Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model.

Mutations in the extracellular matrix gene Fibrillin-2 (FBN2) are related to genetic macular degenerative disorders including age-related macular degeneration (AMD) and early-onset macular degeneration (EOMD). It was reported that the retinal protein expression of FBN2 was reduced in patients with AMD and EOMD. The effect of exogenously supplied fbn2 recombinant protein on fbn2-deficiency-related retinopathy was not known. Here we investigated the efficacy and molecular mechanism of intravitreally applied fibrin-2 recombinant protein in mice with fbn2-deficient retinopathy. The experimental study included groups (all n = 9) of adult C57BL/6J male mice which underwent no intervention, intravitreal injection of adeno-associated virus (AAV) empty vector or intravitreal injection of AAV-sh-fbn2 (adeno-associated virus for expressing short hairpin RNA for fibrillin-2) followed by three intravitreal injections of fbn2 recombinant protein, given in intervals of 8 days in doses of 0.30 µg, 0.75 µg, 1.50 µg, and 3.00 µg, respectively. Eyes with intravitreally applied AAV-sh-fbn2 as compared to eyes with injection of AAV-empty vector or developed an exudative retinopathy with involvement of the deep retinal layers, reduction in axial length and reduction in ERG amplitudes. After additional and repeated application of fbn2 recombinant protein, the retinopathy improved with an increase in retinal thickness and ERG amplitude, the mRNA and protein expression of transforming growth factor-beta (TGF-ß1) and TGF-ß binding protein (LTBP-1) increased, and axial length elongated, with the difference most marked for the dose of 0.75 µg of fbn2 recombinant protein. The observations suggest that intravitreally applied fbn2 recombinant protein reversed the retinopathy caused by an fbn2 knockdown.

Docking-based structural splicing and reassembly strategy to develop novel deazapurine derivatives as potent B-RafV600E inhibitors.

The mutation of B-RafV600E is widespread in a variety of human cancers. Its inhibitors vemurafenib and dabrafenib have been launched as drugs for treating unresectable melanoma, demonstrating that B-RafV600E is an ideal drug target. This study focused on developing novel B-RafV600E inhibitors as drug leads against various cancers with B-RafV600E mutation. Using molecular modeling approaches, 200 blockbuster drugs were spliced to generate 283 fragments followed by molecular docking to identify potent fragments. Molecular structures of potential inhibitors of B-RafV600E were then obtained by fragment reassembly followed by docking to predict the bioactivity of the reassembled molecules. The structures with high predicted bioactivity were synthesized, followed by in vitro study to identify potent B-RafV600E inhibitors. A highly potent fragment binding to the hinge area of B-RafV600E was identified via a docking-based structural splicing approach. Using the fragment, 14 novel structures were designed by structural reassembly, two of which were predicted to be as strong as marketed B-RafV600E inhibitors. Biological evaluation revealed that compound 1m is a potent B-RafV600E inhibitor with an IC50 value of 0.05 µmol/L, which was lower than that of vemurafenib (0.13 µmol/L). Moreover, the selectivity of 1m against B-RafWT was enhanced compared with vemurafenib. In addition, 1m exhibits desirable solubility, bioavailability and metabolic stability in in vitro assays. Thus, a highly potent and selective B-RafV600E inhibitor was designed via a docking-based structural splicing and reassembly strategy and was validated by medicinal synthesis and biological evaluation.

Eutypenoids A-C: Novel Pimarane Diterpenoids from the Arctic Fungus Eutypella sp. D-1.

Eutypenoids A-C (1-3), pimarane diterpenoid alkaloid and two ring A rearranged pimarane diterpenoids, were isolated from the culture of Eutypella sp. D-1 obtained from high-latitude soil of the Arctic. Their structures, including absolute configurations, were authenticated on the basis of the mass spectroscopy (MS), nuclear magnetic resonance (NMR), X-ray crystallography, and electronic circular dichroism (ECD) analysis. The immunosuppressive effects of eutypenoids A-C (1-3) were studied using a ConA-induced splenocyte proliferation model, which suggested that 2 exhibited potent immunosuppressive activities.

Adenovirus-mediated siRNA targeting NOB1 inhibits tumor growth and enhances radiosensitivity of human papillary thyroid carcinoma in vitro and in vivo.

NIN1/RPN12 binding protein 1 homolog (NOB1), a ribosome assembly factor, plays critical roles in tumor progression and development. Previously, we reported that overexpression of NOB1 is correlated with the prognosis of patients with papillary thyroid carcinoma (PTC). Little is known, however, concerning its role in PTC. The aims of the present study were to investigate the association of NOB1 expression with tumor growth and radiosensitivity of human PTC. A recombinant adenovirus expression vector carrying NOB1 was constructed and then infected into the human PTC cell line TPC-1. Cell proliferation, cell cycle distribution, apoptosis, migration and invasion in vitro and tumor growth in vivo were determined after downregulation of NOB1 by RNAi. Additionally, the in vitro and in vivo radiosensitivity of PTC cells was determined by clonogenic cell survival assay and a mouse xenograft model, respectively. The results showed that downregulation of NOB1 expression using RNAi in TPC-1 cells significantly inhibited cell proliferation, migration and invasion and induced cell apoptosis in vitro, and suppressed tumor growth in vivo, as well as enhanced the in vitro and in vivo radiosensitivity of PTC cells. Moreover, our results also showed that downregulation of NOB1 was able to significantly activate constitutive phosphorylation of p38 MAPK, which might contribute to the inhibition of PTC cell growth. These findings suggest that NOB1 may be a potential therapeutic target for the treatment of PTC.

Plasmid-based Stat3-specific siRNA and GRIM-19 inhibit the growth of thyroid cancer cells in vitro and in vivo.

It has been shown that overexpression of signal transducer and activator of transcription 3 (Stat3) contribute to the progression and metastasis of various solid tumors and that silencing Stat3 inhibits tumor growth in several types of cancer. Gene associated with retinoid-IFN-induced mortality 19 (GRIM-19), a Stat3-inhibitory protein, was identified as a potential tumor suppressor associated with growth inhibition and cell apoptosis by targeting the transcription factor Stat3 for inhibition. However, little is known about Stat3 and GRIM-19 roles in the tumor growth of thyroid carcinoma cells. In the present study, we developed a dual expression plasmid that co-expressed Stat3-specific siRNA and GRIM-19 (pSi-Stat3-GRIM-19) and transfected it into SW579 cells (thyroid carcinoma cell line) to evaluate its effects on cell proliferation, cell apoptosis, cell migration and cell invasion in vitro and tumor growth in vivo. Simultaneous expression of pSi-Stat3-GRIM-19 in SW579 cancer cells was found to significantly suppress the proliferation, migration and invasion in vitro and tumor growth in vivo, when compared to the controls either Stat3-specific siRNA or GRIM-19 alone. In conclusion, our data demonstrated that a combined strategy of co-expressed Stat3-specific siRNA and GRIM19 synergistically and more effectively suppressed thyroid tumor growth, and have therapeutic potential for the treatment of thyroid cancer.

Y chromosome interstitial deletion induced Y-STR allele dropout in AMELY-negative individuals.

Short tandem repeat (STR) multiplexes with the amelogenin (AMEL) gene as a gender marker have been used as a routine tool of forensic DNA analysis. It has been reported that AMEL-based gender detection could misidentify a known male as a female due to the dropout of amelogenin Y (AMELY) allele. Other gender markers, such as Y-chromosomal short tandem repeat (Y-STR), may be a substitution of AMEL and help the sex determination. In current study, employing AmpFlSTR® Sinofiler and AmpFlSTR® Y-filer™ PCR Amplification kit, 18 AMELY-negative males were identified. Accordingly, the incidence of the AMELY dropout was 0.227 ‰ (18/79,304) in Chinese population. Sequencing of AMELY allele and analyzing of azoospermia factors region suggested that 3 out of 18 misidentifications were induced by mutations in the primer-binding region of the AMELY, while other 15 sex misidentifications were results of Y chromosome microdeletions with variant lengths. Moreover, variant combination patterns of AMELY dropout and Y-STRs deletions were also observed. Our data suggested that Y-STR locus dropout may indicate more problems, especially in the mixed sample's interpretation. Results of haplogroup prediction showed that seven AMELY dropouts combined with variant Y-STR deletions can be classified as the J2 subdivision, suggesting that some of these Y chromosomes might descend from a common ancestor.

Raphe pallidus modulates Bötzinger complex-induced inhibition of the phrenic nerve activity in rats.

The raphe pallidus (RPa) and Bötzinger complex (BötC) represent two important nuclei which project to spinal phrenic motor neurons. Stimulation of the RPa produces facilitative effects on respiratory activity, whereas stimulation of the BötC induces inhibitory effects on respiratory activity. In the present study, we examined the modulatory effects of serotonergic (5-hydroxytryptamine, 5-HT) RPa neurons on the inhibitory response of the phrenic nerve activity elicited from the BötC in rats. Experiments were performed on spontaneously breathing, urethane-anesthetized adult rats. Either high-frequency stimulation or glutamatergic chemical activation of the RPa region significantly attenuated the BötC-induced inhibition of the phrenic nerve. This attenuation showed a post-stimulation time and intensity dependency. Pharmacological experiments showed that intravenous injection of methysergide, a broad-spectrum antagonist of 5-HT receptors, markedly reduced the respiratory facilitation induced by electrical stimulation of the RPa. Furthermore, microinjections of methysergide into the cerebrospinal fluid around the phrenic motor nucleus (PMN) region at spinal cord segments C4 and C5 significantly decreased the RPa-related attenuation effects on BötC-evoked inhibition of phrenic nerve discharge. These results suggest that RPa serotonergic neurons could modulate the inhibition of phrenic nerve activity induced by BötC. Moreover, as the relevant 5-HT receptors for RPa's modulatory effects are located in the cervical spinal cord, 5-HT may, in part, function as a modulator to suppress the BötC neuronal activity via direct RPa-PMN and BötC-PMN convergent projection pathways to phrenic motoneurons.

[Iris recognize-guided LASIK with Zyoptix System for correction of myopic astigmatism].

OBJECTIVE: To assess the safety, efficacy, predictability, stability, and changes in cylindrical degree and axis after iris recognize-guided LASIK with the Zyoptix 4.01 System for the correction of myopic astigmatism. METHODS: The present study was a prospective, nonrandomized, self-controlled trial. Iris recognize-guided LASIK with the Zyoptix 4.01 System (Bausch & Lomb) was performed in 97 patients (183 eyes) with myopic astigmatism in a 6-month trial. Patients were divided into 3 group according to the pre-operative cylindrical degree: Group 1, -0.50 to -1.00D, 79 eyes; Group 2, -1.25 to -2.00D, 70 eyes; Group 3, 2.00 to 4.00D, 34 eyes. They were also grouped by preoperative astigmatism axis: Group A, with the rule (WTR) astigmatism, 126 eyes; Group B, against the rule (ATR), 34 eyes; Group C, oblique axis astigmatism, 23 eyes. After a Hansatome microkeratome cut, iris recognized wavefront-based excimer ablation (Zyoptix 100) was performed. The degree and axis of astigmatism pre-operative and at various times post-operatively (1 day, 1 month, 3 month and 6 months) were analyzed and compared. RESULTS: At 6 month post-operatively, uncorrected visual acuity (VA) was 1.0 or better in 92.3% of the eyes, and 0.5 or better in all eyes. No eye lost > or = 1 lines of best spectacle-corrected VA (BSCVA) at 6 month post-operatively; 60 eyes gained 1 line of BSCVA, and 22 eyes gained 2 lines. The cylindrical degree of astigmatism decreased from (-1.54 +/- 0.65) D of pre-operation to (-0.26 +/- 0.25) D (6 month, post-operatively), the difference between these two groups was statistically significant. The uncorrected VA (UCVA) and the degree of astigmatism returned to normal in the first week and tended to be stable after 1 month. Six months after the operation, WTR astigmatism decreased from106 eyes (per-operation) to 45 eyes (post-operation). ATR astigmatism decreased from 43 eyes (per-operation) to 31 eyes (post-operation). Oblique astigmatism increased from 34 to 38 eyes. Sixty-nine eyes became non-astigmatism 6 months after the operation. CONCLUSION: Iris recognize-guided LASIK using Zyoptix is an effective and safe procedure for the treatment of myopic astigmatism.

Vascular density is highest in the proximal region of the mouse prostate.

BACKGROUND: The proximal region of the prostatic ducts harbor the prostatic epithelial stem cells. As stem cell niches in other organs are highly vascularized, we determined if the proximal region is more highly vascularized than the remaining regions of the prostate. The effect of androgen on vascular density in the different prostatic regions was also examined. METHODS: Sections from prostates were immunostained with antibodies to CD31, and the vascular density in proximal, intermediate, and distal regions was calculated by image analysis software. Vascular density was compared in prostates from castrated mice that received daily inoculations of testosterone or vehicle alone for 3 days. To examine the role of angiogenic factors in the response to androgen, some animals were also treated with soluble VEGF receptor-2-Fc or Tie-2--Fc fusion proteins, which inhibit the activities of VEGF and angiopoietins, respectively. The endothelial proliferative response to androgen was determined by double staining sections with antibodies to CD31 and Ki-67. RESULTS: In prostates from intact mice, vascular density was highest in the proximal region and lowest in the distal region. Administration of testosterone to castrated mice increased vascular density to the greatest extent in the distal and intermediate regions. The increase in vascular density required VEGF and the angiopoietins. Endothelial cell proliferation was less sensitive to androgen in the proximal region than the remainder of the prostate. CONCLUSIONS: Vascular density is highest in the proximal region of the prostate, but the proximal vessels are less responsive to testosterone.

Vascular endothelial growth factor and angiopoietin are required for prostate regeneration.

BACKGROUND: The regulation of the prostate size by androgens may be partly the result of androgen effects on the prostatic vasculature. We examined the effect of changes in androgen levels on the expression of a variety of angiogenic factors in the mouse prostate and determined if vascular endothelial growth factor (VEGF)-A and the angiopoietins are involved in the vascular response to androgens. METHODS: Expression of angiogenic factors in prostate was quantitated using real-time PCR at different times after castration and after administration of testosterone to castrated mice. Angiopoietins were localized in prostate by immunohistochemistry and in situ hybridization. The roles of VEGF and the angiopoietins in regeneration of the prostate were examined in mice inoculated with cells expressing soluble VEGF receptor-2 or soluble Tie-2. RESULTS: Castration resulted in a decrease in VEGF-A, VEGF-B, VEGF-C, placenta growth factor, FGF-2, and FGF-8 expression after 1 day. In contrast, VEGF-D mRNA levels increased. No changes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hepatocyte growth factor, VEGF receptor-1, VEGF receptor-2, or tie-2 mRNA levels were observed. Administration of testosterone to castrated mice had the opposite effect on expression of these angiogenic factors. Ang-2 was expressed predominantly in prostate epithelial cells whereas Ang-1 was expressed in epithelium and smooth muscle. Inoculation of mice with cells expressing soluble VEGF receptor-2 or Tie-2 blocked the increase in vascular density normally observed after administration of testosterone to castrated mice. The soluble receptors also blocked the increase in prostate weight and proliferation of prostatic epithelial cells. CONCLUSION: VEGF-A and angiopoietins are required for the vascular response to androgens and for the ability of the prostate to regenerate in response to androgens.

Phenomenon of non-associative learning in Hering-Breuer reflex simulated by electrical vagal stimulation in rabbits.

The purpose of this study was to explore learning and memory in the Hering-Breuer (HB) reflex simulated by a 60-second-long electrical stimulation of vagus nerve. The responses of phrenic nerve discharge to electrical stimulation (10-100 Hz, 20-60 muA, pulse duration 0.3 ms, for 60 s) of the vagus nerve were observed in rabbits. The results showed that 60-second-long stimulation of vagus nerve produced classic HB reflex, which is composed of two components - lung inflation reflex that is the inhibition of inspiration, and lung deflation reflex that is the facilitation of inspiration. (1) High frequency stimulation (>/=40 Hz, 60 s) of the central end of vagus nerve induced shortening of the inspiratory phase and lengthening of expiratory duration. The inhibitory effect on phrenic discharge was released gradually during sustained vagal stimulation, indicating the habituation of the inhibition. At the cessation of stimulation, the phrenic discharge showed transient post-stimulus rebound. Low frequency stimulation (<40 Hz, 60 s) of the central end of vagus nerve caused an increase in respiratory frequency (f) and shortening of expiratory duration. The excitatory effect on phrenic discharge was also released gradually during the vagal stimulation. The phrenic discharge returned to control level gradually after the removal of the vagal stimulus, indicating short-term potentiation (STP). (2) The habituation of HB reflex was inversely dependent on stimulus intensity and frequency. With an increase in the stimulus frequency or intensity, the degree of the habituation decreased. On the other hand, with the decrease of stimulation intensity and frequency, the degree of the habituation increased. These data indicate a phenomenon of non-associative learning in HB reflex simulated by vagal stimulation. Neural synaptic plasticity and accommodation may exist in the reflex control of respiration in rabbits.

[Effects of protein tyrosine kinase within the brainstem nucleus tractus solitarius on the ventilatory responses of peripheral chemoreflex].

The aim of the present study was to observe whether protein tyrosine kinase (PTK) within the nucleus tractus solitarius (NTS) was involved in the regulation of ventilatory responses of peripheral chemoreflex. The experiments were performed on anesthetized, immobilized and artificially ventilated rabbits. Peripheral chemoreflex was elicited by ventilating the animal with 10% O2-balance 90% N2. Changes in the peak amplitude and frequency of integrated phrenic nerve activity were observed. The ventilatory responses of peripheral chemoreflex following 0.1 microl microinjection within the NTS of either PTK inhibitor genistein (10 mol/L), AMPA glutamate receptor inhibitor CNQX (10 mol/L),or inactive PTK inhibitor daidzein (10 mol/L) were recorded. The results are as follows: Both genistein and CNQX attenuated the ventilatory responses of peripheral chemoreflex, while no changes occurred following daidzein. The amplitude of integrated phrenic nerve discharge and the phrenic burst frequency were decreased by (-21.77+/-6.93)% and (-24.70+/-7.61)% respectively after administration of genistein. CNQX resulted in similar decreases in the amplitude of phrenic nerve discharge (-27.13+/-7.63)% and the burst frequency (-21.34+/-4.88)%. In addition, the inhibitory effects of CNQX and genistein were the same whether they were applied alone or one after another, indicating that they had no cooperative effects. The results obtained suggest that PTK within the NTS regulates the peripheral chemoreflex control of respiration and that this regulation of PTK may be mediated through the phosphorylation of AMPA receptors in NTS neurons.

[The locus coeruleus modulates the inspiratory inhibition induced by electrical stimulation of the Bötzinger complex].

Experiments were done on urethane anesthetized adult rabbits. Long-train electrical stimulation was delivered to the Bötzinger complex (Böt.C) to observe the changes in the peak amplitude of integrated phrenic nerve activity. Then, a long-train electrical stimulation was delivered to the locus coeruleus (LC) or monosodium glutamate was microinjected into the LC . Within a certain period of time, another long-train electrical stimulation was delivered to the Böt.C to observe the responses of phrenic nerve activity. We investigated whether the LC could modulate the inspiratory inhibition induced by electrical stimulation of the Böt.C. The results are as follows: (1) Within a certain period of time after a long-train electrical stimulation applied at the LC, the inspiratory inhibition produced by electrical stimulation at the Böt.C was significantly attenuated. Comparing with the control stimulation that was only delivered at Böt.C without pre-stimulation of the LC, the inspiratory inhibition was decreased by (28.78+/-19.49)%. (2) Similarly, after chemical stimulation of the LC with microinjection of monosodium glutamate, the inspiratory inhibition produced by electrical stimulation of Böt.C was also significantly attenuated [decreased by (19.18+/-8.06)%]. The results obtained suggest that the LC plays a role in the modulation of the inspiratory inhibition of Böt.C stimulation.

[Sustained electrical stimulation of Bötzinger complex on phrenic nerve discharges in rabbits].

AIM: To observe the effects of sustained electrical stimulation at Bötzinger complex (Böt. c) on phrenic nerve discharges. METHODS: Sustained electrical stimulation (10--50 microA, 40-100 Hz, 0.3 ms, for 15-30 s) of Böt. C on 30 urethane anaesthetized, vagotomized, paralyzed and artificially ventilated rabbits. RESULTS: Sustained electrical stimulation of Bot. C produced the inhibition or "inspiratory off-switch" of phrenic discharge during the stimulation. The inhibition of the phrenic discharges showed intensity and frequency dependence. Habituation was shown during the stimulation, showing the magnitude of the phrenic nerve discharge increased gradually. Post-stimulus rebound exhibited upon the cessation of the stimulation, showing the magnitude of the phrenic activity increased significantly. Short-term memory was shown in the habituation of the phrenic activity. CONCLUSION: Non-associative learning is involved in the central control of respiratory modulation in the Böt. C and synaptic plasticity may exist in the respiratory neurons of Böt. C.

Effects of insulin-like growth factor I on steroidogenic enzyme expression levels in mouse leydig cells.

The role of IGF-I in Leydig cell maturation was studied by evaluation of: 1) steady state levels for nine mRNA species expressed specifically in Leydig cells of 35- and 50-d-old IGF-I-null mice and wild-type controls; 2) protein levels for 17 alpha-hydroxylase/C17-20 lyase, cholesterol side-chain cleavage, and type I 5 alpha-reductase (5 alpha R-1) in Leydig cells by immunocytochemistry; and 3) serum testosterone (T) and testicular interstitial fluid IGF-I levels. Expression levels of all mRNA species associated with T biosynthesis were lower in the absence of IGF-I stimulation. In contrast, androgen-metabolizing enzyme mRNA species had either normal (3 alpha-hydroxysteroid dehydrogenase) or higher expression (5 alpha R-1) levels in IGF-I-null mice (P < 0.05) relative to wild-type controls. None of the mRNA species studied changed developmentally in the mutant, whereas there were increases or decreases between d 35 and 50 in normal controls. Parallel trends were observed for average Leydig cell 5 alpha R-1 immunostaining intensity. T levels in mutants were initially higher during d 14-21, equivalent to normal on d 28, and then failed to increase pubertally, remaining at 30% of control levels (P < 0.01) in 90-d-old adult animals. In normal wild-type mice, interstitial fluid and plasma IGF-I levels were highest (P < 0.05) on d 24, indicating that the action of this growth factor on the testis peaks during pubertal development. These results show that in the absence of IGF-I, there is a failure of adult Leydig cells to mature, and that the reduced capacity for T production is caused by disproportionate expression of T biosynthetic and metabolizing enzymes.

Expression of 11beta-hydroxylase in rat Leydig cells.

11Beta-hydroxy (11beta-OH) derivatives of certain steroids function as inhibitors of 11beta-hydroxysteroid dehydrogenase isoform 1 (11betaHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11beta-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11beta-hydroxylation of steroids and thus producing 11beta-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11beta-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11beta-hydroxylated steroids inhibit Leydig cell 11betaHSD1 activities. 11beta-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expression of 11beta-hydroxylase protein in Leydig cells. Moreover, 11beta-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the K(m) and maximum velocity values were 7.28 +/- 0. 92 microM and 1.13 +/- 0.04 micromol/10(6) cell x h, respectively. When assayed in Leydig cells, several 11beta-hydroxylated steroids were efficient inhibitors of 11betaHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11beta-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11betaHSD1.