.

The novel tubulin-binding drug BTO-956 inhibits R3230AC mammary carcinoma growth and angiogenesis in Fischer 344 rats.

BTO-956 [methyl-3,5-diiodo-4-(4'-methoxyphenoxy)benzoate], a novel tubulin-binding drug and thyroid hormone analogue, was originally found to inhibit human carcinoma cell proliferation in vitro and to have potent growth delay activity in human breast and ovarian carcinoma xenografts in nude mice. Here we report that BTO-956 given to Fischer 344 rats also inhibits corneal angiogenesis and the growth and neovascularization of the R3230Ac rat mammary carcinoma tumor implanted in skin-fold window chambers. Hydron pellets containing recombinant human basic fibroblast growth factor (50 ng) and Sucralfate (20 microg) were implanted into surgically created corneal micropockets (day 0). BTO-956 was administrated by oral gavage (500 mg/kg, twice a day for 6 days) on days 1-6 (controls received vehicle alone). On day 7, rats received retrograde infusions of India ink via the thoracic aorta to visualize the corneal vasculature. Digitized images of slide-mounted corneas from control and treated animals were taken with a microscope. For the tumor growth and angiogenesis study, small pieces of R3230Ac tumor from a donor rat were implanted into surgically prepared window chambers (day 0). BTO-956 was given during days 5-11, and images of the tumors and their vasculature were recorded on day 12. No body weight loss was observed in either study. BTO-956 significantly inhibited corneal angiogenesis (by 50-80%), as assessed by measurements of limbal circumference displaying neovascularization, vessel length, vascularized area, and vascular area density. In the window chamber assay, tumors from treated animals were >50% smaller than tumors in control animals. In addition, vascular length densities in peripheral tumor zones were 30% less in treated compared with control animals. Together, these findings demonstrate that BTO-956 can inhibit angiogenesis induced by a growth factor in the rat cornea and in the peripheral area of implanted tumors, where tumor angiogenesis is most active.

Tamoxifen inhibits angiogenesis in estrogen receptor-negative animal models.

Inhibition of tumor angiogenesis is a therapeutic strategy that can inhibit tumor growth and metastases. The aim of this study was to determine whether the estrogen receptor (ER) ligand drug tamoxifen has antiangiogenic effects. We used three different models of angiogenesis, including measurement of microvessel densities in murine tumors, ex vivo aortic ring assays, and corneal pocket assays. ER-negative fibrosarcoma tumors in tamoxifen-treated ovariectomized rats had significantly less vessel formation compared with untreated animals (median microvessel density, 53.6 versus 94.3 counts/per x 200 field; P = 0.002). Rat aortic rings treated with tamoxifen at several different concentrations demonstrated significantly less vascular sprouting than control rings (P = 0.0001). Corneal pocket assays performed in tamoxifen-treated rats compared with control and estrogen-treated rats demonstrated decreased vascular length (0.88 mm versus 1.26 mm versus 1.47 mm; P = 0.022) and vessel area (21% versus 34% versus 47%; P = 0.018). These three animal models all showed significant inhibition of angiogenesis by tamoxifen and suggest a possible contributory mechanism of ER-independent manipulation by tamoxifen in the treatment and prevention of breast cancer. These studies raise the question as to whether or not newer ER ligand drugs might possess even more potent antiangiogenic effects, which in turn could lead to the broadening of the clinical usefulness of these compounds in a number of diseases. More importantly, these studies suggest that the antiangiogenic effects of tamoxifen are due, in part, to ER-independent mechanisms.

Histopathological analysis of a bladder cancer stalk observed on MRI.

Papillary transitional cell carcinoma of the bladder has a loose connective tissue stalk. For staging of bladder cancer on magnetic resonance imaging (MRI), it is important to clearly separate the cancer from the bladder wall. It is possible to distinguish a stalk from the cancer by the difference of intensity on the using MRI. Sixteen stalks of 20 polypoid bladder tumors on any of the T(2)W(I), dynamic images and delayed enhanced images were demonstrated. Most of the stalks show lower signal intensity than the tumors on T(2)W(I), less enhancement on dynamic images and stronger enhancement on delayed enhanced images. The stalk consisted of fibrous connective tissue, capillary blood vessels, inflammatory cell infiltration and edema. This stalk extended from the bladder wall to the center of the tumor. Some of the superficial muscular bundles were pulled into the stalk. These histopathological findings were compatible with the patterns of signal intensities on MRI. The identification of the stalk of a polypoid tumor may be an important observation to exclude bladder wall invasion by tumor.

Differential sensitivity to ATP-sensitive potassium channel openers of norepinephrine-induced contraction of guinea pig and rat aorta.

Vasorelaxant effects of ATP-sensitive potassium (K(ATP)) channel openers were examined on the tonic phase of vascular contraction induced by norepinephrine (NE) in guinea pig and rat aorta. K(ATP) channel openers, NIP-121 and cromakalim, produced glibenclamide-sensitive and concentration-dependent relaxations in guinea pig and rat aorta preconstricted with NE. However, the vascular relaxations induced by both K(ATP) channel openers were less pronounced in guinea pig aorta than in rat aorta. D-cis-Diltiazem, at the concentration up to 10(-5) M, did not appreciably inhibit the NE-induced contraction of guinea pig aorta, whereas the compound almost completely inhibited the NE-induced contraction of rat aorta at the same concentration. By contrast, sodium nitroprusside relaxed the NE-induced contractions in both guinea pig and rat aorta with similar potencies. These findings suggest that vasorelaxant effects of K(ATP) channel openers on the NE-induced sustained contraction in guinea pig aorta is not attributable to the subsequent inhibition of Ca2+ influx through L-type voltage-gated Ca2+ channels. Lower sensitivity of guinea pig aortic smooth muscle to K(ATP) channel openers is most likely due to the low dependence of NE-induced contraction on the Ca2+ influx in this vascular smooth muscle.

Fenretinide-induced apoptosis of human head and neck squamous carcinoma cell lines.

BACKGROUND: Squamous cell carcinoma of the head and neck (HNSCC) has a high incidence of recurrence and associated second primary malignancy. The retinoid 13-cis-retinoic acid has been shown to be effective as both a chemopreventive and chemotherapeutic agent for HNSCC, but often with treatment-limiting toxicity. The synthetic retinoid fenretinide (N-(4-hydroxyphenyl)retinamide) (HPR) has significant antiproliferative activity against a number of animal and human malignancies and has been used in clinical trials as a chemopreventive agent in patients with breast and prostate cancer and oral leukoplakia. HPR has been shown to have a toxicity profile lower than that for other retinoids used in clinical trials. PURPOSE: The aim of this study was to investigate the effect of HPR on the growth of HNSCC cell lines in vitro. METHODS: Four HNSCC cell lines (JHU-011-SCC, JHU-020-SCC, JHU-022-SCC, and FaDu) were treated with a range of concentrations of HPR for various times. After HPR exposure, cell viability was determined by tetrazolium dye (MTT) colorimetric assay, comparing cell survival with that of untreated control cells. HPR-induced apoptosis was determined by flow-cytometric deoxyribonucleic acid cell-cycle analysis, ultrastructural analysis with electron microscopy, and deoxyribonucleic acid fragmentation detected by gel electrophoresis. RESULTS: HPR caused significant growth inhibition in three of the four HNSCC cell lines in a dose- and time-dependent fashion. In two cell lines (JHU-011-SCC, JHU-020-SCC) a significant antiproliferative effect was achieved between 1 and 2.5 micromol/L HPR after 72 hours of treatment. By deoxyribonucleic acid cell-cycle analysis, electron microscopy, and gel electrophoresis, HPR was shown to induce apoptosis in the JHU-011-SCC and JHU-020-SCC cell lines, but not in the FaDu cell line, which was insensitive to the growth inhibitory effect of HPR. CONCLUSIONS: This study has demonstrated that HPR reduces cell viability in HNSCC cells in vitro at clinically relevant doses, with the growth inhibition occurring through the induction of apoptosis.

Temperature-sensitive effects of potassium channel openers on isolated guinea pig myocardium and aorta.

The effects of K+ channel openers, NIP-121, cromakalim, and pinacidil, on isolated myocardium and aorta were investigated at two different temperatures, 23 degrees C and 37 degrees C. In right ventricular myocardium, NIP-121 shortened the action-potential duration with little influence on other action-potential parameters at 37 degrees C, but not at 23 degrees C. In whole-cell clamped ventricular myocytes, NIP-121 induced a glibenclamide-sensitive outward current at 37 degrees C but not at 23 degrees C. No difference in tissue adenosine triphosphate (ATP) concentration was detected between ventricular myocardia incubated at 37 degrees C and at 23 degrees C. In aortic preparations precontracted with norepinephrine, NIP-121, cromakalim, and pinacidil produced endothelium-independent relaxation at 37 degrees C, which was antagonized by glibenclamide. The vasorelaxant effects were greatly reduced at 23 degrees C. Thus we demonstrated that the effects of K+ channel openers on the myocardium and vascular smooth muscle are temperature sensitive.

Isolation and characterization of matrix associated region DNA fragments in rice (Oryza sativa L.).

To investigate the interactions between chromosomal DNA and nuclear matrices in higher plants, matrix associated regions (MARs) of rice (Oryza sativa L.) DNAs were cloned. First, we prepared nuclear matrices from isolated nuclei by digesting them with EcoRI and then extracting with 2 M NaCl. About 6% of the total DNA remained in the nuclear matrices after this digestion and extraction. The residual DNA fragments in the nuclear matrices were cloned. Some of the cloned DNA fragments showed binding to certain nuclear proteins. One of the MAR fragments contained sequences related to known consensus motifs and a hairpin loop structure. A method is presented for isolation of matrix associated region (MAR) DNAs from plant cells.

Somatic hybrids obtained by fusion betweenPoncirus trifoliata (2x) andFortunella hindsii (4x) protoplasts.

Somatic hybrids were obtained by the symmetric fusion of embryogenic callus cells from tetraploid 'Mame' kumquat [Fortunella hindsii (Champ.) Swing.] and mesophyll cells from diploid trifoliate orange [Poncirus trifoliata (L.) Raf.]. Southern blot analysis of three regenerants revealed that they carried specific rDNA fragments from both fusion partners, thereby confirming their hybridity. In contrast, mitochondrial DNA (mtDNA) and chloroblast DNA (cpDNA) were unidirectionally transmitted from the callus parent without any evidence of recombination. No differences in the restriction fragment patterns of rDNA, mtDNA or cpDNA could be detected among the regenerants. Flow cytometry showed that two regenerants were hexaploids, as expected, but that one was pentaploid, probably due to elimination of chromosomes prior to the regeneration of this plant.

Myocardial and vascular effects of efonidipine in vitro as compared with nifedipine, verapamil and diltiazem.

1. Effects of efonidipine on isolated myocardial and aortic preparations were compared with those of nifedipine, verapamil and diltiazem. 2. All drugs produced concentration-dependent negative chronotropic effects on isolated guinea-pig atrial preparations. The potency order was efonidipine > or = nifedipine > diltiazem > or = verapamil, EC30 values being 3.08 x 10(-8)M, 3.48 x 10(-8)M, 1.27 x 10(-7)M and 1.47 x 10(-7)M, respectively. 3. Nifedipine, verapamil and diltiazem produced concentration-dependent negative inotropic effects on isolated guinea-pig left atrial preparations. The potency order was nifedipine > verapamil > diltiazem, EC30 values being 4.94 x 10(-8)M, 1.49 x 10(-7)M and 8.03 x 10(-7)M, respectively. Efonidipine, even at 1 microM produced no inotropic effect: 10 microM efonidipine decreased the contractile force by about 20%. 4. All drugs concentration-dependently attenuated the KCl-induced contraction of isolated rat aortic ring preparation. The potency order was nifedipine > efonidipine > verapamil > diltiazem, EC30 values being 2.98 x 10(-9)M, 1.24 x 10(-8)M, 3.96 x 10(-8)M and 2.13 x 10(-7)M, respectively. 5. Thus, efonidipine was demonstrated to be a potent vasodilator with negative chronotropic but minimal negative inotropic activity, which may be of benefit in the treatment of cardiovascular disorders.

Studies of marginal zinc deprivation in rhesus monkeys: infant behavior.

Rhesus monkey infants were marginally deprived of zinc (4 ppm diet) from conception and were compared to controls (100 ppm diet) during the first year of life in development of reflexes and motor patterns, mother-infant interaction, delayed response performance, discrimination learning and reversal, and open field behavior. Deficits in amount and variety of behavior were recorded in deprived infants; spontaneous locomotor activity was 50% below control levels in males at 1 mo of age; spontaneous activity was 7-10% lower in both males and females at 3 mo of age; response latencies were 50% lower than controls at 7-9 mo; failure to reach discrimination reversal criterion was seen in 71% of deprived infants as compared to 10% of controls at 10 mo of age; and abnormally low levels of climbing and exploration were seen in two of six deprived infants at 12 mo of age. No abnormalities in the rate of behavioral development or in emotional adaptability were observed. These and other results suggest that syndromes of lethargy, apathy, and hypoactivity are characteristic of behavioral effects of marginal zinc deprivation in primates.

Studies of marginal zinc deprivation in rhesus monkeys. IV. Growth of infants in the first year.

Growth was evaluated in rhesus monkey infants that were the offspring of females given a marginally zinc-deficient diet (4 micrograms/g zinc) from the beginning of pregnancy and through 12 months of postnatal life. These zinc-deficient (ZD) infants were compared to controls whose mothers were fed a complete diet, either ad libitum or pair-fed to zinc-deficient dams, throughout gestation and lactation. Male ZD infants had evidence of growth retardation at birth. In contrast, growth retardation in female ZD infants was not observed until 1 month of age. From 3 to 9 months of age (late lactation and subsequent to weaning) ZD infants attained weights similar to those of the control group. However, analysis of crown-rump and femur length indicated that ZD infants' growth was less than optimal throughout the entire 1st yr of observation. In addition, skinfold thickness was markedly higher in ZD than in control infants in the postweaning period. In the juvenile period (9-12 months of age) both male and female ZD animals fell behind controls in body weight. ZD juveniles were also hypogeusic, as determined by a quinine acceptance test. Low weight ZD infants had reduced somatic growth as reflected in sitting height, long bone growth, head circumference, and limb circumference. Regression analysis indicated that impaired growth rates from 9 to 12 months were associated with both lower food intake and reduced food use efficiency. Plasma zinc concentration was, in general, inversely related to weight gain in both groups during the 1st yr.